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Assessment and Impact of Cognitive Impairment in Multiple Sclerosis: An Overview Cognitive impairment affects 40-60% of patients with multiple sclerosis. It may be present early in the course of the disease and has an impact on a patient's employability, social interactions, and quality of life. In the last three decades, an increasing interest in diagnosis and management of cognitive impairment has arisen. Neuropsychological assessment and neuroimaging studies focusing on cognitive impairment are now being incorporated as primary outcomes in clinical trials. However, there are still key uncertainties concerning the underlying mechanisms of damage, neural basis, sensitivity and validity of neuropsychological tests, and efficacy of pharmacological and non-pharmacological interventions. The present article aimed to present an overview of the assessment, neural correlates, and impact of cognitive impairment in multiple sclerosis. # Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Typically, the disease affects the brain, spinal cord, and optic nerves, with acute inflammation as seen during MS relapses, and variable degrees of chronic inflammation and neurodegenerative processes within the white and gray matter, associated with progressive accumulation of disability. In about 85% of the patients, MS begins as a relapsing-remitting course and secondarily evolves to a progressive stage (secondary-progressive MS) in about 15-30% of patients [bib_ref] Long-term evolution of multiple sclerosis disability in the treatment era, Cree [/bib_ref]. From the onset, about 15% of the patients will develop a primary progressive course [bib_ref] Primary-progressive multiple sclerosis, Miller [/bib_ref]. Most people experience their first symptoms of MS between the ages of 20 and 40. The clinical heterogeneity of MS, as well as the findings of different pathological patterns, suggests that MS may be a spectrum of diseases representing different processes [bib_ref] Enviromental triggers of multiple sclerosis, Kakalacheva [/bib_ref] [bib_ref] Role of puberty in multiple sclerosis risk and course, Chitnis [/bib_ref] [bib_ref] Aloisi, F. B cells and multiple sclerosis, Franciotta [/bib_ref] [bib_ref] T cells in multiple sclerosis and experimental autoimmune encephalomyelitis, Fletcher [/bib_ref]. MS can be clinically categorized in different phenotypes, including clinically isolated syndrome (CIS), relapsing/remitting (RRMS), primary progressive and secondary progressive MS (SPMS), and can be subclassified according to its clinical and radiological activity [bib_ref] Defining the clinical course of multiple sclerosis: The 2013 revisions, Lublin [/bib_ref]. These phenotypes are related to potentially different pathophysiological disease mechanisms, including acute/chronic inflammation, axonal/neuronal loss and gliosis, and variable degrees of tissue repair, as well as plasticity and clinical recovery, mainly related to each individual [bib_ref] T cells in multiple sclerosis and experimental autoimmune encephalomyelitis, Fletcher [/bib_ref] , although these differences have yet to be demonstrated at the molecular level. Clinical symptoms of MS may include motor dysfunction (pyramidal); tremor, dysmetria, or ataxia (cerebellar); diplopia or nystagmus (brainstem); numbness (sensory); urinary/bowel hesitancy, incontinence, or retention; disturbances in vision and cognitive impairment. The latter functional systems can be measured with the Expanded Disability Status Scale (EDSS), which range from 0 (normal neurological examination), to 10 (death due to MS) [bib_ref] Rating neurologic impairment in multiple sclerosis: An expanded disability status scale (EDSS), Kurtzke [/bib_ref] , and although it is the most widely used disability score worldwide, cognitive impairment related to the disease seems fairly underrepresented, even when cognitive and neuropsychiatric symptoms are a major cause of disability, loss of employment, and poor quality of life of patients and their families [bib_ref] How does cognition relate to employment in multiple sclerosis? A systematic review, Clemens [/bib_ref]. Although more than a century ago J.M. Charcot described "marked enfeeblement of the memory" with "conceptions that are formed slowly" in persons with "sclérose en plaques" [bib_ref] In Multiple Sclerosis the History of a Disease, Murray [/bib_ref] , this elegant clinical observation was almost forgotten for more than a hundred years as a remarkable symptom of what is now known as MS. In 1991, S. Rao brought renewed attention to cognitive impairment in MS patients [bib_ref] Cognitive dysfunction in multiple sclerosis: Frequency, patterns, and predictions, Rao [/bib_ref]. Since then, it has been a topic of clinical and basic research, trying to reveal the precise mechanisms behind its presentation, in order to develop effective treatments that include cognitive impairment as an outcome in clinical trials, many of them with unsatisfactory results [bib_ref] Cognitive Deficits in Multiple Sclerosis: Recent Advances in Treatment and Neurorehabilitation, Sokolov [/bib_ref]. The following manuscript is not a systematic review about the topic, but an overview that aims to raise awareness on the cognitive deficits in MS, including the most affected cognitive domains and related neuropsychological batteries for their assessment, their neural correlates with an emphasis on neuroimaging, and a potential therapeutic approach as well as future perspectives. ## Cognitive impairment in multiple sclerosis Cognition represents the function of several neural pathways involved in the processing of information in the brain, including several correlated and interdependent cognitive domains such as executive function, perceptual-motor function, language, learning and memory, complex attention, and social cognition, as defined by the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5). Impairment of individual domains may cause dysfunction of the global cognitive performance [bib_ref] Disorders of cognition, Wooddruff [/bib_ref]. Although impairment in cognitive function occurs in different neurologic diseases, the clinical syndromes, the degree of dysfunction, and related disability, depend on the involvement of different brain structures (cortical or subcortical), the extent of neural damage or number of affected domains, and the patient's previous cognitive reserve and performance. In MS, as a heterogeneous disease, all of the aforementioned characteristics makes it even more difficult to study cognition as a single manifestation of the disease [bib_ref] Treatment of cognitive impairment in multiple sclerosis: Position paper, Amato [/bib_ref] [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravaloti [/bib_ref]. Despite advances in knowledge about the neural basis of cognitive function in MS, there are still key uncertainties concerning what it is called 'normal cognition', and consequently with the assessment of cognitive dysfunction, typically defined as a performance below a chosen threshold in a number of cognitive domains, assessed in a specific neuropsychological test (e.g., 1.5-2 standard deviations below normal of a Z-score of one or more cognitive domains). In these batteries, results are commonly expressed as "intact/preserved" or "impaired" [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref] , and prevalent studies usually differ in cognitive impairment definitions. Almost thirty years ago, in a population-based study performed by S. Rao et al., a 45% frequency of cognitive impairment in MS was found [bib_ref] Cognitive dysfunction in multiple sclerosis: Frequency, patterns, and predictions, Rao [/bib_ref]. Other epidemiological studies reported frequencies of cognitive impairment in patients with MS between 40-70% in North America and Europe [bib_ref] Prevalence of cognitive impairment in newly diagnosed relapsing-remitting multiple sclerosis, Digiuseppe [/bib_ref]. Frequencies of 40-60% have been reported in Latin America [bib_ref] Cognitive and neuropsychiatric disorders among MS patients from Latin America, Vanotti [/bib_ref]. Even though a variety of different methodological approaches and neuropsychological batteries have been used, results are very similar across all reported populations. MS is commonly diagnosed during a patient's most productive life period, and employment years and cognitive impairment supposes a severe impact over a patient's behavior, social functioning, adaptative strategies, and profound functional limitations affecting the activities of daily living and employment [bib_ref] How does cognition relate to employment in multiple sclerosis? A systematic review, Clemens [/bib_ref] [bib_ref] Factors related to difficulties with employment in patients with multiple sclerosis: A..., Schiavolin [/bib_ref]. A large cross-sectional study carried out in nine European countries showed that only 35.8% of MS patients were employed. Low mood and cognitive impairment affecting domains like memory, attention, and slowed information processing speed were reported as frequent determinants of work-related difficulties, but only working memory impairment was responsible for higher unemployment rates [bib_ref] Work-related problems in multiple sclerosis: A literature review on its associates and..., Raggi [/bib_ref]. Employment provides higher quality of life, independence, social participation, personal and professional reaffirmation, monetary income, health insurance, financial support for medication, and in some countries access to work benefits and social security [bib_ref] Predictors of quality of life among multiple sclerosis patients: A comprehensive analysis, Yamout [/bib_ref] , so cognition should be a priority in an era with highly active treatments reducing relapses and new lesions, and even new horizons in preventing accumulation of physical disability with new disease modifying treatments available. A review by Shiavolin et al. concluded that difficulties that people with MS can experience with employment are always secondary outcomes of research, and it is quite difficult to address which factors contribute to reduced work participation. In their review, fatigue, mobility reduction, and cognitive impairment were reported as the main drivers of unemployment, and unemployment was related with worse quality of life scores [bib_ref] Factors related to difficulties with employment in patients with multiple sclerosis: A..., Schiavolin [/bib_ref]. In the same line, social cognition has gained relevance as a non-traditional cognitive domain present in MS since early stages of the disease, a domain that has been related to the capability for developing deep social interactions [bib_ref] Social cognition: Concepts, neural basis and its role in multiple sclerosis, Labbé [/bib_ref]. Recent evidence has shown 20% of social cognition impairment among patients, with a similar distribution for different phenotypes [bib_ref] Social cognition according to cognitive impairment in different clinical phenotypes of multiple..., Dulau [/bib_ref] , and social cognition deficits show a significant correlation with the performance in other cognitive domains as working memory, processing speed, and executive functions [bib_ref] Neuropsychological Correlates of Theory of Mind Deficits in Patients with Multiple Sclerosis, Raimo [/bib_ref] [bib_ref] Communication in Multiple Sclerosis: Pragmatic Deficit and its Relation with Cognition and..., Carotenuto [/bib_ref] [bib_ref] Social Cognition Abilities in Patients with Different Multiple Sclerosis Subtypes, Henry [/bib_ref] and exhibit behavioral impact affecting moral evaluation of other individuals' actions [bib_ref] Elevated moral condemnation of third-PARTY violations in multiple sclerosis patients, Patil [/bib_ref]. Finally, cognitive impairment not only affects patients, but also affects their relationship with their families and is a frequent complaint of higher burden for caregivers [bib_ref] Factors related to difficulties with employment in patients with multiple sclerosis: A..., Schiavolin [/bib_ref]. Mickens et al. studied the mediational effect on the relationships between MS impairments (neurological, cognitive, behavioral, emotional, and functional), unmet family needs (household information, financial, social, support, and health), and caregiver mental health (satisfaction with life, anxiety, burden, and depression) using a structural equation model. They suggested that intervention research on MS caregivers in Latin America may consider focusing on caregiver mental health problems by addressing unmet family needs and teaching caregivers' ways to manage the impairments of the individual with MS [bib_ref] Mediational Model of Multiple Sclerosis Impairments, Family Needs, and Caregiver Mental Health..., Mickens [/bib_ref]. ## Cognitive domains All cognitive domains may be affected in MS, but the most affected ones are episodic memory and information processing speed [bib_ref] Cognitive impairment in multiple sclerosis, Chiaravaloti [/bib_ref] [bib_ref] Cognition in multiple sclerosis, Langdon [/bib_ref]. Working memory, executive function, verbal fluency, and attention have also been widely described [bib_ref] Cognitive dysfunction in multiple sclerosis: Frequency, patterns, and predictions, Rao [/bib_ref] [bib_ref] Validity of the Minimal Assessment of Cognitive Function in Multiple Sclerosis (MACFIMS), Benedict [/bib_ref] [bib_ref] MRI predictors of cognitive outcome in early multiple sclerosis, Deloire [/bib_ref] , with a recent interest in social cognition impairment [bib_ref] Social cognition: Concepts, neural basis and its role in multiple sclerosis, Labbé [/bib_ref] [bib_ref] Social cognition in multiple sclerosis: A systematic review and meta-analysis, Cotter [/bib_ref]. Although clinical phenotypes may differ in the prevalence or severity of cognitive impairment, main determinants are physical disability as measured by EDSS, and patients' age [bib_ref] Age and disability drive cognitive impairment in multiple sclerosis across disease subtypes, Ruano [/bib_ref]. Other individual characteristics such as gender, genetic factors, and cognitive reserve may also play a relevant role [bib_ref] Cognitive impairment in multiple sclerosis, Trenova [/bib_ref]. For a summary of the most frequent cognitive domains affected in MS see [fig_ref] Table 1: Frequency of cognitive impairment in patients with multiple sclerosis [/fig_ref]. ## Learning memory Long-term memory refers to the ability to learn new information and to recall that information at a later time point. It is the most consistently affected cognitive domain in MS patients. Impaired learning of new information seems to be the primary problem [bib_ref] Cognitive impairment in multiple sclerosis, Trenova [/bib_ref] , but the encoding, storing, and retrieval from long-term storage processes of memory seems to be affected in MS patients, so there is still controversy about which of these components of memory is the most influential factor for explaining memory deficits [bib_ref] Memory in multiple sclerosis: Contextual encoding defi cits, Thornton [/bib_ref]. Other factors, such as slow processing speed, susceptibility to interference, executive disfunction, and perceptual deficits can also determine poor learning abilities [bib_ref] Prospective memory in multiple sclerosis, Renell [/bib_ref]. ## Complex attention-information processing Complex attention domain involves sustained attention, divided attention, selective attention, and processing speed [bib_ref] Classifying neurocognitive disorders: The DSM-5 approach, Sachdev [/bib_ref]. MS patients usually present with deficits in information processing efficiency, which refers to the ability to maintain and manipulate information in the brain for a short time period (working memory-executive function) [bib_ref] Brain functional and effective connectivity underlying the information processing speed assessed by..., Silva [/bib_ref] and to the speed with which one can process that information (processing speed-complex attention) [bib_ref] Information processing speed in multiple sclerosis: Past, present, and future, Costa [/bib_ref]. It represents a key cognitive deficit in MS patients and might contribute to the presence of impairment in other cognitive domains [bib_ref] Cognitive impairment in multiple sclerosis, Grzegorski [/bib_ref] [bib_ref] Reduced information processing speed as primum movens for cognitive decline in MS, Van Schependom [/bib_ref]. ## Executive function Executive function is a complex domain which involves goal-directed behavior to adapt individuals to changes and demands of the environment, including planning, decision-making, working memory, responding to feedback, inhibition, and flexibility [bib_ref] Classifying neurocognitive disorders: The DSM-5 approach, Sachdev [/bib_ref] , and is affected in around 20% of MS patients. Some studies claim the difficulty to differentiate executive impairment from information processing, due to most of the tests used to evaluate executive function imply integrity of information processing and are affected by emotional affections such as depression. Leavitt et al. [bib_ref] Does slowed processing speed account for executive deficits in multiple sclerosis Evidence..., Leavitt [/bib_ref] studied executive functions and speed tasks (trail making test and Wisconsin card sorting test) in MS patients versus healthy controls. They found that MS patients score worse than controls, but differences decreased when corrected for information processing. They concluded that slow information processing accounts for executive function deficits in MS patients. The difficulty in assessing a specific domain, such as executive function, may be extrapolated to all other domains, as cognitive abilities are assessed individually in optimal environments, but patients usually struggle with managing multiple goals simultaneously [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. ## Language The language domain includes tasks such as object naming, word finding, fluency, grammar and syntax, and receptive language [bib_ref] Classifying neurocognitive disorders: The DSM-5 approach, Sachdev [/bib_ref]. In MS, language deficits have been less studied than episodic memories or information processing speed. While some articles show intact functionality [bib_ref] Cognitive impairments in relapsing-remitting multiple sclerosis: A meta-analysis, Prakash [/bib_ref] , more recent studies report frequencies of language impairment between 20% and 58% in RRMS or SPMS, respectively [bib_ref] Cognitive and Language Deficits in Multiple Sclerosis: Comparison of Relapsing Remitting and..., Ntoskou [/bib_ref]. The most affected tasks seem to be phonological and semantic fluency, although verbal fluency tests are directly influenced by executive functions, thus many of the deficits have been considered as due to a dysfunctional executive syndrome. ## Social cognition Social cognition, including social perception, empathy and theory of the mind, focuses on how people process, store, and apply information about other people and social situations, guiding social interactions [bib_ref] Social cognition: Concepts, neural basis and its role in multiple sclerosis, Labbé [/bib_ref]. Therefore, it is the sum of these processes that allow subjects of the same species to interact and exchange social codes to obtain information about another's behavior, and about the environment [bib_ref] Social cognition in humans, Frith [/bib_ref]. Its recent inclusion within the six main cognitive domains of the DSM-5, and its association with quality of life and employment, have raised awareness among MS researchers in the last years [bib_ref] Social cognition in multiple sclerosis: A systematic review and meta-analysis, Cotter [/bib_ref]. Social perception has been defined as the ability to perceive information about the mental state of other subjects based on behavioral signals [bib_ref] Direct social perception, mindreading and Bayesian predictive coding, De Bruin [/bib_ref]. Empathy refers to the generation of an emotional response in the observer to situations affecting other subjects (e.g., same or different emotion), and it is an essential component of human emotional experience and social interaction, because when an observed mental state is understood, and affective responses are generated, prosocial and cooperative behaviors can exist [bib_ref] The social neuroscience of empathy, Singer [/bib_ref] [bib_ref] social cognition and autism spectrum disorders, Ruggieri [/bib_ref]. Theory of the mind is the ability to represent the psychological perspective of interacting subjects, requiring an internal theorization about their thoughts and beliefs, emotions, affective states, and feelings [bib_ref] Without a theory of mind one cannot participate in a conversation, Baron-Cohen [/bib_ref]. Recent studies have shown 20-40% of social cognition impairment in MS patients, with similar distribution across phenotypes, greater impact in theory of the mind tasks, as well as in the recognition of certain negative facial emotion expressions [bib_ref] Social cognition according to cognitive impairment in different clinical phenotypes of multiple..., Dulau [/bib_ref] [bib_ref] Social cognition in multiple sclerosis: A systematic review and meta-analysis, Cotter [/bib_ref]. It also seems that social cognition interacts with other cognitive domains, although a distinct patter of association with an exclusive domain (e.g., executive functions) has not been demonstrated [bib_ref] Social cognition in multiple sclerosis: A systematic review and meta-analysis, Cotter [/bib_ref] [bib_ref] Relationship between Social Cognition and traditional cognitive impairment in Progressive Multiple Sclerosis..., Ciampi [/bib_ref]. ## Neuropsychological assessment Cognitive function assessment in MS patients should become a part of everyday clinical practice and as a constant outcome in clinical trials. Ideally, every patient with a diagnosis of MS should undergo a complete neuropsychological assessment and routinely repeat a standardized and validated battery to detect clinically meaningful changes, as well as start a timely and effective treatment, similar to what the Magnetic Resonance Imaging in MS (MAGNIMS) group has proposed for the MRI protocols in the diagnosis and monitoring of the disease [bib_ref] Evidence-based guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple..., Rovira [/bib_ref] [bib_ref] Evidence-based guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple..., Wattjes [/bib_ref]. Nonetheless, this desire from the cognitive research community has many obstacles, including key knowledge gaps and methodological limitations related to the understanding and measurement of cognitive deficits, neuroimaging of neural bases and correlations of deficits, as well as the development of effective treatments [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. Mini-Mental State Examination by Folstein in 1975, which was used for dementia, is not sensitive to MS cognitive disorders [bib_ref] Screening for cognitive impairment in multiple sclerosis. An evaluation of the Mini-Mental..., Beatty [/bib_ref]. The three most frequently used neurocognitive batteries in MS are: (1) The Brief Repeatable Battery of Neuropsychological tests (BRB-N), also known as Rao's battery, (2) the minimal assessment of cognitive function in MS (MACFIMS) introduced by Benedict et al. [bib_ref] Validity of the Minimal Assessment of Cognitive Function in Multiple Sclerosis (MACFIMS), Benedict [/bib_ref] , and (3) the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS), a shorter version that was developed in 2012 by an expert team, and is recommended for small centers with one or few staff members who may not have neuropsychological training [bib_ref] Recommendations for a Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS), Langdon [/bib_ref]. All these screening batteries allow to establish the presence or absence of cognitive dysfunction and the specific domains affected. All of them have similarities and differences but share the fact that they are sensitive, specific, and cover the most frequently affected cognitive domains, and are also reasonably brief. It is important to note that BICAMS should not be used within one month of recovery from relapse or within one month of steroid therapy, and the recommended order of administration is first the Symbol Digit Modalities Test (SDMT), then the California Verbal Learning Test (CVLT-II T1-5), and then the Brief Visuospatial Memory Test Revised (BVMT-R T1-3). In most clinical situations, yearly or bi-annual BICAMS evaluations will be appropriate. Emphasis in testing MS cognitive impairment must be focused on the assessment of the most frequently affected domains, learning/memory, and information processing speed. In this context, experts are encouraging the MS multidisciplinary team (e.g., neurologists, nurses, psychologists, speech therapists, etc.) to be trained to use short MS cognitive assessment batteries, such as the BICAMS [bib_ref] Cognitive dysfunction in multiple sclerosis: Frequency, patterns, and predictions, Rao [/bib_ref]. The subtests that compose the structure of domain specific evaluation of these batteries are shown in [fig_ref] Table 2: Comparison of the three most used neuropsychological batteries in MS [/fig_ref]. For the purpose of this review, we will describe the components of the BICAMS battery, due to its extensive use and validation in many countries, as well as the PASAT as being included as the cognitive test in MSFC, as well as a brief summary of social cognition tasks. ## Information processing speed: symbol digit modalities test (sdmt) When SDMT was first published in 1982, there were precedents of similar formats since 1927 and was adopted by the United States Army to assess precisely the speed of substitution of symbols by digits. SDMT has been used in almost every MS cognitive assessment battery and found to be exceptionally reliable and sensitive to assess information processing speed. The test consists of single digits paired with abstract symbols, with rows of the nine symbols arranged pseudo-randomly. The patient must say (or write) the number that corresponds with each symbol. The SDMT can be completed within 5 min, including instructions, practice, and testing. The SDMT has a reported sensitivity of 82% and a specificity of 60% [bib_ref] Multiple Sclerosis Outcome Assessments Consortium. Validity of the Symbol Digit Modalities Test..., Benedict [/bib_ref]. It is the most sensitive task in MS, with good to excellent reliability, well tolerated by patients, has uniformity across languages, with no floor or ceiling effects, and a preliminary clinically meaningful change of 3-4 points [bib_ref] Multiple Sclerosis Outcome Assessments Consortium. Validity of the Symbol Digit Modalities Test..., Benedict [/bib_ref] , so it is recommended for clinical practice and research [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. ## Episodic verbal memory: california learning verbal test (cvlt) This comprises of a 16-item word list, with four items belonging to each of the four categories, arranged randomly. The list is read aloud five times in the same order to the patient, at a slightly slower rate than one item per second. Patients are required to recall as many items as possible, in any order, after each reading. The CVLT-II T1-5can be completed in 5-10 min. It is recommended for clinical use, and it has high sensitivity with good age and sex adjusted normative data [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. ## Episodic visuospatial memory: brief visuospatial memory test revised (bvmt-r) The BVMT-R T1-3 requires the patient to inspect a 2 × 3 stimulus array of abstract geometric figures. There are three learning trials of 10 s. The array is removed, and the patient is required to draw the array from memory, with the correct shapes in the correct position. It is also recommended for clinical and research use and has high sensitivity, it is time efficient, and is well tolerated by patients. Its main disadvantage is for patients with severe motor impairment [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. ## Working memory: paced auditory serial addition test (pasat-3") The PASAT is a measure of cognitive function that specifically assesses auditory information processing speed and flexibility, as well as calculation ability. Stimulus presentation rates were adapted for use with MS patients by Rao and colleagues in 1989, and the measure has been widely used in MS studies since then. Single digits are presented either every 3" (or every 2" for the optional PASAT-2", which could be a more accurate assessment of information processing speed) and the patient must add each new digit to the one immediately prior to it. The test score is the number of correct sums given (out of 60 possible sums) in each trial. To minimize familiarity with stimulus items in clinical trials and other serial studies, two alternate forms have been developed; the order of these should be counterbalanced across testing sessions [bib_ref] Development of a Multiple Sclerosis Functional Composite as a clinical trial outcome..., Cutter [/bib_ref] [bib_ref] The Multiple Sclerosis Functional Composite Measure (MSFC): An integrated approach to MS..., Fischer [/bib_ref]. Although it has been widely used in clinical research and clinical trials, and it has been included within the MSFC, there are several disadvantages to this test including a limited reliability due to practice effects, susceptibility to ceiling effect, poor tolerability due to a patient's math ability, and test-related anxiety. Therefore, it is not recommended for cognitive monitoring in clinical practice, nor for clinical trials designed with multiple administrations, but it is better used as a putative cognitive processing task to compare results across previous studies [bib_ref] Cognition in multiple sclerosis, State of the field and priorities for the..., Sumowski [/bib_ref]. ## Social cognition The assessment of social cognition in MS include a myriad of tests used in other neurological disorders, for example the Face and Emotion Recognition (e.g., Ekman facesfor social perception, Faux Pas, or Reading the Mind in the Eyes tests for theory of the mind tasks, or compound batteries previously used in other neurological disorders such as in frontotemporal dementia (e.g., Social Emotion Assessment [bib_ref] The sea (social cognition and emotional assessment): A clinical neuropsychological tool for..., Funkiewiez [/bib_ref] [bib_ref] Neural correlates of the mini-SEA (Social cognition and Emotional Assessment) in behavioral..., Bertoux [/bib_ref]. For example, the mini-Social and Emotional Assessment test (mini-SEA) includes the Faux Pas and the Face Emotion Recognition. The Faux Pas is comprised by ten narrative vignettes or short stories in which a character inadvertently hurts or offends another, using Theory of the Mind tasks to infer another's mental state, making attributions to their knowledge, beliefs, and emotions. Half of the vignette test is control stories and the other half includes a principal character who inadvertently hurts or offends another, the 'victim of the Faux Pas'. The subject is expected to recognize the situations in which a Faux Pas is committed, why the leading character did it (cognitive theory of mind, he did not mean it), and how the victim of the Faux Pas must have felt (affective theory of mind, we expect him to recognize that the victim must have had a negative emotion). The Face Emotion Recognition consists of 35 pictures for face affect recognition of basic emotions among a list presented at the bottom of the screen including happiness, sadness, anger, surprise, fear, disgust, and neutral [bib_ref] Neural correlates of the mini-SEA (Social cognition and Emotional Assessment) in behavioral..., Bertoux [/bib_ref]. There is still the need for a consensus statement from expert groups to select those tests with best sensitivity, specificity, and reliability in MS. ## Neural basis of cognitive impairment in ms Underlying neural mechanisms of cognitive damage can be related to the inflammatory and neurodegenerative changes in the MS brain, including grey and white matter structures, both globally and regionally, structurally, and functionally [bib_ref] Multiple sclerosis and cognition: Synaptic failure and network dysfunction, Di Filippo [/bib_ref]. Although one can appreciate some of these changes at a single-subject level [fig_ref] Figure 1: Conventional MRI in a patient with multiple sclerosis and cognitive impairment [/fig_ref] , routine measurements (e.g., brain atrophy) are still not suggested to be used in clinical practice, mainly due to biological changes (e.g., dehydration, pseudo atrophy, etc.), that can exceed the accuracy threshold of current brain analysis software [bib_ref] Evidence-based guidelines: MAGNIMS consensus guidelines on the use of MRI in multiple..., Wattjes [/bib_ref]. On the other hand, a myriad of group-analysis studies have been published trying to unveil the neural basis of cognitive impairment in MS. Differences in the results obtained by various studies may represent biased sample selection and differences between the image technology and software utilized in reported studies. Nonetheless, in vivo studies of neural correlations may contribute to early diagnosis, monitoring, and treatment of cognitive impairment in MS. , and her fatigue severity score was 4 (significative fatigue). She had below normal performance (≤1.5 standard deviation) in verbal and visual episodic memory, and in information processing speed tests, with the diagnosis of cognitive impairment according to the MACFIMS battery. Note the widespread brain volume loss including cortical grey matter, ventricular width, and corpus callosum atrophy. The patient gave her written informed consent to present this data. Structural imaging comprise of measurements of brain volume loss (atrophy), which can include global measurements, such as cross-sectional or longitudinal volumetric or 3D whole brain volume loss using semi-automated software (e.g., brain parenchymal fraction (BPF) [bib_ref] A longitudinal study of brain atrophy and cognitive disturbances in the early..., Zivadinov [/bib_ref] , structural image evaluation using normalization of atrophy SIENAX-SIENA [bib_ref] Accurate, robust and automated longitudinal and cross-sectional brain change analysis, Smith [/bib_ref] [bib_ref] Advances in functional and structural MR image analysis and implementation as FSL, Smith [/bib_ref] , regional measurements of different tissues (e.g., grey matter or white matter volume, also measured by SIENAX), or specific grey or white matter structures (e.g., using voxel-based morphometry or Free Surfer for regional tissue volume loss or cortical thickness, respectively [bib_ref] Measurement and clinical effect of grey matter pathology in multiple sclerosis, Geurts [/bib_ref] , as well as manual/linear or 2D assessments such as the Corpus Callosum Index [bib_ref] Corpus callosum index: A practical method for long-term follow-up in multiple sclerosis, Figueira [/bib_ref] [bib_ref] The relationship between total and regional corpus callosum atrophy, cognitive impairment and..., Yaldizli [/bib_ref] [bib_ref] Corpus callosum atrophy and post-surgical seizures in temporal lobe epilepsy associated with..., Uribe-San-Martín [/bib_ref] , or the third ventricle width, the frontal horn width, and the intercaudate distance [bib_ref] Validation of linear cerebral atrophy markers in multiple sclerosis, Butzkueven [/bib_ref]. From a functional point of view, although positron emission tomography (PET) and single photon emission computed tomography (SPECT) have shown correlations between cerebral blood flow and oxygen use with cognitive impairment in MS patients [bib_ref] Regional cerebral blood flow in multiple sclerosis masured by single photon emission..., Lycke [/bib_ref] [bib_ref] Reduced glucose metabolism in the frontal cortex and basal ganglia of multiple..., Roelcke [/bib_ref] , functional MRI (fMRI) has gained its place among cognitive researchers in assessing the neural correlates of disability in MS, with an special emphasis on early changes, with a potential role for treatment monitoring (e.g., during cognitive rehabilitation) [bib_ref] Functional magnetic resonance imaging in the study of multiple sclerosis, Labbé [/bib_ref]. Also, recent interest has developed in the study of water diffusivity in normal-appearing white matter (regions of the white matter where no lesion is seen in conventional MRI studies), using diffusion tensor imaging (DTI), that can be related both with structural and functional disconnection between different regions of the brain [bib_ref] White matter tract abnormalities are associated with cognitive dysfunction in secondary progressive..., Meijer [/bib_ref]. Other non-conventional and advanced MRI techniques are also in study, including magnetization transfer imaging (MTI), proton MR spectroscopy ( 1 H-MRS), and iron imaging [bib_ref] Advanced Structural and Functional Brain MRI in Multiple Sclerosis, Giorgio [/bib_ref]. Cognitive impairment has been associated with linear measure changes [bib_ref] Validation of linear cerebral atrophy markers in multiple sclerosis, Butzkueven [/bib_ref] , the extent of white matter lesions and lesion load [bib_ref] Elevated moral condemnation of third-PARTY violations in multiple sclerosis patients, Patil [/bib_ref] [bib_ref] MRI predictors of cognitive outcome in early multiple sclerosis, Deloire [/bib_ref] [bib_ref] Relationship between Social Cognition and traditional cognitive impairment in Progressive Multiple Sclerosis..., Ciampi [/bib_ref] , focal cortical lesions [bib_ref] Cortical lesions and atrophy associated with cognitive impairment in relapsing-remitting multiple sclerosis, Calabrese [/bib_ref] , whole brain atrophy [bib_ref] Cognitive impairment in relapsing remitting multiple sclerosis can be predicted by imaging..., Summers [/bib_ref] , diffuse cortical atrophy [bib_ref] Cortical lesions and atrophy associated with cognitive impairment in relapsing-remitting multiple sclerosis, Calabrese [/bib_ref] [bib_ref] Independent contributions of cortical grey matter atrophy and ventricle enlargement for predicting..., Tekok-Kilic [/bib_ref] [bib_ref] Cortical atrophy patterns in multiple sclerosis are non-random and clinically relevant, Steenwijk [/bib_ref] , regional grey matter structures such as thalamus, caudate, putamen, hippocampus, and amygdala, cerebellum and corpus callosum [bib_ref] Multiple sclerosis and cognition: Synaptic failure and network dysfunction, Di Filippo [/bib_ref] [bib_ref] Thalamic-hippocampal-prefrontal disruption in relapsing-remitting multiple sclerosis, Kern [/bib_ref] , as well as with widespread subtle pathological changes in normal-appearing white matter [bib_ref] Independent contributions of cortical grey matter atrophy and ventricle enlargement for predicting..., Tekok-Kilic [/bib_ref] , among others. Neuroanatomical correlates of memory deficits seem to differ across disease stages [bib_ref] Mesial temporal lobe and subcortical grey matter volumes differentially predict memory across..., Sumowski [/bib_ref]. Early brain volume loss is a very precise predictor for the presence of cognitive impairment years later [bib_ref] Cognitive impairment in relapsing remitting multiple sclerosis can be predicted by imaging..., Summers [/bib_ref] [bib_ref] Independent contributions of cortical grey matter atrophy and ventricle enlargement for predicting..., Tekok-Kilic [/bib_ref] , and although hippocampal atrophy was not significantly seen in patients with CIS suggestive of MS compared with healthy controls in a study using DTI, hippocampal fractional anisotropy was significantly decreased in CIS patients, and mean diffusivity was correlated with verbal episodic memory performance [bib_ref] Hippocampal microstructural damage correlates with memory impairment in clinically isolated syndrome suggestive..., Planche [/bib_ref]. An interesting study showed that a predictive model of cognitive performance in MS should include bilateral posterior cingulate cortex and bilateral temporal pole cortical thickness, overall white matter lesion load, normal-appearing white matter integrity, and age, reaffirming the multifactorial etiology of MS cognitive impairment [bib_ref] Cortical atrophy patterns in multiple sclerosis are non-random and clinically relevant, Steenwijk [/bib_ref]. Episodic memory has been correlated with total grey matter and regional cortical structures (e.g., left precuneus [bib_ref] Relationship between episodic memory and volume of the brain regions of two..., Aladro [/bib_ref] ; visuospatial memory has been associated with brain MRI total lesion area, T1 lesion, and FLAIR lesion volume, BPF, third ventricular width, and right superior frontal atrophy, among others [bib_ref] Frontal cortex atrophy predicts cognitive impairment in multiple sclerosis, Benedict [/bib_ref] [bib_ref] Thalamic atrophy and cognition in multiple sclerosis, Houtchens [/bib_ref] ; verbal episodic memory has been associated with total and regional hippocampal atrophy, total lesion load and BPF [bib_ref] Frontal cortex atrophy predicts cognitive impairment in multiple sclerosis, Benedict [/bib_ref] [bib_ref] Thalamic atrophy and cognition in multiple sclerosis, Houtchens [/bib_ref] [bib_ref] Regional hippocampal atrophy in multiple sclerosis, Sicotte [/bib_ref] [bib_ref] Hippocampal dysfunction is associated with memory impairment in multiple sclerosis: A volumetric..., González Torre [/bib_ref] ; information processing speed has been correlated with thalamus, whole grey matter atrophy, and third ventricle width [bib_ref] The thalamus and multiple sclerosis: Modern views on pathologic, imaging, and clinical..., Minagar [/bib_ref] , cerebellum atrophy [bib_ref] Information processing speed impairment and cerebellar dysfunction in relapsing-remitting multiple sclerosis, Ruet [/bib_ref] [bib_ref] Posterior lobules of the cerebellum and information processing speed at various stages..., Moroso [/bib_ref] , as well as with less white matter integrity, and increases in functional connectivity [bib_ref] White matter tract abnormalities are associated with cognitive dysfunction in secondary progressive..., Meijer [/bib_ref] ; executive disfunction has been associated with frontal lobe structural and functional damage [bib_ref] Executive function in multiple sclerosis. The role of frontal lobe pathology, Foong [/bib_ref] [bib_ref] Diffusion MRI-based cortical complexity alterations associated with executive function in multiple sclerosis, Muhlert [/bib_ref] and with dorsolateral prefrontal, orbitofrontal, anterior cingulate, and insular areas [bib_ref] Brain activity, regional gray matter loss, and decision-making in multiple sclerosis, Weygandt [/bib_ref] , as well as with thalamic structural and functional changes; PASAT-3" scores have been correlated with cortical and subcortical structures such as bilateral precuneus, posterior cingulate, caudate putamen, and cerebellum [bib_ref] Structural MRI correlates of PASAT performance in multiple sclerosis, Matias-Guiu [/bib_ref] , and acute changes in PASAT score with no physical changes (EDSS) have been associated with presence of acute gadolinium enhancing lesions [bib_ref] Poor PASAT performance correlates with MRI contrast enhancement in multiple sclerosis, Bellmann-Strobl [/bib_ref] , with similar results observe with transient SDMT changes [bib_ref] Isolated cognitive relapses in multiple sclerosis, Pardini [/bib_ref] , proposing that patients could also experience "cognitive relapses". Concerning social cognition, when assessing regional gray matter atrophy in a cohort of progressive MS patients with social cognitive impairment, significant loss was seen within bilateral cortical regions of orbitofrontal, insula and cerebellum, and right regions of fusiform gyrus, and precuneus [bib_ref] Relationship between Social Cognition and traditional cognitive impairment in Progressive Multiple Sclerosis..., Ciampi [/bib_ref] , while functional MRI studies have shown increased activation in the posterior cingulate cortex and precuneus for the identification of anger and disgust faces, and greater activity in the occipital fusiform gyri, the anterior cingulate, and the inferior frontal cortex for the recognition of neutral faces [bib_ref] Cognitively preserved MS patients demonstrate functional differences in processing neutral and emotional..., Jehna [/bib_ref]. Also, increased lesion volume has been correlated with lower success in face emotion recognition [bib_ref] Disconnection mechanism and regional cortical atrophy contribute to impaired processing of facial..., Mike [/bib_ref]. When assessing theory of the mind tasks, it seems that a disconnection syndrome, caused by white matter lesions, could also be one of the possible mechanisms underlying this specific impairment [bib_ref] Disconnection mechanism and regional cortical atrophy contribute to impaired processing of facial..., Mike [/bib_ref] [bib_ref] Cognitive dysfunctions in multiple sclerosis "a multiple disconnection syndrome, Calabrese [/bib_ref] [bib_ref] Social cognition impairments in relapsing-remitting multiple sclerosis, Henry [/bib_ref] [bib_ref] Impairment of social cognition in multiple sclerosis: Amygdala atrophy is the main..., Batista [/bib_ref] [bib_ref] Deficits in Social Cognition: An Unveiled Signature of Multiple Sclerosis, Chalah [/bib_ref]. Other regions of interest associated with cognitive performance in multiple cognitive domains include the thalamus, as a relay station or cortico-subcortical and cortico-cortical networks [bib_ref] Thalamic-hippocampal-prefrontal disruption in relapsing-remitting multiple sclerosis, Kern [/bib_ref] (e.g., global cognitive disfunction, information processing speed, attention, verbal memory, spatial memory, verbal fluency, executive function) [bib_ref] Thalamic atrophy and cognition in multiple sclerosis, Houtchens [/bib_ref] [bib_ref] Clinical significance of atrophy and white matter mean diffusivity within the thalamus..., Benedict [/bib_ref] [bib_ref] Thalamus structure and function determine severity of cognitive impairment in multiple sclerosis, Schoonheim [/bib_ref] [bib_ref] Connectivity-based parcellation of the thalamus in multiple sclerosis and its implications for..., Bisecco [/bib_ref] ; the cerebellum, as a historically understudied region for cognitive performance (attention, working memory, information processing speed, etc.) [bib_ref] Relationship between Social Cognition and traditional cognitive impairment in Progressive Multiple Sclerosis..., Ciampi [/bib_ref] [bib_ref] Information processing speed impairment and cerebellar dysfunction in relapsing-remitting multiple sclerosis, Ruet [/bib_ref] [bib_ref] Posterior lobules of the cerebellum and information processing speed at various stages..., Moroso [/bib_ref] ; and the corpus callosum, the main white matter tract of the brain (e.g., information processing speed, working memory, verbal fluency, etc.) [bib_ref] The relationship between total and regional corpus callosum atrophy, cognitive impairment and..., Yaldizli [/bib_ref] [bib_ref] Corpus callosum atrophy and post-surgical seizures in temporal lobe epilepsy associated with..., Uribe-San-Martín [/bib_ref]. Finally, we would like to highlight advances of fMRI and cognitive research in MS [bib_ref] Functional magnetic resonance imaging in the study of multiple sclerosis, Labbé [/bib_ref] , although there has been some controversies about the real meaning of fMRI results, it seems that early changes can be seen, even in cognitively preserved patients, including higher recruitment of non-related areas, such as supplementary motor cortex during working memory tasks [bib_ref] Novel fMRI working memory paradigm accurately detects cognitive impairment in multiple sclerosis, Nelson [/bib_ref] or by changes in activity properties of regions highly related to cognitive functions, as centrality measures of the default mode network [bib_ref] Increased default-mode network centrality in cognitively impaired multiple sclerosis patients, Eijlers [/bib_ref] , changes that may be used as a biomarker for neurocognitive rehabilitation [bib_ref] A randomised controlled trial of efficacy of cognitive rehabilitation in multiple sclerosis:..., Campbell [/bib_ref] especially, resting state fMRI [bib_ref] Reproducibility of resting state connectivity in patients with stable multiple sclerosis, Pinter [/bib_ref] [bib_ref] Improvement of spasticity following intermittent theta burst stimulation in multiple sclerosis is..., Boutiere [/bib_ref] [bib_ref] Classic Block Design "Pseudo"-Resting-State fMRI Changes After a Neurorehabilitation Program in Patients..., Pareto [/bib_ref]. MS specific disease modifying therapies such as the injectables interferon beta, glatiramer acetate; oral agents such as fingolimod, teriflunomide, or dimethyl fumarate; and monoclonal antibodies such as natalizumab, alemtuzumab, and ocrelizumab, have shown significant benefits in reducing the annualized relapse rate and MRI activity (new T2 or gadolinium enhancing lesions), with a more discrete efficacy over reducing disability progression or the brain atrophy rate. However, their specific impact on cognitive impairment remains unclear, mainly because most phase III clinical trials established cognitive impairment as a secondary or tertiary outcome measure. Comparative efficacy on cognitive outcomes across trials is even more difficult, because of the different neuropsychological batteries used, the varied methods for evaluation and outcome analysis, and the differences between populations included in the trials. Pivotal interferons and glatiramer acetate clinical trials did not include cognitive evaluation as primary endpoints. Intramuscular interferon beta 1a versus placebo included neuropsychological evaluation as a secondary outcome measure and showed 52.7% improvement compared with 29% in the placebo group [bib_ref] Neuropsychological effects of interferón beta 1 a in relapsing multiple sclerosis, Fisher [/bib_ref] , including processing speed and episodic memory outcomes. In the COGIMUS (Cognitive Impairment in Multiple Sclerosis) study, subcutaneous interferon 1a protected RRMS patients from general cognitive decline when reevaluated at 3 [bib_ref] Effects of immunomodulatory treatment with subcutaneous interferon beta-1a on cognitive decline in..., Patti [/bib_ref] and 5 years [bib_ref] Subcutaneous interferon β-1a may protect against cognitive impairment in patients with relapsing-remitting..., Patti [/bib_ref] after therapy onset. Regarding interferon beta 1b, Pishkin reported only improvement of delayed visual reproduction performance [bib_ref] Improved delayed visual reproduction test performance in multiple sclerosis patients receiving interferon..., Pliskin [/bib_ref] , and the Betaferon/Betaseron in Newly Emerging Multiple Sclerosis for Initial Treatment (BENEFIT) trial revealed that in patients with CIS interferon beta 1b had beneficial effects on working memory, and the effects remained over 8 years [bib_ref] Longterm impact of interferon beta-1b in patients with CIS: 8-year follow-up of..., Edan [/bib_ref]. Glatiramer Acetate trials, while included BRB-N evaluation, did not show significant differences versus placebo [bib_ref] Development of a Multiple Sclerosis Functional Composite as a clinical trial outcome..., Cutter [/bib_ref]. The GOLDEN Study using once-daily oral fingolimod was compared with interferon beta 1b using a trial design that included cognitive impairment as the primary outcome. This study showed improvement in cognitive function (BRB-N and DKEFS) in both treatment arms, favoring fingolimod on MRI parameters [bib_ref] Accurate, robust and automated longitudinal and cross-sectional brain change analysis, Smith [/bib_ref] , although some baseline imbalances may have favored the interferon beta 1b arm, according to the authors. In a patient-reported outcomes study, evaluating global satisfaction in switching treatment from several disease modifying drugs to teriflunomide and using SDMT to measure cognitive impairment, results showed that patients and physicians reported stability of cognition in a 48-week period [bib_ref] Neuropsychologic status in multiple sclerosis after treatment with glatiramer, Weinstein [/bib_ref]. Natalizumab pivotal studies showed that it can reduce the risk of progressive working memory impairment by 43% compared with placebo [bib_ref] Additional efficacy endpoints from pivotal natalizumab trials in relapsing-remitting MS, Weinstock-Guttman [/bib_ref]. In a long-term observational study by Jacques et al., natalizumab was reported to preserve cognition over 7 years of continuous therapy using a computed test and the SDMT. No patient showed evidence of sustained cognitive deterioration over a 24-month period [bib_ref] Cognitive evolution in natalizumab-treated multiple sclerosis patients, Jacques [/bib_ref]. In a study including 21 patients during a 15-month follow-up period, alemtuzumab showed stable cognitive function using an extensive neuropsychological battery [bib_ref] Alemtuzumab improves cognitive proccesing speed in active multiple sclerosis-A longitudinal observational study, Riepl [/bib_ref]. Ocrelizumab has shown improvement in MS Functional Composite score (a composite measure of walking speed, upper-limb movements, and cognition assessed by PASAT) compared with interferon beta 1a [bib_ref] Ocrelizumab versus Interferon Beta-1a in Relapsing Multiple Sclerosis, Hauser [/bib_ref]. ## Cognitive impairment-specific treatment The use of acetylcholinesterase inhibitors (AChEI) in multiple sclerosis patients remains controversial. Few studies in a small number of MS patient populations reported contradictory results. While Krupp in 2004 reported the positive impact of donepezil in verbal learning and memory in a cohort of 69 patients [bib_ref] Donepezil improved memory in multiple sclerosis in a randomized clinical trial, Krupp [/bib_ref] , the same investigator reported no significant effect in 2011, which included 120 MS patients [bib_ref] Multicenter randomized clinical trial of donepezil for memory impairment in multiple sclerosis, Krupp [/bib_ref]. It is important to stress that long term treatments with AChEI compels one to be aware of the side effects. Regarding memantine, similar contradictory findings were reported in a small number of studies prevailing negative outcomes for this drug [bib_ref] Memantine for cognitive impairment in multiple sclerosis: A randomized placebo-controlled trial, Lovera [/bib_ref]. Amphetamines significantly improved visuospatial memory and verbal memory [bib_ref] L-amphetamine improves memory in MS patients with objective memory impairment, Sumowski [/bib_ref] , fampridine has shown to be able to improve cognitive fatigue, alertness, psychomotor speed, and verbal fluency [bib_ref] Positive effects of fampridine on cognition, fatigue and depression in patients with..., Broicher [/bib_ref] [bib_ref] Short-term impact of fampridine on motor and cognitive functions, mood and quality..., Pavsic [/bib_ref] , while no benefit on learning were found using modafinil [bib_ref] Cognitive effects of modafinil in patients with multiple sclerosis: A clinical trial, Ford-Johnson [/bib_ref]. ## Non-pharmacological interventions ## Neuropsychological rehabilitation Only recently, neuropsychological rehabilitation has been established as a useful therapeutic tool. Multidisciplinary and cognitive-behavioral interventions, computer-assisted training and combinations of the above, have been showing consistently better results [bib_ref] Cognitive Deficits in Multiple Sclerosis: Recent Advances in Treatment and Neurorehabilitation, Sokolov [/bib_ref] [bib_ref] Pharmacological and Non-pharmacological Therapies of Cognitive Impairment in Multiple Sclerosis, Millera [/bib_ref] , especially when tailored to individual needs. Evidence-based conclusions have only recently become stronger in regards to which interventions may have real benefits for MS patients. In a recent review article and meta-analysis including literature from 2007 to 2016, only one intervention received support for a practice standard in verbal learning and memory (modified Story Memory Technique-mSMT [bib_ref] An RCT to treat learning impairment in multiple sclerosis: The MEMREHAB trial, Chiaravalloti [/bib_ref] , two computer programs received support as a practice guideline for attention and multicognitive domains (Attention Process Training-APT [bib_ref] Computer-assisted rehabilitation of attention in patients with multiple sclerosis: Results of a..., Amato [/bib_ref] and RehaCom [bib_ref] A low-cost cognitive rehabilitation with a commercial video game improves sustained attention..., De Giglio [/bib_ref] , and several studies provided support for the practice option in attention, learning, and memory [bib_ref] Evidenced-Based Cognitive Rehabilitation for Persons with Multiple Sclerosis: An Updated Review of..., Goverover [/bib_ref]. ## Exercise To date, numerous publications have shown the positive impact of physical exercise on different clinical parameters, but evidence remains to be demonstrated, as clinical trials have shown equivocal results [bib_ref] Pragmatic intervention for increasing self-directed exercise behaviour and improving important health outcomes..., Carter [/bib_ref] [bib_ref] Effects of exercise on fitness and cognition in progressive MS: A randomized,..., Briken [/bib_ref]. A systematic review by showed that a few comparable studies did not yield a significant positive impact of physical exercise on cognitive impairment outcomes [bib_ref] Evidence-Based Review of Exercise, Physical Activity, and Physical Fitness Effects on Cognition..., Sandroff [/bib_ref]. Another systematic review of the impact of yoga also failed to show the effect of this discipline in cognitive impairment [bib_ref] Langhorst J Yoga for Multiple Sclerosis: A Systematic Review and Meta-Analysis, Cramer [/bib_ref]. This maybe the result of collectively insufficient well-designed research, and again, the fact that cognitive impairment is maintained as a secondary outcome. The cognitive effects of physical exercise in MS is gaining hype among cognitive researchers, as one relevant intervention both in preventing and improving cognitive outcomes, although clear results, as well as doses and regimens (e.g., aerobic versus weight training), are still missing [bib_ref] Cognitive Deficits in Multiple Sclerosis: Recent Advances in Treatment and Neurorehabilitation, Sokolov [/bib_ref]. # Conclusions In the last three decades, increasing knowledge in the field of cognitive impairment in MS has arisen. From defining the most sensitive neuropsychological tests and compound batteries for clinical practice and research, to better understanding the neural correlates in specific populations with assistance from conventional-structural and non-conventional/functional neuroimaging, better and more effective treatment, rehabilitation, and prevention strategies are being proposed. Cognitive function assessment should be included in the standard clinical evaluation and clinical trials involving MS patients, and treatment strategies should be implemented as supported by current evidence. Limitations are still present, especially due to the validation and standardization of both diagnostic and therapeutic tools. Due to the devastating impact over the working status, social interaction, and self-care of MS patients, improvement in all the aforementioned areas, as well as education to patients, families, and the community should be stated as a priority, and an unmet need. [fig] Figure 1: Conventional MRI in a patient with multiple sclerosis and cognitive impairment. Baseline MRI (A: Sagittal T1, B: Axial FLAIR, C: Coronal FLAIR) from a 15-year-old female with fulminant MS (Marburg variant, EDSS 8.0). After initial aggressive treatment in 2012, including myeloablation with cyclophosphamide, the patient remained asymptomatic without disease modifying treatment, until a second supratentorial motor relapse in 2015, confirming her MS diagnosis and beginning fingolimod. Since then, no relapses or new T2/enhancing lesions have appeared, and she had an EDSS 1.0 by the time of the second MRI in 2018 (D: Sagittal T1, E: Axial FLAIR, F: Coronal FLAIR). Her Mini-Mental State Examination was 30 (normal), Beck Depression Inventory 4 (without depression) [/fig] [table] Table 1: Frequency of cognitive impairment in patients with multiple sclerosis (MS) by cognitive domain. [/table] [table] Table 2: Comparison of the three most used neuropsychological batteries in MS. [/table]

Mini-review: Angiotensin- converting enzyme 1 (ACE1) and the impact for diseases such as Alzheimer’s disease, sarcopenia, cancer, and COVID-19 Ageing has been associated with comorbidities, systemic low-grade of inflammation, and immunosenescence. Hypertension is the most common morbidity and antihypertensives are used for more than 50%. Angiotensin-converting enzyme 1 inhibitors (ACEi) and angiotensin II receptor blockers (ARB) control blood pressure but also seem to play a role in comorbidities such as Alzheimer's disease, sarcopenia and cancer. The impact of anti-hypertensives in comorbidities is due to the expression of renin-angiotensin system (RAS) in several tissues and body fluids. Angiotensin-converting enzyme 1 (ACE1) has been linked to oxidative stress, metabolism, and inflammation. The levels and activity of ACE1 are under genetic control and polymorphisms have been correlated with susceptibility to Alzheimer's disease. In addition, some results found that ACEi and ARB users present delayed cognitive decline and reduced risk of dementia. Regarding to sarcopenia, RAS has been linked to the catabolic and anabolic pathways for muscle mass maintenance. In some studies, older adults using ACEi were highly benefited by exercise training. In cancer, RAS and its products have been shown to play a role since their inhibition in animal models modulates tumor microenvironment and improves the delivery of chemotherapy drugs. Clinically, the incidence of colorectal cancer is reduced in patients using ACEi and ARB. During the pandemic COVID-19 it was found that ACE2 receptor plays a role in the entry of SARS-CoV-2 into the host cell. ACE1 genotypes have been linked to an increased risk for COVID-19 and severe disease. In some studies COVID-19 patients taking ARB or ACEi presented better outcome. # Introduction Ageing is a complex process which has been associated with comorbidities, systemic lowgrade of inflammation, and changes in the frequency/function of immune cells (immunosenescence) [bib_ref] Ageing and myeloid-derived suppressor cells: Possible involvement in immunosenescence and age-related disease, Bueno [/bib_ref] [bib_ref] Parameters of the immune system and vitamin D levels in old individuals, Alves [/bib_ref] [bib_ref] Immunosenescence: participation of T lymphocytes and myeloid-derived suppressor cells in aging-related immune..., Alves [/bib_ref]. A comorbidity with high impact in older individuals is hypertension which can affect 27% of individuals younger than 60 years and 74% of older adults with more than 80 years [bib_ref] Hypertension in adults across the age spectrum: Current outcomes and control in..., Lloyd-Jones [/bib_ref]. Hypertension in the older adults has been linked to an increased risk of ischemic and hemorrhagic strokes, vascular dementia, Alzheimer's disease, coronary artery disease, atrial fibrillation, chronic kidney disease and retinal diseases [bib_ref] Effect of treating isolated systolic hypertension on the risk of developing various..., Perry [/bib_ref] [bib_ref] Pulse pressure and risk for myocardial infarction and heart failure in the..., Vaccarino [/bib_ref] [bib_ref] Results of the pilot study for the hypertension in the very elderly..., Bulpitt [/bib_ref] [bib_ref] Cardiovascular risk factors for Alzheimer's disease, Rosendorff [/bib_ref]. Non-pharmacological approaches includes healthy diet, physical activity, non-smoking, avoidance of high intake of alcohol, among others. However, pharmacological interventions can be required, and the benefits on cardiovascular outcomes can be reached by thiazide diuretics, angiotensin-converting enzyme inhibitor (ACEi), angiotensin II receptor blockers (ARB), and calcium channel blocker (CCB). [reviewed in]. In this mini-review it is not our goal to discuss hypertension and its treatment, since there are excellent articles on the field [bib_ref] Challenges in the management of hypertension in older populations, Pont [/bib_ref] [bib_ref] Multi-trajectories of systolic and diastolic hypertension and coronary heart disease in middle-aged..., Li [/bib_ref] and the topic itself would require a new whole article. Instead, our aim is to discuss angiotensin-converting enzyme 1 (ACE1) expression and the possible link with age-related diseases such as Alzheimer's disease, sarcopenia, cancer, and COVID-19. In addition, we will describe findings on how ACE1 inhibition/blockade can interfere with the outcome of older patients from these conditions. However, it has to be pointed that the benefits observed by the use of anti-hypertensives in some age-related diseases can be linked, at least in part, to the control of high blood pressure. The angiotensin-converting enzyme 1 (ACE1) is a dipeptidyl carboxylase which can be inhibited leading thus to the reduction of blood pressure via the renin-angiotensin system (RAS). In a summary definition, RAS is composed by a vasoconstrictor, pro-inflammatory ACE1/AngII/AT 1 R axis, and a vasodilating anti-inflammatory ACE2/ Ang1-7/MasR axis. In addition to the blood pressure control, ACE1 and its peptide substrates affect cardiovascular and renal function, hematopoiesis, reproduction, and the immunity [bib_ref] A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting..., Bernstein [/bib_ref] [bib_ref] Update on RAAS modulation for the treatment of diabetic cardiovascular disease, Bernardi [/bib_ref]. ACE1 expression has been observed not only in tissues since its soluble form was found in urine, serum, seminal fluid, amniotic fluid, and cerebrospinal fluid [bib_ref] Angiotensin converting enzyme: Implications from molecular biology for its physiological functions, Hooper [/bib_ref]. Because of its wide expression in several tissues and body fluids, ACE1 has been associated to some diseases development/ progression. Recently it has been shown, mainly in experimental models, that ACE1 can interfere with several processes in the organism through mechanisms such as oxidative stress, metabolism, and inflammation [bib_ref] Enalapril and captopril enhance glutathione-dependent antioxidant defenses in mouse tissues, De Cavanagh [/bib_ref] [bib_ref] Physiology and pathophysiology of the adipose tissue renin-angiotensin system, Engeli [/bib_ref] [bib_ref] Angiotensin II and endothelin induce inflammation and thereby promote hypertension-induced endorgan damage, Muller [/bib_ref]. It has also been suggested that ACE1 influences age-related diseases (i.e., Alzheimer's disease-AD, sarcopenia, cancer) [bib_ref] The ectopeptidases CD10, CD13, CD26, and CD143 are upregulated in gastric cancer, Carl-Mcgrath [/bib_ref] [bib_ref] Physical function is weakly associated with angiotensin-converting enzyme gene I/D polymorphism in..., Yoshihara [/bib_ref] [bib_ref] Angiotensin I-converting enzyme gene plays a crucial role in the pathology of..., Zhang [/bib_ref] [bib_ref] Dysregulation of ACE-1 in normal aging and the early stages of Alzheimer's..., Maclachlan [/bib_ref]. ACE1 levels are under genetic control and many studies have focused on insertions and deletions polymorphisms in intron 16 of the ACE gene as a marker for a functional polymorphism. Many single nucleotide polymorphisms have been detected in the ACE1 gene in recent years and the search for the locations of functional polymorphisms currently represents a topic of extensive investigation. For example, it has been shown that ACE1 polymorphisms are correlated with susceptibility to ADbut the associated mechanisms are still poorly understood. In addition, it was found recently that in normal ageing, ACE1 expression is increased in brain homogenates and this expression is unchanged in the early stages of AD [bib_ref] Dysregulation of ACE-1 in normal aging and the early stages of Alzheimer's..., Maclachlan [/bib_ref] Regarding sarcopenia, [bib_ref] Physical function is weakly associated with angiotensin-converting enzyme gene I/D polymorphism in..., Yoshihara [/bib_ref] found a weak correlation between ACE1 polymorphism and physical function in aged individuals. In cancer (gastric or colorectal), the same patient presented higher expression of ACE1 in tumor microenvironment than in healthy tissues [bib_ref] The ectopeptidases CD10, CD13, CD26, and CD143 are upregulated in gastric cancer, Carl-Mcgrath [/bib_ref] [bib_ref] Angiotensin I-converting enzyme gene plays a crucial role in the pathology of..., Zhang [/bib_ref]. In the immune system, our group found that ACE1 (CD143) is expressed not only in all subsets of CD4 + and CD8 + T cells (naive, central memory, effector memory, and effector memory re-expressing RA), but also in B cells (naive, unswitched memory, switched memory, double negative) and in myeloid cells from PBMC of ageing individuals. Within the immune system, ACE1 has been shown to have both beneficial and detrimental effects, due to its role in signalling pathways [bib_ref] Rediscovering ACE: Novel insights into the many roles of the angiotensin-converting enzyme, Gonzalez-Villalobos [/bib_ref]. In older adults with significant hypertension it was found that ACE1 inhibitors (ACEi) interfere with cytokines secretion by stimulated T cells in culture [bib_ref] Effect of converting enzyme inhibitor captopril on T cell functions in essential..., Shasha [/bib_ref]. In PBMC from controls, it was also observed that ACEi in culture suppress the production of cytokines by monocytes and T cells, confirming the role of ACE in inflammatory signalling pathways [bib_ref] Captopril and lisinopril suppress production of interleukin-12 by human peripheral blood mononuclear..., Constantinescu [/bib_ref]. These findings suggest a role played by ACE1 in the agerelated chronic diseases via inflammation, but the associated mechanisms are not completely understood yet. It is important to know that the effects of the treatment of individuals with ageassociated conditions and/or diseases with ACEi is strictly dose-, host-, and disease-dependent. SARS-CoV-2 has been the cause of more than 6 million of deaths worldwide (WHO, 2023) and as the first step for the entry into the host cell the virus binds the human angiotensin-converting enzyme (ACE) 2 receptor. Moreover, polymorphisms of ACE1/ACE2 and inhibition/ blockade of ACE1 have been associated with patient outcome from COVID-19. Therefore, considering the major impact of infections on aged adults, mainly in those with chronic diseases this mini-review will focus on ACE1 and ACEi and their possible correlation with important age-related conditions such as Alzheimer's disease, sarcopenia, cancer and SARS-CoV-2 infection. Figure 1. ## Ace1 and cognition/dementia/alzheimer's disease The extension of life expectancy has been related to an increase in cases of dementia and loss of cognitive functions. The most common type of dementia in older adults is AD which has been characterized by reduced synaptic strength, synaptic loss, and neurodegeneration. The hallmark of this disease is the accumulation of senile plaques and neurofibrillary tangles that are respectively associated with changes in the cleavage in amyloid precursor protein (APP)/production of the beta-amyloid (Aβ) and tau protein hyperphosphorylation [bib_ref] Aging and Alzheimer's disease pathology, Sengoku [/bib_ref]. However, it has been proposed that AD clinical symptoms cannot be explained only by the neuropathology of abnormal protein accumulation. As the AD pathogenesis is not completely understood, the current treatments lead to a modest effect in cognition, and therefore it is desirable to develop therapies that can modify the disease development and progress. A potential target is the RAS which has been suggested to play a role in AD. ACE inhibitors (ACEi) or blockers of Angiotensin II receptors (ARB) have been shown to delay the cognitive decline and reduce the risk of dementia [reviewed in [bib_ref] Brain reninangiotensin system as novel and potential therapeutic target for Alzheimer's disease, Loera-Valencia [/bib_ref] ]. [bib_ref] Associations of angiotensin converting enzyme-1 and angiotensin II blood levels and cognitive..., Yasar [/bib_ref] found that cognitively normal older adults (mean age 67.8 years) with elevated ACE1 serum levels at the study entry, presented a worse 1 year evolution in processing speed and work memory suggesting a role played by RAS in neurodegenerative Frontiers in Aging frontiersin.org diseases. This hypothesis is reinforced by findings of circulating AngII in regions without blood brain barrier (BBB) and the expression of RAS components in brain. Moreover, ACE1 (acts in the generation of AngII) is expressed in the frontal and temporal lobe and its expression is altered in patients with AD [bib_ref] Angiotensin converting enzyme in Alzheimer's disease increased activity in caudate nucleus and..., Arregui [/bib_ref]. In opposition, MacLachlan et al. found that in normal ageing, human brain homogenates presented increase of ACE1 and AngII protein levels and decrease of ACE1 activity whereas in early stages of AD it was observed increased ACE1 activity and unchanged ACE1 and AngII protein levels [bib_ref] Dysregulation of ACE-1 in normal aging and the early stages of Alzheimer's..., Maclachlan [/bib_ref]. In post-mortem brain of older adults (78.5 years) it was found that ACE1 activity correlated inversely with ACE2 activity in AD. Brain homogenates displayed significantly reduced activity of ACE2 which was related to the disease stage. ACE2 activity correlated inversely with total insoluble amyloid β (Aβ) levels, β-secretase activity and protein tau (ptau) load. Authors suggested that the imbalance between classical ACE1 axis and the regulatory ACE2 axis of RAS plays a role in AD pathogenesis. In older adults with memory complaints or AD and no antihypertensive treatment, it was found that ACE1 levels and activity in cerebral spinal fluid (CSF) and serum were reduced in patients with AD. Lower ACE1 in CSF correlated with lower CSF Aβ levels, indicating more brain pathology. Reduced ACE1 in CSF was also linked to lower CSF tau and ptau levels [bib_ref] The association of angiotensin-converting enzyme with biomarkers for Alzheimer's disease, Jochemsen [/bib_ref]. Regarding patients using anti-hypertensives, [bib_ref] The use of angiotensinconverting enzyme inhibitors and diuretics is associated with a..., Yasar [/bib_ref] evaluated women (70-80 years) for 9 years and observed that the use of ACEi was associated with 59%-85% lower risk of impairment in speed of processing, executive functioning, and verbal memory. In another study, the use of ACEi during 3 years of follow-up showed that anti-hypertensives with BBB crossing status were associated with 65% less decline in instrumental activities of daily living per year compared to other anti-hypertensive drugs. Anti-hypertensives with no BBB crossing status were associated with increased risk of dementia by 20% per year of medicament exposure. In cognitively normal hypertension-treated older adults (65-84 years and follow-up of 3.5 years) the use of BBB crossing ACEi was associated with a reduced risk of mild cognitive impairment. In a meta-analysis (14 cohorts/6 countries) with a total of 12,849 (50-90 years) individuals it was shown that anti-hypertensive BBB crossing drugs were associated with a better memory recall over a follow-up of 3 years.evaluated hypertensive patients using at the time of Alzheimer's diagnostic centrally acting ACEi (C-ACEi, n = 1,207), non-centrally acting ACEi (NC-ACEi, n = 143) and 3,910 using neither. Improved cognition was observed in patients using C-ACEi over the first 9 months after diagnosis in comparison with NC-ACEi. Long-term differences were not found in cognition and survival between the groups. In conclusion, anti-hypertensive medications reduce the risk of cognitive impairment but it remains unclear whether the benefits are reached via blood pressure control alone or via action on the RAS components. However, [bib_ref] Angiotensin-converting enzyme inhibitors and Alzheimer's disease progression in older adults: Results from..., Soto [/bib_ref] found in patients (mean age 77.8 years) with AD and using ACEi that the slow rate of cognitive decline over 4 years of follow-up was independent of hypertension at baseline or developed subsequently. In addition, human cortical neuron cell line cultured with Losartan displayed decline in the activation of tau kinases, production of p-tau and reactive oxygen species [bib_ref] Renin-angiotensin-system increases phosphorylated tau and Reactive Oxygen Species in human cortical neuron..., De Dios [/bib_ref] suggesting reduction of neurotoxicity and neuron death which are causes of dementia. ## Ace1 and sarcopenia/exercise Essential daily activities can be impaired in older adults because of physical limitations. During the aging process muscle mass can be ## Figure 1 The renin-angiotensin system (RAS): ACE -angiotensin converting enzyme, AT1R -Angiotensin II type 1 receptor, AT2R -Angiotensin II type 2 receptor. Frontiers in Aging frontiersin.org reduced and the remaining muscle may present reduced quality. Sarcomeric proteins, collagen, fibers and myocites relies on the balance between catabolic and anabolic pathways for muscle mass maintenance. In addition, the replacement of muscle fibers by fibroblasts, extracellular matrix proteins, and conective tissue caracterizes the histological quality and quantity of the muscle. The renin-angiotensin system (RAS) modulates all these processes via protein turnover, cellular apoptosis, and collagen metabolism [bib_ref] Fibrotic response induced by angiotensin-II requires NAD(P)H oxidaseinduced reactive oxygen species (ROS)..., Cabello-Verrugio [/bib_ref]. Decreased physical function in the ageing population has been related with disability, loss of independence, higher risk of cardiovascular morbidity and mortality [bib_ref] Association of long-distance corridor walk performance with mortality, cardiovascular disease, mobility limitation,..., Newman [/bib_ref] [bib_ref] Gait speed and survival in older adults, Studenski [/bib_ref]. In addition, chronic diseases (i.e., hypertension) could exacerbate the process of sarcopenia in aged individuals [bib_ref] Low-intensity exercise as a modifier of physical frailty in older adults, Brown [/bib_ref] [bib_ref] The effects of multidimensional home-based exercise on functional performance in elderly people, Nelson [/bib_ref]. In hypertensive middle-aged patients and also in healthy ageing males it was shown an inverse correlation between blood pressure and ACE1 activity in vastus lateralis muscle suggesting that ACE1 levels in muscle are influenced by hemodynamic factors [bib_ref] Skeletal muscle angiotensin-converting enzyme and its relationship to blood pressure in primary..., Reneland [/bib_ref]. However, in spite of ACE1 activity identification in vastus lateralis muscle, no correlation was found between these measurements and age [bib_ref] Angiotensin-converting enzyme in human skeletal muscle. A simple in vitro assay of..., Reneland [/bib_ref]. The evaluation of ACE1-mRNA transcripts in vastus lateralis muscle by [bib_ref] Angiotensinconverting enzyme gene expression in skeletal muscle in patients with chronic heart..., Schaufelberger [/bib_ref] found no difference in ACE1-mRNA transcripts between hypertensive patients and control individuals nor differences in ACE1 gene expression was observed due to ACEi treatment. More recently it has been suggested that ACE1 inhibitors (ACEi) could improve physical function not only via regulation of blood pressure but also through direct effects on body composition and secreted factors [bib_ref] Angiotensinconverting enzyme inhibition intervention in elderly persons: Effects on body composition and..., Carter [/bib_ref]. In older adults the muscle wasting linked to AngII is due to the decrease of the growth factor 1 (IGF-1) and since the use of ACEi was capable to elevate the levels of IGF-1, it has been suggested a beneficial role played by ACEi [bib_ref] Effects of ACE-inhibition on IGF-1 and IGFBP-3 concentrations in older adults with..., Giovannini [/bib_ref]. evaluated older adults (70-89 years old) with mild to moderate functional deficits and found that ACEi users were highly benefited by 12 months exercise training than non-users. In opposition,observed that in functionally impaired older individuals (n = 170, mean age-75 years), ACEi did not enhance the effect of 20 weeks exercise training. In agreement,found that even though older patients (79-82 years old) not taking ACEi presented a negative correlation between muscle power/muscle contraction and ACE activity, patients taking ACEi showed no consistent association with handgrip strength, muscle power, muscle contraction velocity, and functional performance. [bib_ref] ACE inhibitors, statins and thiazides: No association with change in grip strength..., Witham [/bib_ref] followed 639 individuals (mean age 65 years) during 4.4 years and observed no difference in grip strength change per year in ACEi users. However, in patients with knee osteoarthritis, a study of 8 years follow-up of 4,295 individuals (mean age 61.2 years) showed that ACEi use (12.8% of participants) was correlated with a reduced risk of frailty. Preliminary findings have also indicated beneficial effects of other RAS inhibitors such as ARBs and renin inhibitors, mostly because of their inhibitory effects on local inflammation and oxidative stress. Sarcopenia diagnostic is complex and should consider not only muscle loss and impaired function, but also how the age-related decline in cognition affects for example grip strength and gait speed. The results from ACEi effects on muscle mass and function are controversial and are probably linked to differences in the enrolled populations, parameters used to assess muscle mass and strength, period of intervention and follow-up, anti-hypertensive used and protocols of physical activity. Further studies are required to support the evidence of ACEi and ARB beneficial effect in preventing sarcopenia. ## Ace1 and cancer Cancer is highly incident in ageing individuals, and considering that age is a risk factor for hypertension and requires the use of antihypertensive drugs, these pharmacological treatments have been studied to determine whether they interfere with cancer development and progression [bib_ref] Nonclassic endogenous novel [corrected] regulators of angiogenesis, Ribatti [/bib_ref]. Renin-angiotensin system (RAS) and its products have been linked to tumor growth since AT2, the active fraction derived from angiotensin activity is associated to cellular growth, angiogenesis induction, and activation of AT1R receptor. Intracellular signalling pathways of AT1R are linked to fibroblasts growth factor, epidermal growth factor, tumor growth factor (TGF-) beta, platelets-derived growth factor, nitric oxide synthase, protein kinase C, angiopoiethin 2, and metalloprotease [reviewed in [bib_ref] The renin-angiotensin system and cancer: Old dog, new tricks, George [/bib_ref] ]. Experimental studies have linked the RAS to cancer development. In addition, ACEi or ARBs inhibit experimental tumor growth [reviewed in [bib_ref] The renin-angiotensin system and cancer: Old dog, new tricks, George [/bib_ref] ]. Although the antitumoral mechanism associated with ACEi, and ARBs has not been elucidated yet, it has been proposed a role for Ang II-dependent signalling in inhibiting angiogenesis, inflammation, and cellular proliferation [bib_ref] Angiotensin II, cell proliferation and angiogenesis regulator: Biologic and therapeutic implications in..., Escobar [/bib_ref]. Inhibition of the RAS pathway, indeed, actively modulates the tumor microenvironment, modifies the tumor stroma, reduces the stiffness of the matrix and ultimately improves the delivery of chemotherapeutic drugs, suggesting that ACEi and ARBs could provide a complementary treatment in cancer patients. A change in the immune milieu, and in the modulation of macrophages, CD8 + T cells and T regulatory cells has also been reported [bib_ref] Angiotensin II, cell proliferation and angiogenesis regulator: Biologic and therapeutic implications in..., Escobar [/bib_ref]. In human lung adenocarcinoma, it was observed that genes encoding for ACE and for the angiotensin II receptor were repressed whereas genes encoding for angiotensinogen were overexpressed [bib_ref] Alterations in gene expression of components of the renin-angiotensin system and its..., Goldstein [/bib_ref]. In opposition, AGTR1 (encode AT1 angiotensin II receptor) mRNA was increased in estrogen receptor-positive breast cancer and hormone-independent prostate cancer. Data with aggregated results of randomized controlled trials (RCT) aim to evaluate the possible increased risk of developing cancer in patients using ACEi or ARBs. In five RCT it was observed a discrete increase in the risk of cancer with the use of ARBs. Patients with cancer such as breast, prostate and lung presented a higher occurrence of new lung cancer if receiving ARB. However, in a meta-analysis of 70 RCT (324,168 participants) it was not found any difference in the risk of cancer with ARB or ACEi versus placebo . The study of 15 multicenter double-blind RCT with 138,769 participants and a follow-up of 12 months showed no site-specific cancer incidence (lung, breast, prostate) in patients using ARB versus controls. In a systemic review (meta-analysis-12 publications) Datzman et al. (2019) evaluated ARB and carcinogenesis as primary outcome and concluded that there is no correlation Frontiers in Aging frontiersin.org between ARB therapy and the increase in the risk of lung cancer. However, a large cohort in Caucasian patients showed an augmented risk of lung cancer (14% after 5 years) associated with ACEi therapy and increase related to years of using the medicaments. Studies in patients with advanced non-small cell lung cancer (NSCLC) showed no detrimental effect of RAS-blockers in the patient outcome [bib_ref] Renin-Angiotensin system blockers may prolong survival of metastatic non-small cell lung cancer..., Aydiner [/bib_ref] [bib_ref] Effect of angiotensin system inhibitors on survival in patients receiving chemotherapy for..., Menter [/bib_ref]. ## Ace1 and colorectal cancer Colorectal cancer (CRC) is highly incident in older individuals and a significant percentage of patients suffers from cardiovascular diseases. Data from 2005 to 2008 with 12,648 metastatic CRC patients showed that 52% were 65 years old or more and 48.3% were hypertensive. In this study antibiotics and anti-hypertensives were the most used medicaments (61.7% and 49.7% respectively) [bib_ref] Comorbid conditions in patients with metastatic colorectal cancer, Fu [/bib_ref]. The use of anti-hypertensive drugs (ACEi and ARBs) have been extensively evaluated in CRC because there is more convincing evidence of cancer risk reduction in patients taking these medicaments. A meta-analysis (6 studies, 113,048 patients) performed by [bib_ref] Angiotensinconverting enzyme inhibitors/angiotensin receptor blockers therapy and colorectal cancer: A systematic review..., Dai [/bib_ref] found that the incidence of CRC was significantly reduced in patients using ACEi/ ARB than in non-users. Moreover, this study showed that patients using ACEi/ARB presented a better outcome in CRC. In patients with negative CRC colonoscopy, the use of ACEi or ARB (for at least 180 days) was evaluated and correlated with tumor development between 3 and 36 months after the negative diagnosis. In 3 years of follow-up, patients using ACEi/ARB (n = 30,856, 61-78 years) showed significantly lower incidence of CRC than non-users [bib_ref] The therapeutic potential of angiotensin-converting enzyme and angiotensin receptor inhibitors in the..., Asgharzadeh [/bib_ref]. A retrospective study with 13,982 patients (65 years or more) diagnosed with CRC found that the use of ACEi, beta blockers and thiazide diuretics was associated with reduced mortality. It was also found correlation between adherence to therapy (antihypertensive) and decreased specific mortality to CRC. The authors suggested that these drugs could be used in addition to the anti-tumor therapy for the treatment of CRC in stages I-III [bib_ref] ACE (Angiotensin-Converting enzyme) inhibitors/angiotensin receptor blockers are associated with lower colorectal cancer..., Cheung [/bib_ref] [bib_ref] Associations between initiating antihypertensive regimens on stage I-III colorectal cancer outcomes: A..., Balkrishnan [/bib_ref]. [bib_ref] Increased tumor response to neoadjuvant therapy among rectal cancer patients taking angiotensin-converting..., Morris [/bib_ref] evaluated patients with rectal cancer treated with surgery and adjuvant radiology and found that users of ACEi/ARB presented a significant positive correlation between anti-hypertensive drugs and complete pathological response after therapy. Authors concluded that ACEi/ARB can modulate the tumor response to neoadjuvant therapy em patients with in situ rectal cancer in advanced stage. [bib_ref] Exposure to ACEI/ ARB and β-blockers is associated with improved survival and..., Engineer [/bib_ref] found that patients (60-65 years) with stage III CRC, taking ACEi/ARB drugs or beta blockers and treated with chemotherapy or radiotherapy presented decrease in the tumor progression, less hospitalization, and reduced mortality in comparison with nonusers. In contrast, observed that patients taking ACEi or ARB, and with metastasis in regional lymph node, history of adenocarcinoma, neoadjuvant therapy, and rectal resection presented reduced survival when compared with non-users. The small number of patients and the more severe disease could be the reason for the difference in Zeman's results. In summary, the association between the use of antihypertensive drugs and the risk and prognosis of cancer remains inconclusive. In colorectal cancer, the benefit of medicaments such as ACEi and ARB are more evident and some authors suggest that these medicaments could be used in association with chemotherapy, radiotherapy, and check-point blockade with the aim to improve the efficacy of the anti-tumor therapy. ## Ace1 and covid-19 Considering that COVID-19 and other infectious diseases have a major impact in aged adults, mainly in those with chronic diseases (i.e., hypertension and users of ACEi or ARBs) the association between SARS-CoV-2 infection/outcome and renin-angiotensin system (RAS) has been proposed. On cell surface, SARS-CoV-2 binds the human angiotensinconverting enzyme (ACE)2 receptor and the transmembrane protease (TMPRSS)2 contributes for the fusion between the virus membrane and the cell membrane with subsequent entry of the virus into the host cell. ACE2 and TMPRSS2 are expressed extensively in organs such as lung, heart, kidney, gastrointestinal tract, among others and have been linked to the SARS-CoV-2 widespread infection [bib_ref] Coronavirus disease 2019 and cardiovascular system: A narrative review, Kwenandar [/bib_ref] [bib_ref] COVID-19 pathophysiology: A review, Yuki [/bib_ref] [bib_ref] Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential..., Zou [/bib_ref] [bib_ref] Computational analysis of TMPRSS2 expression in normal and SARS-CoV-2-infected human tissues, Cao [/bib_ref] [bib_ref] Anosmia/hyposmia is a good predictor of coronavirus disease 2019 (COVID-19) infection: A..., Hariyanto [/bib_ref]. Using systematic review with metaanalysis, several studies have shown some level of correlation between polymorphisms of RAS-related genes and increased risk of developing severe COVID-19 [bib_ref] Genetic polymorphisms associated with susceptibility to COVID-19 disease and severity: A systematic..., Dieter [/bib_ref] [bib_ref] The association of ACE1, ACE2, TMPRSS2, IFITM3 and vdr polymorphisms with COVID-19..., Dobrijevic [/bib_ref] [bib_ref] Systematic review and metaanalysis of human genetic variants contributing to COVID-19 susceptibility..., Gupta [/bib_ref]. [bib_ref] Angiotensin-converting enzyme (ACE) 1 gene polymorphism and phenotypic expression of COVID-19 symptoms, Yamamoto [/bib_ref] in a Medline database search found a higher link between ACE1 DD genotype and severe COVID-19 whereas small studies show that the ACE1 II genotype is a risk factor. Confirming the association between ACE and COVID-19, it was found that patients (n = 1,686, mean age 65.6 years) infected with SARS-CoV-2 and taking ARB or ACEi at hospital admission required less use of ventilation and vasopressors compared with non-users. However, this association was observed only for males and authors suggested that sex-based differences in RAS dysregulation may explain these results since males presented higher plasma ACE1 and AngII than females at baseline and early period of admission [bib_ref] Renin-angiotensin system pathway therapeutics associated with improved outcomes in males hospitalized with..., Rocheleau [/bib_ref]. In another study it was evaluated whether the use of ACEi/ARB (outpatient and inhospital) had any association with COVID-19 mortality. It was found that these medicaments were independently associated with a reduced risk of in-hospital mortality. Moreover, African American patients whose display higher prevalence of ACE D allele and consequent more severe COVID-19, showed a significant reduction in in-hospital mortality [bib_ref] In-hospital use of ACE inhibitors/angiotensin receptor blockers associates with COVID-19 outcomes in..., Li [/bib_ref]. In summary, there are few results until now, but they suggest that the individual genetic background and the treatment with ACEi or ARB are associated with a better clinical outcome in COVID-19 disease. More studies are needed, but using the findings obtained until now it will be possible to stratify the individual risk for disease severity and maybe to include ACEi or ARB therapy for those patients with higher risk of worst outcome. ## Concluding remarks ACE1 expression has a wide tissue and body fluids distribution and interferes with several biological processes. Frontiers in Aging frontiersin.org During the ageing process, ACE1 expression/activity is changed in several tissues which has been linked to age-related diseases (Alzheimer's, sarcopenia, cancer). In addition, ACE expression in lymphoid and myeloid cells of older individuals has been recently shown suggesting that ACE can also influence immunity. Old individuals are more likely hypertensive and use angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs) and these medicaments can somehow interfere with the outcome from age-related diseases. Some results on cognition impairment and dementia have shown a reduced risk on these conditions in individuals using ACEi or ARBs. In contrast, for muscle wasting, muscle power, sarcopenia and frailty there are contradictory results regarding the benefit of using ACEi or ARBs. In carcinogenesis, the dual colorectal cancer CRC -ACEi or ARBs has been widely studied since the benefits of these drugs for the patient outcome are reported in several clinical trials. In SARS-CoV2 infection the virus entry into the host cells relies on ACE2 expression and some preliminary results show the better outcome of patients taking ACEi or ARB. The precise mechanisms that link the use ACEi or ARBs and age-related diseases are unknown yet but it is possible to stratify each patient risk for disease development/progression and in addition to evaluate ACE polymorphism. Data from each patient could be used to decide whether or not to include ACEi or ARB to other therapies (precision medicine). # Author contributions VB and DF contributed equaly for this mini-review. They searched on literature, discussed the main points of the theme, and wrote the review. Funding CAPES PrInt UNIFESP no 88881.310735/2018-01. ## Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ## Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Burkholderia species in human infections in Mexico: Identification of B. cepacia, B. contaminans, B. multivorans, B. vietnamiensis,B. pseudomallei and a new Burkholderia species BackgroundBurkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described.Methodology/Principal findingsIn this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia. # Introduction second group comprised 26 strains isolated in Hospital Infantil de México Federico Gómez (HIMFG) from children with pneumonia and initially identified as B. cepacia. These strains were included in this study to identify them at the species level. The type species B. cepacia ATCC 25416 T , B. contaminans LMG 23361 T , B. multivorans LMG 13010 T , B. vietnamiensis TVV75 T and B. cenocepacia J2315 T and reference strain B. cenocepacia TAtl-371 were included in the analyses. The strains were regularly kept in LB Petri dishes at room temperature and preserved with 35% glycerol at -70˚C. ## 16s rrna and atpd gene amplification and phylogenetic analysis Total DNA was isolated by growing the strains in 5 mL LB and incubating overnight at 30˚C with shaking (120 rpm). DNA was obtained using the cetyl trimethyl ammonium bromide (CTAB) method according to Moore and Dowhan. The 16S rRNA gene was amplified with primers 27F/1492R. The atpD gene was amplified according to Baldwin et al.. The PCR fragments were sequenced at Macrogen Inc. (https://www.macrogen.com) using the same primers for amplification. The sequences were analyzed and assembled with Chroma-sPro 2. ## Polymerase chain reaction amplification of the box dispersed-repeated motif (box-pcr) analysis All strains were analyzed with BOX-PCR using the BOXA1R primer. Cycling conditions were 95˚C for 5 min and then 35 cycles of 95˚C for 1 min, 63˚C for 1 min and 72˚C for 6 min and a final elongation cycle of 10 min at 72˚C. The BOX patterns were observed in 1% agarose gels. ## Whole-genome sequencing and analysis Selected strains were grown in 40 mL LB and incubated overnight at 30˚C. The total DNA was isolated using the method of Moore and Dowhan. The genome sequence was obtained at Novogene (https://en.novogene.com/) using Illumina Platform PE150 with a 350-bp insert DNA library. For the genome assembly, the quality control analysis was carried out with FastQC to 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), checking for the presence of adapters and length of reads. The clipping and filtering of the information was performed with Trimmomatiq 0.39, keeping only those sequences with phred quality greater than 28. All samples were assembled with an optimized method of on de Bruijn graphs and automatic adjustment of the K value with the SPAdes 3.14 program. Some of the assemblies underwent a cleaning process to remove spurious sequences using Blast 2.9.0comparing to a Burkholderia genome bank. Metrics such as N50 and misassembles were obtained using QUAST 5.0.2. Annotation was performed using the standard operating procedure at DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4). Average Nucleotide Identity (ANI) calculations were obtained with JSpeciesWS, using ANIm (based on the MUMmer algorithm). The digital DNA-DNA hybridization (dDDH) was calculated in silico with the Genome-to-Genome Distance Calculator (GGDC 2.1) using the BLAST method. Results were based on recommended formula 2 (identities/HSP length), which is independent of genome length and appropriate for incomplete genomes. # Phylogenomic analysis A phylogenomic analysis was carried out to determine the relatedness of Burkholderia strains with Burkholderia species using the program up-to-date bacterial core gene (UBCG), which is based in the phylogenetic analysis of 92 core genes, that were previously selected from the analysis of 1429 species available at EzBioCloud (https://www.ezbiocloud.net/), representing 28 phyla, including Burkholderia species. Single-nucleotide polymorphism (SNPs) was analyzed with strain 294H and compared to other B. pseudomallei strains. The analysis was performed with maximum parsimony based on core SNPs with Parsnp, a component of the Harvest 1.3 software (https://github.com/marbl/harvest). ## Multilocus sequence typing (mlst) analysis MLST allele sequences were analyzed by BLAST searches using the sequenced genomes with the Bcc and B. pseudomallei PubMLST Databases. ## Antibiotic susceptibility All strains were tested with the VITEK2 system for antibiotic susceptibility. , including those from HGMGG and HIMFG, and the type species of Paraburkholderia unamae used as an external species, showed that the strains were not clearly grouped with any of the species from the Bcc, despite the high similarity found with the 16S rRNA gene analyses, except for a group from HIMFG that was close to B. pseudomallei/B. mallei strains (S1 Since the phylogenetic analysis with 16S rRNA genes has low taxonomic resolution, which is in line with several reports, a phylogenetic analysis was performed with atpD gene, using the same number of strains. The atpD gene was used because it is one of the genes listed in the PubMLST for the taxonomical analysis of Bcc (https://pubmlst.org/ organisms/burkholderia-cepacia-complex). Phylogenetic analysis of the atpD gene gave a better resolution, showing that the strains from HGMGG formed a large cluster, and the strains from HIMFG clustered in few groups but none clearly close to any Burkholderia species (S2 . The concatenation of both 16S rRNA and atpD genes to seek a better resolution and identification of the strains, resulted in a large group containing all strains from HGMGG. Strains 184D and 662D from HIMFG were close to B. vietnamiensis, with similarity of 99.28 and 99.93%, respectively, and 99.2% similar to each other. Other HIMFG strains, such as 500H and 501H, grouped with B. cenocepacia but the 16S rRNA showed more similarity to B. contaminans (99.85%). The strains identified as B. pseudomallei/B. mallei were intermingled with these species. The positioning of the rest of the strains from HIMFG was not clear, showing no association to any particular Burkholderia species. # Results ## Bacterial identification and phylogenetic analysis # Box-pcr analysis Since the resolution in the analysis of 16S rRNA and atpD genes was poor in general, the BOX profiles were analyzed visually to determine clonality and to define possible Burkholderia subgroups among the isolates. Twelve BOX patterns were found among the HGMGG and HIMG strains; as expected, none of these patterns was shared among the strains from both hospitals. Strains collected from HGMGG displayed four different patterns with pattern I present in the largest number of strains (31 strains). Strains from HIMFG displayed eight different patterns, with pattern VIII present in 10 strains. At least one strain with each BOX pattern was selected for genome sequencing to determine the Burkholderia species they belonged to. The results suggested that many of the isolates were clonal and that an internal outbreak might have happened at HGMGG. # Whole-genome analysis The genomes of 12 selected strains were analyzed with the ANI and dDDH tools, considering that cutoff values of 95-96 and 70%, respectively, as the delineation of a species. pattern XI as B. vietnamiensis (ANI 98.46% and dDDH 90.50%). The strain with the BOX pattern XII (strain 500H) was close to B. cenocepacia, but, according to the genome comparative analysis, it belongs to "B. servocepacia" (ANI 97.88% and dDDH 83.00%). However, this bacterial species has not yet been validated, and therefore it is not present in the LPSN. The genome sizes of the clinical strains were in the range of 6.5-8.8 Kbp, which is a genome size expected for bacteria in the Burkholderia genus. More genome features are presented in S2A phylogenomic analysis was performed with concatenating 92 genes with the UBCG pipeline. In this analysis, the type strain and four to five reference strains from each of . servocepacia" species were included. The reference strains were selected on the basis of sharing more than 70% similarity by dDDH to the type strain to avoid using incorrectly classified species. The topology of the tree showed that all strains analyzed in this study were grouped with the respective speciesand the strain 500H was clustered with "B. servocepacia" strains. A dendrogram based on maximum parsimony from analysis of core SNPs for strain 294H was carried out comparing with a number of B. pseudomallei strains that were isolated from patients with melioidosis living in different locations. Since strain 294H had the same BOX pattern with the rest of the B. pseudomallei strains analyzed in this study, this strain was randomly selected for genome sequencing and for SNPs analysis. The result showed that strain 294H clustered with B. pseudomallei strains that were isolated from people living in the US who had a history of travelling to Mexico and other countries in the American continent. Strain 294H was close to a strain isolated from a person with melioidosis living in Illinois, US, who previously had travelled to Mexico. This result shows the presence of the etiologic agent of melioidosis. # Mlst analysis The strains' genomes were analyzed in the PubMLST Database to determine whether they correspond to a defined sequence type (ST) or they represented a new ST. The B. cepacia strains belonged to a single ST, which was ST 9 (S2 . . Since Burkholderia sp. 500H represents a novel species, the ST will be established once the species is proposed. These results showed that new STs were identified in some of the strains in this work. ## Antibiotic susceptibility Analysis with the VITEK2 system showed that all strains were resistant to ticarcillin/clavulanic acid, 87% were sensitive to trimethoprim/sulfamethoxazole, 94% were sensitive to meropenem, 82% were sensitive to levofloxacin and only 28% were sensitive to ceftazidime. These results showed that most of the strains analysed here are susceptible to elective antibiotics, although a small proportion are resistant, underscoring the relevance of defining the antibiotic sensitivity of all Burkholderia isolated in clinical settings. # Discussion Bcc and B. pseudomallei are widely distributed in the world, but the evidence of their presence in Mexico is scarce, both in clinical settings and in the environment. Identification of these microorganisms at the species level is very problematic. Species from the Bcc can share up to 100% similarity in 16S rRNA gene sequences, prompting to the use of more efficient tools, such as MLSA and ultimately genome sequencing. Our analysis of Burkholderia strains isolated from human infections in two different hospitals in Mexico City showed that the identification system is poor, since all the strains were identified as B. cepacia, but we found that many belong to other Burkholderia species and even other genera. The proper identification of a bacterial species is important to provide the appropriate treatment, which is relevant in Burkholderia species as they are highly resistant to many antibiotics. For example, all strains studied were resistant to ticarcillin/clavulanic acid, but most of the strains were sensitive to meropenem. B. pseudomallei is efficiently treated with ceftazidime, meropenem or trimethoprim/sulfamethoxazole, and the B. pseudomallei strains studied here were sensitive to these antibiotics as well. Accurate identification of B. pseudomallei might lead to successful treatment of the patient. Unfortunately, this this did not happen in two fatal cases in northern Mexico; since the melioidosis caused by B. pseudomallei is barely known in Mexico, correct antibiotic treatment was not provided to the children, who died within a few hours after the admission to a hospital. The analysis of 16S rRNA gene sequence showed poor resolution for the position of the Burkholderia strains in the phylogenetic tree. This is expected in this genus and with this gene. But when the atpD gene sequence was added to the analysis, the resolution of the bacterial positioning improved significantly, and the strains from HGMGG and HIMFG formed different clusters. Only the strains from HGMGG clustered with B. cepacia. This species is not typically recognized with highly distribution in clinical settings, as B. cenocepacia and B. multivorans are . To group the strains and select some of them for genome sequencing, BOX-PCR was used, revealing 12 BOX patterns. One strain from each profile was selected for genome sequencing. The ANI and dDDH results confirmed that the strains from HGMGG belonged to B. cepacia, while the strains from HIMFG were identified as B. contaminans, B. multivorans, B. vietnamiensis and B. pseudomallei. A novel species in the Bcc was also found among the strains from HIMFG. In Mexico, B. cepacia has been found in several hospitals; however, the identification methodology is not always mentioned, and when automated systems, as used in this study, might not accurately identify the strains . Although most of the strains from HGMGG were confirmed as B. cepacia, other strains had been mistakenly identified as this species, but belonged to other genera, such as Pseudomonas, Stenotrophomonas, and Providencia. B. cenocepacia and B. multivorans are Bcc species largely distributed in clinical settings, mainly affecting immunosuppressed patients. In Mexico, B. multivorans strains have not been reported in clinical settings; this is the first report describing this species in Mexico. B. contaminans is considered an emerging CF pathogen in several countries [6], but not in Mexico. B. vietnamiensis strains have been isolated previously in Mexico, but from the environment. A number of these strains were obtained from maize, coffee and sorghum rhizosphere and rhizoplane in different geographic regions of Mexico. B. vietnamiensis has been proposed as a plant growth-promoting rhizobacterium (PGPR), but its use in agriculture is ethically incorrect since it is an opportunistic pathogen of the Bcc and has been found in patients with CF. The presence of B. vietnamiensis in Mexican soils may represent a reservoir for the infection with this microorganism in immunosuppressed patients. Scientific and agricultural communities around the world are encouraged to stop proposing the use of Bcc strains as a PGPRs, as they are opportunistic pathogens. By using genome sequencing, we corroborated the identification of strain 294H as B. pseudomallei. This species is not well known in Mexico as few cases have been detected since 1958. The disease produced by B. pseudomallei, melioidosis, is present in Mexico, but it is a neglected disease because it is not accurately identified and reported. Recent cases with fatal outcomes, suggest that greater awareness of this bacterium and the disease is needed to prepare the health care staff to deal with the disease. Since the distribution of B. pseudomallei is worldwide, an increasing interest has emerged in the origin of B. pseudomallei strains. Several methods have been used to explore this origin, such as MLST and SNPs. Some studies using SNPs have shown the association of clinical B. pseudomallei strains with environmental isolates, establishing an epidemiological link to understand the source of infection. Our analysis with SNPs showed that strain 294H grouped with a strain that was isolated from a melioidosis patient in the US (Illinois) who had travelled to Mexico. Originally, the strain IL2014 formed a group with strains CA2009, MX2013, MX2014 and TX2015, all associated with clinical cases in patients residing in or visited Mexico. However, strain 294H split this group and clustered closely with IL2014. This result show that more studies about the isolation of more B. pseudomallei strains are necessary to understand the origin of melioidosis infections both in Mexico but also in the US, since many US citizens travel for pleasure or work to Mexico. Moreover, since the US seems not endemic of melioidosis and many strains were isolated from people travelling to Mexico, our country might be endemic for B. pseudomallei. All Burkholderia genomes were analyzed in the MLST database to determine whether they belonged to known STs or represented new ones. This database includes more than 100 different microbial species and genera, and it is intended to address several functional questions, such as activities of different variants that lead to key phenotypes. The Bcc PubMLST database functions to study the epidemiology and diversity of Bcc species. The B. cepacia strains from this study belonged to a single type, which was ST 9. This ST does not seem to be widely distributed since the database contains only two strains identified as ST 9: one isolated in Canada in 1997 (abdomen from non-CF patient) and a second isolated in Spain in 2009 (sputum from a CF-patient). The strains identified as B. contaminans were placed within ST 102 and ST 482. These STs are better represented, including 39 strains and 23 strains, respectively. The strains from B. multivorans and B. pseudomallei represented new STs; the former was assigned ST 1867 and the latter as ST 1872. However, the number of STs might be underestimated since not all Bcc species are present in the PubMLST database and the distribution of Bcc in the environment and clinical setting is poorly known in Mexico. However, we believe that the Mexican ST's might be endemic of Mexico, according to our previous analysis, but more studies should be carried out. Additionally, two strains close to B. cenocepacia were found. By analyzing the genome of strain 500H, which shared the same BOX pattern with strain 501H, we found that it belonged to the novel species "B. servocepacia" . The proposal of "B. servocepacia" was based on a comparison among many B. cenocepacia strains. The result of this analysis showed that B. cenocepacia could be divided in two major groups, and a few other strains might still represent novel species. Therefore, "B. servocepacia" was proposed and strain TAtl-371 was selected as the type strains. However, the species name of "B. servocepacia" has not been validated following the rules of the International Code of Nomenclature of Prokaryotesand is not listed at the LPSN. Thus, we decided to maintain the name Burkholderia sp. According to the identification of the strains, and taking into account the hospital of origin, the isolation source of the strains and the age of the person from whom the strains were isolated, it seems that no correlation exists. Only B. cepacia was identified from HGMGG but different species were obtained from HIMFG. The range of age was different in the patients from each hospital, but the isolation source might be important since in HGMGG most of the strains were isolated from bronchus secretions and in HIMFG from pharyngeal exudates. Considering these results, the potential diversity of Burkholderia species in Mexico is not yet well understood. The distribution of species from the Bcc in Mexico is barely known, both in clinical settings and in the environment. The latter is important as a reservoir for potential infection in immunosuppressed patients which could explain the presence of Bcc and B. pseudomallei in human infections. The correct identification of Burkholderia species might help to provide accurate treatment for immunosuppressed patients.

Does trocar-guided tension-free vaginal mesh (Prolift™) repair provoke prolapse of the unaffected compartments? Introduction and hypothesis The objective of this study was to assess the effect of the tension-free vaginal mesh (Prolift™) procedure on the non-treated and initially unaffected vaginal compartments. Methods This prospective observational cohort study involved 150 patients who underwent a Prolift™ procedure. Pelvic organ prolapse (POP) quantification and evaluation of prolapse symptoms with validated questionnaires was performed pre-operatively and 6 and 12 months postoperatively. Primary outcome was the rate of POP stage ≥II in the non-treated vaginal compartments. Results Twenty-three percent of all patients developed a de novo POP stage ≥II in the untreated compartment. This occurred in 46% and 25% of patients after an isolated anterior and isolated posterior Prolift™, respectively. Conclusion Tension-free vaginal mesh treatment of one vaginal compartment seems to provoke the development of vaginal prolapse in initially unaffected vaginal compartments, particularly after an isolated anterior Prolift™ procedure. # Introduction Pelvic organ prolapse (POP) has a high prevalence in parous women [bib_ref] A 25-year experience with 519 anterior colporrhaphy procedures, Beck [/bib_ref]. A wide variety of abdominal and vaginal surgical techniques is available for the treatment of POP, indicating lack of consensus on the optimal treatment. The choice of the type of operation depends on multiple factors such as site and severity of prolapse, additional symptoms, and the surgeon's preference and capability. In order to prevent recurrence, the use of synthetic meshes and biological grafts in pelvic reconstructive surgery has increased considerably in recent years. The results of prolapse repair with synthetic mesh are promising, with success rates ranging from 71-100% [bib_ref] The use of graft materials in vaginal pelvic floor surgery, Huebner [/bib_ref]. Mesh exposure, infection, dyspareunia, constipation, urgency, urge urinary incontinence, and urinary retention are reported as adverse effects, as well as bladder and rectal injury and bleeding during surgery. Since 2005, we performed an ongoing prospective observational cohort study to evaluate the anatomical and functional efficacy as well as morbidity of POP repair with a tension-free Vaginal Mesh Kit (Prolift™ Ethicon, Somerville, NJ, USA). During the systematic follow-up of these patients, the clinical impression rose that mesh treatment of one vaginal compartment provoked the development of vaginal prolapse in other initially unaffected compartments. In a previous study, reported prolapse in the non-mesh compartment after a Prolift™ procedure in 15.5% of cases [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref]. In contrast, only 5.3% of patients who underwent conventional prolapse surgery without mesh needed repeat prolapse surgery within 5 years in the compartment that previously appeared well-supported [bib_ref] Epidemiologic evaluation of reoperation for surgically treated pelvic organ prolapse and urinary..., Clark [/bib_ref]. The aim of this article, therefore, is to evaluate the anatomical effect of Prolift™ treatment and its impact on prolapse symptoms on the non-treated and initially unaffected vaginal compartments. # Material and methods In two Dutch centers (Radboud University Nijmegen Medical Centre and Reinier de Graaf Group Delft) specializing in pelvic organ dysfunction and surgery, prolapse repair with the Prolift™ system was performed since September 2005. After obtained informed consent, consecutive patients with a recurrent POP stage ≥II and those with a primary POP stage ≥III were enrolled in this prospective observational cohort study. Surgery was performed by four gynecologists who were trained for the Prolift™ procedure as described in the paper by Fatton et al. prior to enrolment of patients in this study [bib_ref] Transvaginal repair of genital prolapse: preliminary results of a new tension-free vaginal..., Fatton [/bib_ref]. As recommended by these authors, a midline incision was made which included full thickness of the fibromuscular wall of the vagina. The vagina was closed without resection of any vaginal tissue with a continuous running Vicryl 2.0 suture. No simultaneous hysterectomy or T-incisions were made in order to reduce the chance of mesh exposure and erosions [bib_ref] Transvaginal repair of genital prolapse: preliminary results of a new tension-free vaginal..., Fatton [/bib_ref]. POP was quantified pre-operatively and during followup at 6 and 12 months postoperatively according to the pelvic organ prolapse quantification (POP-Q) system, as recommended by the International Continence Society [bib_ref] The standardization of terminology of female pelvic organ prolapse and pelvic floor..., Bump [/bib_ref]. Subjective symptoms were obtained pre-operatively and during follow-up visits at 6 and 12 months postoperatively with the standard urogynecological questionnaire of the Dutch Pelvic Floor Society (a disease-specific quality of life questionnaire which among others contains the Urogenital Distress Inventory (UDI). The questionnaire as a whole has been validated for the Dutch language [bib_ref] Measuring health-related quality of life in women with urogenital dysfunction: the urogenital..., Van Der Vaart [/bib_ref]. Since the aim of this study was to specifically evaluate the anatomic effect of Prolift™ treatment on the non-treated and initially unaffected vaginal compartments, we only used the domain score on genital prolapse of the UDI. We choose to include only this domain since vaginal bulging symptoms are the only symptoms that are consistently associated with vaginal prolapse [bib_ref] Pelvic organ prolapse, Jelovsek [/bib_ref]. Primary outcome of this study was the rate of POP stage ≥II in the non-treated and initially unaffected compartments. Secondary outcomes were UDI scores on the domain of genital prolapse, anatomical success in the mesh-treated compartments, defined as POP stage≤I, as well as morbidity. Data on patient and surgical characteristics are presented as numbers with corresponding percentages or medians with range. Comparison between proportions was performed using the Pearson's chi-square test. Mean domain scores and standard deviations were calculated on the UDI domain genital prolapse. Differences in means between baseline and 12-month follow-up were tested with the paired sample t test and differences in means between different groups were tested with the independent sample t test. Data were analyzed with the Statistical Package for the Social Sciences, version 16.0. A p value of <0.05 was considered significant. # Results Since the start of our study, 297 patients underwent the Prolift™ procedure and were registered in our database. On January 1, 2009, 196 patients had completed a follow-up of at least 12 months. Patients with a total Prolift™ procedure (46 patients) for post-hysterectomy vaginal vault prolapse, in whom, by definition, all three compartments are restored, were excluded from this study, leaving 150 patients for analysis. At baseline, POPQ data of 147 patients were complete and these patients were eligible for anatomic analysis. Two patients could not visit the hospital for the 12-month follow-up, leaving 145 patients for follow-up analysis. At follow-up, 127 patients (88%) had completed the validated questionnaire. Patient and surgical characteristics are presented in [fig_ref] Table 1: Patient and surgical characteristics [/fig_ref]. Median age was 64 years (range 32-89). In those patients who underwent a primary repair, median age was 71 years (range 55-89). Ninety-five patients (63%) underwent prior POP surgery, 28 (19%) of those underwent more than one prior prolapse repair. Thirty-five patients (23%) underwent an anterior Prolift™ repair, 80 (54%) a posterior Prolift™ repair, and 35 (23%) a combined anterior and posterior Prolift ™ repair. Three (2%) bladder perforations occurred, all during dissection for an anterior Prolift™ procedure. In two of these patients, the anterior mesh was not placed, but the procedure was converted into a conventional anterior colporrhaphy. One superficial serosa lesion of the rectum occurred, without a perforation. The mesh procedure was continued, since the rectum wall was intact. One patient (1%) had significant hemorrhage (>500 ml) and five patients (3%) developed a postoperative hematoma. Ten patients (7%) had temporary postoperative urinary retention, which resolved spontaneously in all cases within 11 days. In 15 patients (10%), a mesh exposure was detected, six cases at the 6-month follow-up visit and nine at the 12-month follow-up visit. Most of these patients were asymptomatic. Only one patient complained of pain and one other of de novo dyspareunia, but none of them had vaginal discharge or signs of infection. The size of these mesh exposures varied from 2 to 20 mm. All mesh exposures were easily excised and covered with vaginal mucosa in a minor day-care procedure after initial treatment with local estrogens. POP stages per compartment are shown in [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref] , divided into three categories: those who underwent a solitary anterior Prolift™, a solitary posterior Prolift™, or a combined anterior and posterior Prolift™ procedure. In [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref] , the patients who underwent concomitant, nonmesh surgery are included. Patients who underwent repeat surgery before 12 months follow-up were considered failures. In [fig_ref] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at... [/fig_ref] , the effect on the non-operated compartment is shown in patients without concomitant surgery. UDI scores on the domain of genital prolapse are shown in. ## Results after anterior prolift™ Anatomical success at 1 year in the anterior compartment after anterior Prolift™ repair was 89% [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref]. Sixteen patients (46%) had a post-operative POP stage II or III of the posterior compartment, of whom three patients had already undergone repeat surgery with a posterior Prolift™ before the 12 months follow-up visit, resulting in a 54% "success" rate. In [fig_ref] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at... [/fig_ref] , deterioration and improvement of the posterior vaginal wall (Bp) is shown. Twenty-six patients underwent an anterior Prolift™ without concomitant posterior colporrhaphy or sacrospinous fixation. In three patients (12%), point Bp improved with 1 cm. However, in 16 patients (62%), the posterior wall deteriorated (1-4 cm) and in 12 patients (46%), a de novo POP stage ≥II of the posterior compartment was diagnosed at the 12-month follow-up visit. Five patients (14%) had a post-operative POP stage II or III of the apical compartment resulting in 86% "success" rate [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref]. In [fig_ref] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at... [/fig_ref] , it is demonstrated that in five patients (19%) the apical compartment deteriorated, and in three patients (12%) a de novo POP stage ≥II of the apical compartment was detected. These three patients also developed a de novo prolapse in the posterior compartment. Pre-operatively, there was one patient with an apical compartment POP stage III. Due to scar tissue, it was not possible to perform any other surgery then an isolated anterior Prolift™. At 12 months, the apical compartment of this patient was classified as POP stage II. In two patients, a pre-operative POP stage II of the apical compartment resolved without concomitant intervention. [formula] 2 (1-11) 1 (1-10) 2(2-6) 2 (1-11) Hospital stay (days) a 4 (2-11) 4 (2-6) 4 (3-11) 4 (2-11) Complications Bladder perforation b 3 (8%) 0 0 3 (2%) Rectum serosalesion b 0 1 (1%) 0 1 (1%) Hemorrhage >500 ml b 0 0 1 (3%) 1 (1%) Repeat surgery for postoperative hemorrhage b 0 1 (1%) 1 (3%) 2 (1%) Hematoma b 2 (6%) 1 (1%) 0 3 (2%) Urinary retention b 5 (14%) 3 (4%) 2 (6%) 10 (7%) Mesh exposure b 2 (6%) 9 (11%) 4 (11%) 15 (10%) a median (range) b number of patients (%) [/formula] In conclusion, of the 26 patients who underwent an anterior Prolift™ without concomitant surgery, 12 (46%) developed a de novo POP stage ≥II in the non-mesh, nontreated compartment. Genital prolapse symptoms were evaluated with UDI domain genital prolapse. In all patients that underwent an anterior Prolift™, this score improved significantly. However, patients that developed de novo prolapse had a significantly higher bother score at 12 months as compared to patients without de novo prolapse. Anatomical success in the posterior compartment in patients who underwent an isolated posterior Prolift™ was 90% at 12 months [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref]. Thirty-one patients (40%) had a post-operative POP stage II or III in the anterior compartment, of whom one patient was treated with an anterior Prolift™ before the 12-months follow-up visit resulting in a 60% "success" rate. In [fig_ref] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at... [/fig_ref] , deterioration and improvement of the anterior vaginal wall (Ba) is shown after a posterior Prolift™. Sixtyfive patients underwent no concomitant surgery. Deterioration occurred in 35% of them. In 16 patients (25%), a de novo POP stage II or III of the anterior compartment was diagnosed at 12 months. Five patients (8%) had a POP stage ≥II of the apical compartment at 12 months. In only two patients (3%), this was a de novo apical prolapse. These two patients were also diagnosed with a de novo anterior compartment prolapse. In conclusion, of the 65 patients with a posterior Prolift™ without concomitant surgery, 16 (25%) developed a de novo POP stage ≥II in the non-mesh, non-treated compartment. In all, patients that underwent a posterior Prolift™, the UDI score on the domain genital prolapse improved significantly. Patients that developed de novo prolapse showed a higher bother score at 12 months as compared to patients without de novo prolapse. The difference, however, was not statistically significant (p=0.06). De novo POP after an isolated posterior Prolift™ was diagnosed less often compared to de novo POP after an isolated anterior Prolift™ (25% vs. 46%) (p=0.044). ## Results after a combined anterior and posterior prolift™ Anatomical success at 12 months in patients with a combined anterior and posterior Prolift™ procedure was 79% for the anterior compartment, 91% for the posterior compartment, and 76% for the apical compartment [fig_ref] Table 2: POP stage at baseline and at 12 months [/fig_ref]. In [fig_ref] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at... [/fig_ref] , deterioration and improvement of the apical compartment is shown. In only two patients (6%), a deterioration of the apical compartment was detected, though these patients were still classified as having POP stage I, which, by definition, is not regarded as a failure. Despite an improvement in centimeters compared to baseline, however, eight patients (24%) were still diagnosed with POP stage ≥II of the apical compartment, of which two had additional surgery within 1 year; one laparoscopic cervicosacropexy, and one vaginal hysterectomy with a high Mc Call procedure. In all these eight patients, it was the uterus that had descended, not a vaginal vault. In conclusion, none of the 35 patients with a combined anterior and posterior Prolift™ developed a de novo prolapse. However, in eight patients (24%), the improvement of the apical compartment prolapse was insufficient. In all patients that underwent an anterior+posterior Prolift™, the UDI score on the domain genital prolapse improved significantly. Patients who were still diagnosed with POP ≥II showed a higher bother score as compared to patients with a POP <II. The difference, however, was not statistically significant (p=0.06). In summary, 28 patients (23%) who underwent a Prolift™ procedure (anterior, posterior or combined) were diagnosed with de novo POP stage ≥II in the untreated compartment, and they all had higher bother scores on the UDI domain genital prolapse compared to patients without de novo prolapse. Six patients (4%) had to undergo repeat surgery in the previously non-mesh-treated compartment within the first year of follow-up. # Discussion The symptomatic de novo POP rate in the non-mesh-treated compartment appeared to be alarmingly high, particularly after an isolated anterior Prolift™. Our de novo POP rate in the non-treated compartment (23%) appeared higher than previously reported by Raalte et al. (15.5%) [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref]. One explanation for this difference could be that our study population consisted of a higher number of patients with prior prolapse surgery compared to the population described by Raalte et al. (63% vs. 45%) [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref]. The study of Clark et al. on conventional vaginal prolapse surgery demonstrated a repeat surgery rate in the untreated compartment in only 5.3% of patients after 5 years, whereas in our study, this rate was already 4% within the first year [bib_ref] Epidemiologic evaluation of reoperation for surgically treated pelvic organ prolapse and urinary..., Clark [/bib_ref]. De novo POP in the non-mesh-treated compartment was diagnosed less common after a posterior Prolift™ than after an anterior Prolift™ (25% vs. 46%). Previous studies demonstrated that restoration of DeLancey's level I support diminishes the rate of anterior and posterior wall prolapse [bib_ref] The relationship between anterior and apical compartment support, Summers [/bib_ref] [bib_ref] The role of apical vaginal support in the appearance of anterior and..., Lowder [/bib_ref]. The position of the arms of the posterior Prolift™ through the sacrospinous ligaments ensures level I support, which potentially results in less de novo POP after a posterior Prolift™ compared to an anterior Prolift™. A recent study, using three-dimensional models generated from magnetic resonance pelvic imaging in women with normal pelvic support, demonstrated that an anterior mesh mainly offers level II support and not enough level I support, since the upper part of the vagina lies well above and posterior to the distal suspension points of the anterior mesh [bib_ref] The relationship between superior attachment points for anterior wall mesh operations and..., Larson [/bib_ref]. Since the posterior Prolift™ procedure does provide adequate level I support and the anterior Prolift™ mainly provides level II support, this could explain why we detected an almost twofold higher de novo POP rate after an isolated anterior compared to an isolated posterior Prolift™. We found no de novo POP in the apical compartment after a combined anterior and posterior Prolift™. However, if the uterus was left in situ, the improvement of the apical compartment was still insufficient (POP stage II or III) in eight cases (24%). Our previously published results, after a total Prolift™ procedure with one continuous mesh for post-hysterectomy vaginal vault prolapse, were significantly better (failure rate 9%) [bib_ref] Trocar-guided total tension free vaginal mesh repair of post hysterectomy vaginal vault..., Milani [/bib_ref]. In order to prevent possible descent of the uterus, the French Tension-free Vaginal Mesh group advised us, during the study, to remove a little part of the mesh at the level where this is fixated to the uterus to ensure a more adequate and, thus, higher suspension of the uterus. In the mesh-treated compartments, the success rates of 89% (95% CI 78-99) after an anterior mesh and 90% (95% CI 83-97) after a posterior mesh were comparable with other reports on Prolift™ with 1 year follow-up [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref] [bib_ref] Trocar-guided transvaginal mesh repair of pelvic organ prolapse, Elmér [/bib_ref]. In the present study, the number of complications was comparable as well with previous reports [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref] [bib_ref] Transvaginal repair of genital prolapse: preliminary results of a new tension-free vaginal..., Fatton [/bib_ref] [bib_ref] Trocar-guided transvaginal mesh repair of pelvic organ prolapse, Elmér [/bib_ref]. Although reported no patients with mesh exposure, our incidence of mesh exposure (10%) was comparable with an exposure rate of 11% described by Elmer [bib_ref] One-year anatomic and quality-of-life outcomes after the prolift procedure for treatment of..., Raalte [/bib_ref] [bib_ref] Trocar-guided transvaginal mesh repair of pelvic organ prolapse, Elmér [/bib_ref]. Despite the fact that mesh exposures rarely caused severe complaints and could be easily excised and covered with vaginal mucosa, it remains an important concern for the future, since the follow-up in this study of 1 year is still relatively short, and the exposure rate might still rise. The high success rates in the mesh-treated compartments and the high rates of (de novo) symptomatic prolapse in the unaffected/untreated compartments could indicate that we have to change our surgical strategies. Urogynecologists have to realize that placing a mesh in one compartment can provoke or deteriorate a prolapse in any other compartment. It seems logical to be "more liberal" in using mesh, also, in none or minor affected compartments. Whether such a strategy will improve results without an increase in morbidity, such as mesh exposure, mesh retraction and dyspareunia remains to be seen. Providing adequate level I support without the use of more mesh is another surgical strategy, for example adding a sacrospinous hysteropexy or a sacrospinous fixation as a preventive measure of de novo POP to an anterior Prolift™ procedure. Other effective surgical alternatives are the modified Manchester procedure or vaginal hysterectomy with high Mc Call procedure to restore or prevent apical compartment prolapse and, thus, ensuring adequate level I support [bib_ref] The effectiveness of surgical correction of uterine prolapse: cervical amputation with uterosacral..., De Boer [/bib_ref]. In our opinion, the strengths of this study are the high number of patients with adequate follow-up of 12 months, the prospective data collection, and the use of validated instruments of measurement, such as POP-Q and urogynecological questionnaires. A relative drawback is the lack of a control group. A control group with conventional nonmesh surgery might answer the question whether prolapse in the previously unaffected compartments is more often provoked by mesh surgery than by conventional surgery. # Conclusion Our data suggest that mesh treatment of only one vaginal compartment does provoke the development of POP in other initially unaffected compartments. The development of de novo POP stage ≥II in previously unaffected compartments is almost twice as high after an isolated anterior Prolift™ than after an isolated posterior Prolift™. In case of a POP stage ≥II of the anterior compartment and a stage I of the posterior and/or apical compartment, we suggest to add level I support by a conventional sacrospinous hysteropexy. If there is no uterus in situ, we would suggest considering a total Prolift™ procedure since the results of this treatment are highly effective as reported earlier by us [bib_ref] Trocar-guided total tension free vaginal mesh repair of post hysterectomy vaginal vault..., Milani [/bib_ref]. We feel that patients should be counseled about this strategy and about the pros and cons of additional mesh surgery. They should also be made aware of the potential risk on secondary surgery if no additional treatment is performed initially. [table] Table 1: Patient and surgical characteristics [/table] [table] Table 2: POP stage at baseline and at 12 months [/table] [table] Table 3: Effect on non-treated compartment at 12 months Ba most descendant point at anterior vaginal wall, C cervix or vaginal apex, Bp most descendant point at posterior vaginal wall [/table]

The ‘Shape-Shifter’ Peptide from the Disulphide Isomerase PmScsC Shows Context-Dependent Conformational Preferences # Introduction Chameleon sequences, amino acid sequences that adopt different secondary structure conformations in different protein structures, are currently attracting considerable interest [bib_ref] ChSeq: A database of chameleon sequences, Li [/bib_ref]. Studies of these sequences in different contexts firstly provides an understanding of the fundamental determinants of protein structure and dynamics [bib_ref] On the use of sequence homologies to predict protein-structure-identical pentapeptides can have..., Kabsch [/bib_ref] [bib_ref] Analysis of chameleon sequences and their implications in biological processes, Guo [/bib_ref]. Secondly, the properties of these sequences can give insights into protein folding diseases, such as Alzheimer's and transmissible spongiform encephalopathies, where a native protein structure undergoes a major conformational change to form amyloid fibrils [bib_ref] Discordant and chameleon sequences: Their distribution and implications for amyloidogenicity, Gendoo [/bib_ref] [bib_ref] Chameleon sequences in neurodegenerative diseases, Bahramali [/bib_ref]. Finally, these sequences have the potential to be utilized as tools in protein engineering and design, for example in stimulus-responsive peptide systems which undergo a conformational transition prompted by changes in the external environment [bib_ref] Design and application of stimulus-responsive peptide systems, Chockalingam [/bib_ref]. Chameleon sequences are also relevant in intrinsically disordered proteins (IDPs) in the context of short molecular recognition features (MoRFs) which can adopt different conformations upon binding to different partner proteins [bib_ref] Flexible nets: Disorder and induced fit in the associations of p53 and..., Oldfield [/bib_ref] [bib_ref] Understanding protein non-folding, Uversky [/bib_ref] [bib_ref] Exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding, Hsu [/bib_ref] [bib_ref] Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions, Oldfield [/bib_ref]. Recently, a so called 'shape-shifter' peptide, has been identified in Proteus mirabilis suppressor of copper sensitivity protein C (PmScsC), a homo-trimeric protein disulphide isomerase [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. Each monomer unit consists of an N-terminal trimerization stalk and a thioredoxin-fold catalytic domain linked by an eleven-residue peptide motif. Three crystal structures of PmScsC have been determined under different crystallization conditions: compact, transitional and extended [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. The compact and transitional crystals were obtained using a 2.85 M sodium malonate well solution at pH 5.8 while the extended crystals were obtained using a 0.1 M HEPES well solution at pH 8. The structures of the trimerization stalk and catalytic domains are essentially unchanged between these three forms but the linker peptide adopts a helical, strand or loop conformation resulting in significant changes in the quaternary structure of the trimer. In the extended structure the linker is helical and the catalytic domain is rotated and positioned away from the trimerization domain [fig_ref] Figure 1: Cont. [/fig_ref] while in the compact form the catalytic domains are positioned close to the trimerization stalk and the linker forms a connecting loop [fig_ref] Figure 1: Cont. [/fig_ref]. The transitional crystal structure contains monomers adopting both the compact and extended conformations along with an intermediate conformation in which the linker forms a short β-strand hydrogen bonding to the catalytic domain positioned above the trimerization stalk [fig_ref] Figure 1: Cont. [/fig_ref]. The eleven-residue linker peptide has been called a 'shapeshifter' peptide based on its ability to adopt different secondary structures and to influence the relative orientations of the domains that it links [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. A variant of PmScsC in which the eleven-residue linker is deleted, PmScsC∆Linker, lacks disulphide isomerase and dithiol oxidase activities [bib_ref] Engineered variants provide new insight into the structural properties important for activity..., Furlong [/bib_ref]. In this variant, a mixture of monomer, dimer and trimer are present in solution, whereas only trimer is observed for the wild-type protein. A crystal structure of the PmScsC∆Linker trimer shows that the three catalytic domains are packed closely together. This suggests that the linker peptide plays an important role as a spacer between the trimerization stalk and catalytic domains which enables a stable and functional trimer to form. Replacement of the linker in PmScsC by a rigid helical peptide also leads to the loss of disulphide isomerase activity showing that dynamical motion is essential for the mechanism of PmScsC [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. In light of the important role of the shape-shifter linker peptide in the function of PmScsC and its interesting conformational properties, we have characterised this linker peptide in solution. In particular, experimental NMR studies and molecular dynamics (MD) simulations have been used to study a peptide with a sequence corresponding to residues 38-50 (the eleven-residue linker and one additional residue at each end) of PmScsC. The X-ray structures of PmScsC have also been analysed to identify intermolecular interactions involving the linker peptide sequence. The results highlight the important role that quaternary interactions can play in determining conformational properties. In panel e the linker peptide sequence in molecule C is shown in blue and residues 117-120 in molecule D with which it makes intermolecular contacts are shown in orange. There is a salt bridge between Asp 41 in chain C and Lys 120 in molecule D. Note that the coordinates of residues 46-47 are missing from chain C. # Materials and methods ## Sample preparation The thirteen-residue linker peptide, with sequence KKADEQQAQFRQA, was purchased from Pepceuticals Limited (Leicester, England). The N-and C-termini were modified with acetyl and amide groups, respectively. Peptide samples for NMR were 2.5 mM in either 0.01 M sodium phosphate buffer (95% H 2 O/5% D 2 O) at pH 7 or a 50:50 mixture of deuterated trifluoroethanol (TFE) and 0.01 M sodium phosphate buffer at pH 7. A small amount of 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) was added as a chemical shift reference. ## Nmr spectroscopy and nmr data analysis NMR experiments were carried out at 288 K using 500, 600 and 950 MHz spectrometers equipped with Bruker Avance consoles. The 500 and 600 MHz spectrometers were equipped with TCI CryoProbes and the 950 MHz spectrometer with a room temperature probe. Resonance assignments for the peptide in H 2 O and in 50% TFE were obtained using 2D [bib_ref] ChSeq: A database of chameleon sequences, Li [/bib_ref] NMR data were processed using NMRPipe [bib_ref] NMRPipe-A multidimensional spectral processing system based on Unix Pipes, Delaglio [/bib_ref] and analysed using CcpNmr Analysis [bib_ref] The CCPN data model for NMR spectroscopy: Development of a software pipeline, Vranken [/bib_ref]. Spin system information was obtained from analysis of the COSY, TOCSY and 1 H-13 C HSQC spectra; the latter contained both one-bond and two-bond 1 H-13 C correlations. Sequence specific assignments were obtained from analysis of HN(i)-HN(i+1), Hα(i)-HN(i+1) and Hβ(i)-HN(i+1) NOEs. [bib_ref] ChSeq: A database of chameleon sequences, Li [/bib_ref] H and 13 C chemical shifts were referenced using DSS, and 15 N chemical shifts were referenced indirectly. The resonance assignments in water and in 50% TFE have been deposited in the BMRB with deposition codes 50399 and 50400, respectively. Analysis of NMR chemical shifts to obtain secondary structure predictions was carried out using the Secondary Structure Propensities (SSP) software [bib_ref] Sensitivity of secondary structure propensities to sequence differences between alpha-and gamma-synuclein: Implications..., Marsh [/bib_ref]. The SSP scores were calculated with the default averaging window of 5 residues but also without averaging (m = 1). The ∆C α -∆C β and ∆H α secondary shifts were also calculated using SSP. All structural figures were rendered using PyMOL. ## Md simulations Molecular dynamics simulations were performed using the GROMOS bio-molecular simulation software [bib_ref] Architecture, implementation and parallelisation of the GROMOS software for biomolecular simulation, Schmid [/bib_ref] with the GROMOS 54A7 force field [bib_ref] Definition and testing of the GROMOS force-field versions 54A7 and 54B7, Schmid [/bib_ref]. Four 100 ns MD simulations of the linker peptide were performed and one 20 ns MD simulation of the protein trimer. The initial structures for the peptide simulations were taken from residues 38-50 in the X-ray structures of PmScsC [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref] with protein data bank codes 4xvw (molecule A; loop), 5id4 (helix) and 5idr (molecules A; irregular helix and B; β-strand). Note that there is a regular pattern of NH(i)-CO(i-4) hydrogen bonds along the length of the peptide in the 5id4 structure but in the 5idr molecule A structure the hydrogen bonding pattern seen is not that of a regular α-helix with four missing NH(i)-CO(i-4) hydrogen bonds in the centre of the peptide and one NH(i)-CO(i-3) hydrogen bond characteristic of a 3 10 helix (45 NH-42 O). The initial structure for the protein simulation was the 5id4 X-ray structure. The peptide or protein molecules were solvated in rectangular boxes and minimum image periodic boundary conditions were applied. The minimum solute-box wall distance was set to values ranging from 0.9 to 1.4 nm depending upon the starting conformation, yielding 3647 (4xvw and 5id4 peptide simulations), 4996 (5idr molecule B peptide simulation), 5256 (5idr molecule A peptide simulation) and 58,785 (5id4 protein simulation) SPC water molecules [bib_ref] Interaction models for water in relation to protein hydration, Berendsen [/bib_ref]. To compensate for the overall positive charge of the solute, one and three Clions were included in the peptide and protein simulations, respectively. For each of the simulations an equilibration scheme comprising five 20 ps simulations of 60 K, 120 K, 180 K, 240 K and 298 K was used at constant volume. During the first 80 ps of this equilibration the solute atoms were harmonically restrained to their positions in the initial structures with force constants of 25,000, 2500, 250 and 25 kJ mol −1 nm −2 at temperatures of 60 K, 120 K, 180 K and 240 K, respectively. Following this equilibration, all simulations were performed at a temperature of 298 K and a pressure of 1 atm using the weak-coupling algorithm [bib_ref] Molecular dynamics with coupling to an external bath, Berendsen [/bib_ref] , with relaxation times of τ T = 0.1 ps and τ p = 0.5 ps and an isothermal compressibility of 4.575 × 10 −4 (kJ mol −1 nm −3 ) −1 . Solute and solvent were separately coupled to the heat bath. The SHAKE algorithm [bib_ref] Numerical integration of cartesian equations of motion of a system with constraintsmolecular..., Ryckaert [/bib_ref] was used to constrain bond lengths of the solutes and the rigid geometry of the solvent molecules, with a relative geometric tolerance of 10 −4 allowing for an integration time step of 2 fs. The centre of mass motion of the system was removed every 1000 time steps. Non-bonded interactions were calculated using a triple-range cut-off scheme with cut-off radii of 0.8 nm and 1.4 nm. Interactions within 0.8 nm were evaluated every time step and intermediate interactions were updated every fifth time step. To account for the influence of the dielectric medium outside the 1.4 nm cut-off sphere, a reaction-field force [bib_ref] A generalised reaction field method for molecular dynamics simulations, Tironi [/bib_ref] with a dielectric permittivity ε RF = 61 for water was used. The solute configurations were saved for analysis every 5 ps. In the analysis of the simulation trajectories hydrogen bonds were identified according to a geometric criterion: a hydrogen bond was assumed to exist if the hydrogen-acceptor distance was smaller than 0.25 nm and the donor-hydrogen-acceptor angle was larger than 135 - . Conformational clustering was performed using the algorithm of Daura et al. [bib_ref] Folding-unfolding thermodynamics of a beta-heptapeptide from equilibrium simulations, Daura [/bib_ref] and the backbone N, CA and C atoms of residues 3-11. The atom positional RMSD cut off used to determine the structures belonging to a single cluster was 0.15 nm. Trajectory structures lying 10 ps apart were used. # Results 2D 1 H-1 H COSY, TOCSY, NOESY and natural abundance 1 H-13 C and 1 H-15 N HSQC experiments were used to assign the spectrum of the linker peptide in aqueous solution at pH 7 [fig_ref] Figure 1: Cont. [/fig_ref]. The secondary chemical shifts for the peptide under these conditions were all small (∆C α -∆C β in the range −0.76 to +0.55 ppm and ∆H α in the range −0.06 to +0.07 ppm) [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. The chemical shift data were analysed using the Secondary Structure Propensities (SSP) algorithm [bib_ref] Sensitivity of secondary structure propensities to sequence differences between alpha-and gamma-synuclein: Implications..., Marsh [/bib_ref]. All residues gave low SSP scores in the range of +/− 0.15 when applying the default 5-residue averaging window [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. Without this averaging, slightly larger SSP scores are obtained with a small preference for extended structure at the N-and C-termini and a small preference for helix at residues Asp 41, Glu 42, Ala 45 and Gln 46. Overall, the SSP scores of~0.10 indicate that only~10% of the conformational ensemble populated would be adopting a regular helical conformation. The unstructured, random coil nature of the linker peptide in aqueous solution was further confirmed by the observation of only intraresidue and sequential NOE's and by 3 J HNα coupling constants in the range 6.7-8.1 Hz [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. Predictions from Agadir [bib_ref] Elucidating the folding problem of helical peptides using empirical parameters, Munoz [/bib_ref] for the sequence were in good agreement with these results, predicting approximately 3% overall helicity for the peptide in aqueous solution. The peptide was also characterised in trifluoroethanol (TFE) solution. A titration monitored using CD spectroscopy showed no cooperative transition on the addition of TFE to the peptide solution, and 50% TFE (v/v) was needed to induce a significant helical population [fig_ref] Figure 3: The four central structures show the loop [/fig_ref]. This is in contrast to peptides which have a considerable helical propensity in aqueous solution, where often significant increases in helicity are seen on the addition of low concentrations of TFE [bib_ref] Trifluoroethanol and colleagues: Cosolvents come of age. Recent studies with peptides and..., Buck [/bib_ref]. NMR studies were performed in 50% TFE solution (v/v) and the secondary chemical shifts ∆C α -∆C β were in the range 1.6-2.7 ppm for residues 40-46 [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. Analysis of the chemical shift data using the SSP approach [bib_ref] Sensitivity of secondary structure propensities to sequence differences between alpha-and gamma-synuclein: Implications..., Marsh [/bib_ref] gave scores in the range 0.42-0.74 for residues 40-46 meaning that approximately half the ensemble of conformers populated adopt a helical conformation [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. The residues towards each terminus have lower SSP scores in the range −0.14 to +0.30. The helical propensity in 50% TFE solution was confirmed by the observation of H α (i)-H β (i+3) and H α (i)-H N (i+3) NOE's along the peptide sequence. The 3 J HNα coupling constant values measured under these conditions were in the range 4.2-6.8 Hz [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref]. In general, the measured 3 J HNα was larger than the value of 3.9 Hz typically seen for a regular helix in a protein but for residues 40-46 the J value was below the value predicted for a random coil. The NOE and coupling constant data are consistent with the estimate of 50% helical conformers from SSP. The NMR studies in solution have been complemented by molecular dynamics (MD) simulations. Firstly, four 100 ns simulations were run for the linker peptide (residues 38-50) in aqueous solution using starting coordinate sets from the different structures the peptide adopts in the reported X-ray structures [fig_ref] Figure 3: The four central structures show the loop [/fig_ref] , column in the centre): loop from the compact structure, regular helix from the extended structure, and β-strand and irregular helix from the transitional structure. The hydrogen bonds present in the starting coordinate sets are summarised in and the Φ, Ψ torsion angles are shown in [fig_ref] Figure 4: Structural changes observed in a 20 ns molecular dynamics simulation of the... [/fig_ref] in the Supplementary Materials. The arrows indicate the main trajectories whose conformers contribute to the cluster. Two or more adjacent residues with Φ, Ψ torsion angles in the α or β region of the Ramachandran plot are shown in red and green, respectively. In each case the peptide is orientated with the N-terminus to the left. . Hydrogen bonds in the linker peptide in the X-ray structures used as starting coordinate sets for the MD simulations and their percentage populations in the combined trajectory of the four 100 ns peptide MD simulations. Only hydrogen bonds in the X-ray structures, or with a population of at least 20% in the simulations, are included. Note that residue 201 is in the catalytic domain, in the region that forms the β-sheet with the linker peptide in the transitional X-ray structure. In all four of the MD simulations run, the linker peptide showed significant deviations from its starting structure, adopted a wide range of conformations and sampled a number of different hydrogen bonds during the simulations . The peptide conformations in the four 100 ns trajectories have been taken together (40,000 structures) and conformational clustering was performed (see Methods Section 2.3). Seven main clusters were identified to which 75% of the peptide structures belong [fig_ref] Figure 3: The four central structures show the loop [/fig_ref] and . The two largest clusters each contained approximately 20% of the peptide structures. In cluster 1 the peptide adopts a distorted hairpin type structure, which enables formation of a 40NH-49O hydrogen bond (92% population) while in cluster 2 the structures show a high population of a helical hydrogen bond in the centre of the peptide (45NH-41O 89%,) The structures in clusters 1 and 2 are derived almost exclusively from the MD trajectories starting from the loop and irregular helix structures, respectively. Clusters 3 and 5 contain structures which mostly have helical hydrogen bonds only in the middle or at the N-terminus of the peptide, respectively. Structures in these clusters result from several different trajectories [fig_ref] Figure 3: The four central structures show the loop [/fig_ref] , . In contrast, cluster 4 contains structures which have helical hydrogen bonds along the length of the linker peptide. This cluster contained 11% of the 40,000 structures analysed and hence is similar to the level of helicity suggested by the SSP score for the peptide in aqueous solution. It is interesting to note that~50% of structures in cluster 4 result from the MD trajectory that starts from the β-strand peptide conformation [fig_ref] Figure 3: The four central structures show the loop [/fig_ref] , . Except for the structures in cluster 4, where in general the Φ, Ψ torsion angles are typical of a regular helix, residues with Φ, Ψ torsion angles in both the α and β regions of the Ramachandran plot are seen, although hydrogen bonds characteristic of a β-strand conformation cannot form in such a short peptide fragment alone. ## Hydrogen To gain further understanding into the conformations of the peptide seen in the different crystal structures of PmScsC, the intermolecular contacts, hydrogen bonds and salt bridges were analysed, both within the trimer and between other molecules in the asymmetric unit, using the Protein Interfaces, Surfaces and Assemblies service (PISA) [bib_ref] Inference of macromolecular assemblies from crystalline state, Krissinel [/bib_ref] and the Evolutionary, Protein-Protein Interface Classifier (EPPIC) webserver [bib_ref] Automated evaluation of quaternary structures from protein crystals, Bliven [/bib_ref]. In all the crystal structures, the N-terminal region of the three molecules in the trimer come together to form an irregular three-helix coiled coil with hydrogen bonds and salt bridges between the chains involving the side chains of [fig_ref] Figure 1: Cont. [/fig_ref]. Finally, the linker peptide in molecule C forms a loop and the coordinates of residues 46-47 are absent in the crystal structure. Here, the linker peptide sequence forms contacts with residues 117-120 of molecule D in a neighbouring trimer, with a salt bridge between Asp 41 in chain C and Lys 120 in chain D [fig_ref] Figure 1: Cont. [/fig_ref]. Under solution conditions, any hydrogen bonds between molecules in different trimers will clearly be missing. In addition, any intramolecular or inter-monomer hydrogen bonds involving side chain groups on the surface of the protein are expected to be fluctuating with short lifetimes [bib_ref] On the Use of Side-Chain NMR Relaxation Data to Derive Structural and..., Smith [/bib_ref]. Consequently, the quaternary structure of the trimer is likely to be an ensemble of conformers, rather than any one of the different structures seen in crystals predominating in solution. An indication of this was seen in a short 20 ns simulation of the trimer of the full PmScsC protein run starting from the extended structure seen in the 5id4 crystal structure [fig_ref] Figure 1: Cont. [/fig_ref]. Here, all three linker peptides started in a regular helical conformation. However, even in the short simulation time the N-terminal region of the linker peptide in molecule B started to unfold with residues 38-40 adopting extended β-strand like conformations [fig_ref] Figure 4: Structural changes observed in a 20 ns molecular dynamics simulation of the... [/fig_ref] , and a number of short-lived hydrogen bonds involving side chains of residues in the linker region being observed. In addition, the quaternary structure of the trimer became much more compact with the catalytic domains moving towards each other to fill space that was occupied by symmetry related molecules in the crystal structure. # Discussion The linker peptide in PmScsC was initially identified as a shape-shifting motif which had potential for plug-and-play applications in protein engineering [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. The studies of this peptide sequence alone in solution reported here show that it is essentially disordered and the different conformations it adopts in crystal structures of PmScsC are a consequence of different intermolecular hydrogen bonding patterns and crystal contacts formed under a variety of crystallization conditions which differ in pH and ionic strength [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. The compact and transitional crystals were obtained at a pH as low as 5.8 while the extended crystals were obtained at a pH as high as 8. However, this variation in pH should not affect the ionization state of the arginine, aspartic acid, glutamic acid and lysine side chains in the peptide which should maintain an overall net charge of +1 between pH 5.8 and pH 8. In addition, none of the other residues involved in interactions that stabilize the different linker peptide conformations should be affected by the pH range used. Chameleon sequences are usually recognised as those that adopt different secondary structure when found in different overall protein sequences. In the case of the PmScsC linker, however, the protein sequence is the same and it is the quaternary intermolecular interactions in an assembly of molecules which are promoting the different conformations observed for the linker peptide. The ability of this shape-shifting motif to adopt α-helix, β-strand and loop conformations is reminiscent of 'one-to-many' molecular recognition features (MoRFs) found in intrinsically disordered proteins. These are segments of a disordered protein that are able to adopt different conformations when they undergo 'disorder-to-order' transitions upon interaction with multiple binding partners [bib_ref] Flexible nets: Disorder and induced fit in the associations of p53 and..., Oldfield [/bib_ref] [bib_ref] Understanding protein non-folding, Uversky [/bib_ref] [bib_ref] Exploring the binding diversity of intrinsically disordered proteins involved in one-to-many binding, Hsu [/bib_ref] [bib_ref] Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions, Oldfield [/bib_ref]. For example, residues 374-388 in the C-terminus of p53 are disordered in the absence of any binding partners but upon interaction with cyclin A2, sirtuin, CREB binding protein and S100ββ this region adopts a coil, a β-strand, a coil and an α-helix structure, respectively [bib_ref] Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions, Oldfield [/bib_ref]. The β-strand conformation formed in molecule B in the transitional crystal structure [fig_ref] Figure 1: Cont. [/fig_ref] has relevance for our understanding of protein misfolding and in particular protein aggregation and amyloid formation. This is a clear demonstration of a system where intermolecular interactions, involving both the protein backbone and also charged and polar side chains in hydrogen bond and salt bridge formation, together with van der Waals interactions between more hydrophobic atoms, can stabilise very different conformational properties in a given protein sequence and lead to the formation of short regions of β-sheet. Short sequences, such as that of the linker peptide, in an overall protein sequence could then provide nucleation sites for the overall conversion of a soluble folded protein into amyloid aggregates. Indeed, previous studies have shown that even proteins with a highly helical native structure, such as myoglobin and cytochrome c 552 [bib_ref] Amyloid fibrils from muscle myoglobin-Even an ordinary globular protein can assume a..., Fandrich [/bib_ref] [bib_ref] Amyloid fibril formation by a helical cytochrome, Pertinhez [/bib_ref] , can form amyloid fibrils when the native state is destabilized but conditions favour non-covalent interactions such as hydrogen bonds [bib_ref] Designing conditions for in vitro formation of amyloid protofilaments and fibrils, Chiti [/bib_ref]. In the extended crystal structure, the linker peptide adopts a helical conformation connecting the stalk and catalytic domains [fig_ref] Figure 1: Cont. [/fig_ref]. The observation of a long helix connecting two domains in a crystal structure which converts into a flexible loop region in solution, as reported here, has also been seen in a number of other systems. For example, the crystal structure of calcium-bound calmodulin showed two domains linked by a long, rigid central helix [bib_ref] Structure of calmodulin refined at 2.2 Åresolution, Babu [/bib_ref] [bib_ref] Structure of a recombinant calmodulin from Drosophila-Melanogaster refined at 2.2-Åresolution, Taylor [/bib_ref]. However, NMR studies demonstrated that in solution the central helix is disrupted near its midpoint giving a flexible linker between the two domains [bib_ref] Backbone dynamics of calmodulin studied by 15 N relaxation using inverse detected..., Barbato [/bib_ref]. Disruption of a long α-helix connecting two domains in a crystal structure is also seen for the virulence factor Mip protein in solution [bib_ref] Domain motions of the Mip protein from Legionella pneumophila, Horstmann [/bib_ref]. Another example is the ribosomal protein L12 where crystallographic studies identified two different dimerization modes [bib_ref] Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein..., Wahl [/bib_ref]. This ribosomal system also has a hinge interdomain linker which is unstructured in solution but helical in the crystal structures [bib_ref] From structure and dynamics of protein L7/L12 to molecular switching in ribosome, Bocharov [/bib_ref] [bib_ref] The enigmatic ribosomal stalk, Liljas [/bib_ref] as seen in PmScsC. In all of these examples, the flexibility of the region identified as a linker or hinge in solution has been recognised to have functional importance [bib_ref] The role of dynamic conformational ensembles in biomolecular recognition, Boehr [/bib_ref]. These regions not only give independent motion to the different domains they connect but also allow for the domains to bind to a variety of different target peptides, proteins or substrates [bib_ref] Domain motions of the Mip protein from Legionella pneumophila, Horstmann [/bib_ref] [bib_ref] Solution structure of a calmodulin-target peptide complex by multidimensional NMR, Ikura [/bib_ref]. In the case of PmScsC, the flexibility may be important in allowing bound misfolded substrate to access a broad folding landscape so allowing for disulphide bond shuffling [bib_ref] A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance, Furlong [/bib_ref]. This is in agreement with previously published experimental observations that the deletion or replacement of the linker peptide with a rigid helical peptide abolishes the disulphide isomerase activity of PmScsC [bib_ref] Engineered variants provide new insight into the structural properties important for activity..., Furlong [/bib_ref]. Interestingly, sequence comparisons have identified two other DsbA-like bacterial disulphide isomerases, L. pneumophilia DsbA2 and C. crescentus ScsC, that contain a peptide sequence that may be indicative of intrinsic disorder in between their N-terminal oligomerization and C-terminal catalytic domains [bib_ref] The atypical thiol-disulfide exchange protein alpha-DsbA2 from Wolbachia pipientis is a homotrimeric..., Walden [/bib_ref]. Thus, it appears that the intrinsic disorder propensity of the PmScsC linker peptide, rather than its ability to adopt different rigid structures, is of key functional importance in PmScsC and potentially in other related bacterial disulphide isomerases. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11050642/s1, [fig_ref] Figure 1: Cont. [/fig_ref] Overlay of the 1 Hα-13 Cα region of the natural abundance 1 H-13 C HSQC spectra of the shape-shifter peptide in H 2 O and in 50% TFE. [fig_ref] Figure 2: Summary of the NMR data for the linker peptide [/fig_ref] Overlay of the natural abundance 1 HN-15 N HSQC spectra of the shape-shifter peptide in H 2 O and in 50% TFE. [fig_ref] Figure 3: The four central structures show the loop [/fig_ref]. Far-UV CD spectra collected for the shape-shifter peptide as a function of TFE concentration. [fig_ref] Figure 4: Structural changes observed in a 20 ns molecular dynamics simulation of the... [/fig_ref] : Phi and psi torsion angles for residues 35-60 in PmScsC. : The results of clustering the 40000 conformers taken from the four 100 ns MD simulations of the linker peptide. [fig] Figure 1: Cont. [/fig] [fig] Figure 2: Summary of the NMR data for the linker peptide. (a) Secondary shifts ∆C α -∆C β . (b) Secondary shifts ∆H α . (c) The Secondary Structure Propensities (SSP) scores. (d) The 3 J HNα coupling constant values.In each panel the data for the linker peptide in water and in 50% TFE solution are shown with a filled and open bar or symbol, respectively. In panel b the dashed lines indicate the 0.1 ppm cut off usually used for secondary structure identification. In panel c the bars show the SSP scores without averaging and the solid and dashed lines show the SSP scores averaged over a 5-residue window for the peptide in water and 50% TFE solution, respectively. The predicted coupling constant values for a random coil, taking into account the character of the preceding residue, are indicated with an x symbol in panel d[26]. [/fig] [fig] Figure 3: The four central structures show the loop (4xvw, molecule A), helix (5id4), β-strand (5idr molecule B) and irregular helix (5idr molecule A) conformations of the linker peptide in crystal structures which were used as starting structures for the MD simulations. The secondary structure indicated is that present in the intact protein trimer. Structures 1-7 show the conformations at the centres of the seven main clusters identified in the combined four 100 ns MD simulations of the linker peptides. [/fig] [fig] Figure 4: Structural changes observed in a 20 ns molecular dynamics simulation of the extended structure trimer of PmScsC. (a) Cartoon representation of the trimer after 20 ns MD simulation. The linker peptide is shown in blue in all three molecules. Gln 43 and Gly 188 from molecule B are shown in blue and red, respectively. (b) An expansion showing residues 38-50 and 174-193 from molecule B. Following partial unfolding of the helical linker peptide, the side chain Nε2 of Gln 43 and the backbone CO of Gly 188 form a hydrogen bond which is not found in the starting structure. (c) Phi (open square and solid line) and psi (filled circle and dotted line) torsion angles for residues 35-60 in chains A (black), B (red) and C (blue) in the trimer at the end of the 20 ns MD simulation. Residues 38 and 39 adopt extended β-strand phi/psi angles in chain B after the 20 ns simulation. [/fig] [fig] Author: Contributions: Conceptualization, L.J.S. and C.R.; methodology, L.J.S. and C.R.; formal analysis, L.J.S., C.W.G. and C.R.; investigation, L.J.S., C.W.G. and C.R.; resources, L.J.S. and C.R.; data curation, L.J.S. and C.R.; writing-original draft preparation, review and editing, L.J.S. and C.R.; supervision, L.J.S. and C.R.; funding acquisition, L.J.S. and C.R. All authors have read and agreed to the published version of the manuscript. Funding: The Bruker console and probe upgrades to the 500 and 600 and 950 MHz NMR spectrometers were funded by the Wellcome Trust (Ref: 075330 for 500 and Ref:095872 for 600), and with support from the Wellcome Trust Institutional Strategic Support Fund, the John Fell Fund and the EPA Cephalosporin Fund in Oxford. We acknowledge the use of the Advanced Research Computing (ARC) facility at the University of Oxford in carrying out some of this work, http://www.arc.ox.ac.uk. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The NMR resonance assignments are openly available in the BioMa-gRes Bank with deposition codes 50399 and 50400. [/fig]

Hippo Pathway and YAP Signaling Alterations in Squamous Cancer of the Head and Neck Head and neck cancer affects the upper aerodigestive tract and is the sixth leading cancer worldwide by incidence and the seventh by cause of death. Despite significant advances in surgery and chemotherapy, molecularly targeted therapeutic options for this type of cancer are scarce and long term survival rates remain low. Recently, comprehensive genomic studies have highlighted the most commonly altered genes and signaling pathways in this cancer. The Hippo-YAP pathway has been identified as a key oncogenic pathway in multiple tumors. Expression of genes controlled by the Hippo downstream transcriptional coactivators YAP (Yes-associated protein 1) and TAZ (WWTR1, WW domain containing transcription regulator 1) is widely deregulated in human cancer including head and neck squamous cell carcinoma (HNSCC). Interestingly, YAP/TAZ signaling might not be as essential for the normal homeostasis of adult tissues as for oncogenic growth, altogether making the pathway an amenable therapeutic target in cancer. Recent advances in the role of Hippo-YAP pathway in HNSCC have provided evidence that genetic alterations frequent in this type of cancer such as PIK3CA (phosphatidylinositide 3-kinase catalytic subunit alpha) overexpression or FAT1 (FAT atypical cadherin 1) functional loss can result in YAP activation. We discuss current therapeutic options targeting this pathway which are currently in use for other tumor types.protein 1) is the main downstream effector of the pathway and acts as a transcription cofactor regulating the expression of genes involved in cell proliferation, pro-survival and cell migration signals, all of which contribute to the pro-tumorigenic phenotype[12]. Recent evidence shows that some types of tumors could rely on YAP transcriptional regulation[13,14]. This opens the way for strategies targeting YAP as a new therapeutic option for the treatment of cancer.Head and Neck Cancer: Today´s Problems and NeedsHead and neck cancer arises in the upper aerodigestive tract (lips, oral cavity, salivary glands, larynx, nasopharynx, hypopharynx and oropharynx), and is the sixth most common cancer type worldwide and the seventh by cause of death[15]. Current figures are discouraging-nearly 900,000 people are diagnosed with this type of cancer every year[15]and only about half of them will survive the first five years[16]. Most of these tumors are head and neck squamous cell carcinomas (HNSCCs) that develop in the outer layer of the skin and in the mucous membranes of the tract. The specific locations affected by HNSCC are the oral (including lips) and nasal cavities, paranasal sinuses, pharynx and larynx. Classical risk factors for HNSCC are smoking and heavy alcohol consumption. Moreover, patients with genetically inherited diseases such as Li-Fraumeni or Fanconi anemia show increased susceptibility to the development of this type of cancer. However, the etiology of this disease is gradually changing in the Western world where infection with high-risk human papillomavirus (HPV) is the cause of a rising number of these tumors despite reduction in cigarette smoking rates[17]. HPV positive tumors particularly affect the oropharynx and show a better prognosis. They differ from HPV negative tumors not only in their etiology, localization and prognosis, but also in their molecular characteristics[3,[18][19][20]. Tumors with HPV infection do not display mutation of classical tumor suppressors and gene expression patterns reveal that they constitute a different subtype within HNSCC [3].Molecular Alterations in HNSCCInactivation of the p53 pathway is a widespread molecular event in HNSCC. Among HPV negative tumors, 92% of cases present an inactivation (mutation or deletion) of the tumor suppressors TP53 (tumor protein 53, p53) and/or CDKN2A (which encodes for p16 and p14arf)[3]. In HPV positive tumors, p53 pathway inactivation is achieved by the viral oncoproteins E6 and E7[21]. However, therapeutic strategies aimed to reactivate p53 function are not yet available in the clinical setting.Signaling pathways regulated by growth factors, such as EGFR (epidermal growth factor receptor) and PI3K/AKT (phosphatidylinositide 3-kinase; v-akt murine thymoma viral oncogene homolog), are frequently affected in HNSCC. Both pathways are interconnected and promote cell survival and proliferation, PI3K/AKT/mTOR being the most commonly altered in HNSCC[3,22]. Within this pathway, the PIK3CA (phosphatidylinositide 3-kinase catalytic subunit alpha) gene, which codes for the p110α catalytic subunit of PI3K, is the main oncogene in human cancer, conferring cells growth advantage, evasion of apoptosis and invasion capacities[21,23]. Activating mutations of PIK3CA have been found in approximately 20% of HNSCC, and increase in PIK3CA copy number and/or overexpression is present in up to 40% of the cases[3]. Overexpression of the PIK3CA gene is a poor prognosis factor in HNSCC and is associated with the activation of YAP[24]. In contrast to other tumors, mutations in EGFR are not frequent in HNSCC (≤5%)[3]. Instead, an increase in copy number and/or expression of the gene has been associated with poor prognosis, metastasis and resistance to radio and chemotherapy[25]. EGFR is the target of the monoclonal antibody Cetuximab, the only growth factor-specific targeted therapy currently used for the treatment of HNSCC[26].Alteration of the cadherin-like protein tumor suppressor FAT1 (FAT atypical cadherin 1) is a recurrent event (>10%) in human cancer[10]. Across the different cancers sequenced by The Cancer Gene Atlas (TCGA) Consortium, HNSCC is the tumor type that bears the highest rate of alterations in this gene. More than 25% of HNSCC tumors bear FAT1 mutation or deletion, approximately twice the frequency of EGFR alteration in this cancer type[27]. Despite these facts, the molecular mechanisms # Introduction Head and neck cancer is a high-incidence poor-prognosis tumor for which molecularly targeted therapeutic options are limited. This has a detrimental impact on the overall survival of patients. Part of the problem is attributable to a significant delay in the molecular characterization of this type of cancer compared to others. However, over recent years, comprehensive studies including genomic, transcriptomic and clinical data of head and neck tumors have identified the most commonly altered genes and signaling pathways in this cancer. They have also established the existence of different molecular subtypes based on transcriptomics, and gene expression signatures associated with poor outcome or drug resistance have been reported. At present, we need to make an effort to translate this knowledge into the identification of actionable molecules and pathways, that is, clinically and druggable relevant targets that help to broaden and guide head and neck cancer therapeutics. Recently, the Hippo-YAP pathway has been identified as a relevant oncogenic signaling pathway altered across a wide variety of tumor typesincluding head and neck cancer. YAP (Yes-associated that contribute to tumor development in the context of loss of FAT1 function are poorly understood. Recently, FAT1 has been identified as a Hippo pathway regulator in HNSCC. Loss of FAT1 hampers the formation of the multimeric Hippo signaling complex leading to unrestrained YAP activity and tumor progression. Thus, YAP and its regulation may be a neglected therapeutic option for HNSCC. ## Current therapies in hnscc Existing therapeutic efforts to treat HNSCC include surgery, radiation, chemotherapy and combinations thereof. Despite significant advances, mainly in surgery and radiation procedures, long term survival rates remain alarmingly low and most of the patients who experience recurrent or metastatic disease die within a year of diagnosis. The chemotherapeutic arsenal available to treat this cancer is insufficient and is based on the use of drugs that widely target DNA (Cisplatin, Fluorouracil) or microtubules (Docetaxel, Paclitaxel). To date there are only two molecularly-based treatments approved for HNSCC, the abovementioned anti-EGFR antibody Cetuximab, and the monoclonal anti-PD-L1 (programmed cell death 1 ligand 1) receptor antibodies Pembrolizumab and Nivolumab. Cetuximab was approved for the treatment of HNSCC in 2006, and over time the figures show that the survival improvement of this therapy is modest and only a small group of patients show long-term benefit. It took a decade for the next targeted anti-cancer therapy to come into play. Immune checkpoint inhibitors were introduced in 2016 to treat HNSCC; however, long-term solid evidence regarding the benefit of this therapy is still lacking. One of the main problems in the field of targeted therapies in HNSCC is the complete lack of biomarker-based patient selection to allow stratification into subgroups with different therapeutic options, even for the aforementioned molecularly based therapies. The presence of high-risk HPV in oropharyngeal cancer is the only molecular marker currently used for risk stratification. New approaches specifically targeting critical molecular pathways are needed to overcome low survival rates in HNSCC. Along these lines, EGFR and PI3K/AKT/mTOR pathway inhibitors in clinical use for other tumor types, such as Erlotinib and Afinitor, are under preclinical evaluation in HNSCC cancer. Furthermore, the specific class I PI3K p110α catalytic subunit inhibitor Alpelisib, that just gained FDA approval for the treatment of breast cancer patients with mutations in PIK3CA [33], the gene coding for this protein, is under preclinical evaluation in HNSCC. However, no PIK3CA mutation-based patient stratification is being considered in clinical trials with HNSCC patients at this point. ## Hippo pathway and yap signaling Hippo-YAP is one of the canonical oncogenic signaling pathways recently analyzed within the framework of the TCGA PanCancer Atlas initiative, which covers >9000 samples from 33 cancer types. A major role for this pathway in the control of cell growth and organ size was uncovered more than a decade ago with the molecular and phenotypical characterization of the Hippo gene in Drosophila melanogaster. The biological relevance of the pathway is highlighted by the fact that it is evolutionarily conserved from flies to mammals, where orthologs for the different components of the Hippo pathway have been described. Working as a switch, the multimeric core complex of the Hippo pathway integrates signaling from different upstream cues to control the activity of a downstream effector nuclear transcriptional module . This nuclear module activates the transcription of genes involved in cell proliferation and survival. In fully differentiated tissues and organs, cell-cell contact inhibition works to restrain cell growth. Under these circumstances, the Hippo pathway is usually active (Hippo ON) and the serine/threonine kinases that make up the Hippo upstream core complex phosphorylate and sequester the downstream transcriptional effectors in the cytosol, thus hampering the transcription of target genes. In the absence of cell-cell contact or under cell-growth requiring physiological situations, such as normal tissue repair and renewal or embryo development, the mechanisms blocking gene transcription can be switched off, leading to the activation of genes involved in cell proliferation and survival (Hippo OFF). ## Figure 1. Schematic representation summarizing the main extrinsic (cell-cell contact and growth factors) signals that regulate the Hippo-YAP (Yes-associated protein) pathway. The Hippo-YAP pathway works as a cellular switch. Cell-cell contact promotes the activation of the MST/LATS (mammalian STE20-like protein kinase/large tumor suppressor kinase) core Hippo kinases by different signaling complexes that typically act as scaffolds promoting their phosphorylation. In turn, activated LATS phosphorylates YAP, which is then targeted to ubiquitin mediated proteosomal degradation or to cytosolic sequestration by binding to 14-3-3 protein, thus preventing its nuclear translocation and switching off the expression of its target genes. In the absence of cell-cell contact or in the presence of growth signals, the components of the Hippo core complex are not active. Non-phosphorylated YAP can translocate to the cytosol binding to TEAD (TEA domain transcription factor) family transcription factors (among others) and switching on the expression of genes involved in cell proliferation, survival and migration. ## Components of the hippo pathway As mentioned above, the Hippo pathway is composed of two main elements, one responsible for the integration of the different stimuli which then controls the activity of a downstream effector element. In mammalians, the cytosolic multimeric signaling complex is formed by the serine and threonine kinases MST1 and MST2 (mammalian STE20-like protein kinase 1 and 2, also STK4 and 3), and LATS1 and LATS2 (large tumor suppressor kinase 1 and 2). MST1/2 and LAST1/2 interact with the adaptor proteins SAV1 (salvador family WW domain containing protein 1) and MOB1 (MOB kinase activator 1), respectively. When the Hippo pathway is active, the Drosophila Hippo ortholog MST1/2 phosphorylates and activates LATS1/2, which in turn phosphorylates the transcriptional coactivators YAP and its dimerizing partner TAZ. The approved gene symbol for YAP in humans is YAP1 and is expressed in two isoforms, YAP1-1 and YAP1-2. TAZ is the transcriptional co-activator with a PDZ binding domain; also known as WWTR1, WW domain containing transcription regulator 1. YAP1-1, YAP1-2 and TAZ are similar in their structure (TAZ is a paralog of YAP, arising from the duplication of a common gene), consequently they might retain similar functions and many aspects of their regulation are shared. These factors differ mainly in the number of WW domains responsible for binding to regulatory proteins such as LATS1/2 and AMOT, or downstream transcription factors such as RUNX1/2 (Runt-related transcription factor 1 and 2) or TBX5 (T-box transcription factor X5), and in the number of residues phosphorylated by LATS1/2 kinases (five in YAP and four in TAZ. Additionally, YAP contains sites that can be phosphorylated by c-Abl/Scr/Yes and by JNK kinases or methylated by Set7 histone methyltransferases that are not present in TAZ. These differences could account for some of the particularities observed in the regulation and/or functions of these factors in certain tissues or contexts. ## Yap signaling The phosphorylation of YAP and TAZ by LATS1/2 kinases of the Hippo pathway core complex promotes YAP/TAZ interaction with 14-3-3 proteins and their retention in the cytosol, or YAP/TAZ ubiquitination and subsequent degradation. Either mechanism causes YAP/TAZ nuclear exclusion which impedes the transcription of target genes. Canonical targets of this pathway include genes involved in cell adhesion and epithelial to mesenchymal transition (EMT), development, cell cycle regulation, survival and stemness (GTGF, CYR61, COL4A3, ITGB2, CCNE2, CDK2, BIRC5 and SOX9 among others). The largest YAP/TAZ regulated gene transcription signature was accomplished using oral squamous cell carcinoma (OSCC) cell lines. This signature revealed that, at least in this tumor type, YAP has a more prominent transcriptional role than TAZ. It also revealed that YAP canonical targets such as CTGF (connective tissue growth factor) or CYR61 (cysteine rich angiogenic inducer 61) do not show significant changes in their expression with respect to tumor grade or stage . . Selection of the top 100 genes (by fold change gene expression) regulated by YAP and TAZ in oral squamous cell carcinoma (OSCC) cell linesthat are involved in different molecular processes or pathways relevant to head and neck squamous cell carcinoma (HNSCC) progression. Genes also present in the consensus Cordenonsi Yap signatureare also shown. Note that none of the genes shared with Cordenonsi signature increase in HNSCC with grade or stage. Gene signatures are from the MSigDB (Molecular Signature Data Base. Broad Institute). Abbreviations for these genes are shown in Supplementary Table S1. ## Genes that increase their expression with tumor grade/stage in hnscc [formula] BLM CDC6 DIAPH3 DSCC1 DTL EXO1 HELLS MCM6 MYBL1 CCNE2 CDC25A CDC6 CENPI CENPK GINS1 GINS2 LMNB1 MCM10 MCM6 POLE2 ATAD2 CCNE2 CENPK DIAPH3 DSCC1 DTL ESCO2 FANCI GINS1 HELLS POLE2 RAD51AP1 CENPK FAM111B MCM6 HELLS MCM6 ANKRD1 AXL CTGF CYR61 DDAH1 FSTL1 SLIT2 THBS1 [/formula] In the absence of MST1/2-LATS1/2 activation, YAP and TAZ are able to translocate to the nucleus where they need to associate with DNA-binding transcription factors to regulate gene expression, since they cannot bind directly to DNA. The main transcription factors mediating YAP/TAZ activity are those belonging to the TEA domain transcription factor (TEAD) family, comprised of TEAD1 to TEAD4. In fact, YAP function can be abolished in the absence of TEADs or if the YAP-TEAD interaction is impaired. YAP and TAZ can bind to transcription factors other than TEADs, such as AP-1 (activator protein 1), the intracellular domain (ICD) of ERBB4 (erb-b2 receptor tyrosine kinase 4), Smads, transcription factors of the RUNX family and p73. Binding to these factors can occur in YAP-TAZ-TEAD complexes, thus further modulating transcriptional activity. It has been reported that the association of YAP-TAZ-TEAD with AP-1 activates genes involved in S-phase entry in epithelial cells. Similarly, the ICD of ERBB4 interacts with YAP and TEAD to promote migration in breast cancer cell lines. More recently, the discovery of the interaction of the coactivator bromodomain-containing protein (BRD4) with YAP-TAZ-TEAD1 to enhance the transcription of cancer related genes opened the way for new therapeutic strategies targeting BRD proteins to inhibit YAP activity in tumors. Binding of YAP/TAZ to Smads links Hippo and transforming growth factor-β (TGF-β) pathways, thus connecting processes such as cell density to responsiveness to TGF-β. It is worth mentioning that in some tumors, such as gastric and breast cancer, RUNX factors have a tumor suppressor activity through the formation of RUNX-YAP-TEAD complexes that reduce the transcription of YAP-TEAD target genes, thus abolishing YAP oncogenic activity. A tumor suppressor role for YAP has been reported. In response to DNA damaging drugs YAP can translocate to the nucleus and interact with p73 promoting its stabilization and subsequent acetylation by p300, this leads to the transcription of p73 proapoptotic target genes p53AIP1 and BAX. Furthermore, in HNSCC cell lines, overexpression of ∆Np63 repressed YAP expression as well as expression of other apoptotic genes promoting cell survival, whereas YAP silencing in this context enhanced proliferation, survival, migration and resistance to cisplatin. These data are in discrepancy with those obtained in OSCC cell lines, showing that YAP silencing or inhibition of YAP phosphorylation and its subsequent degradation promote cell growth, migration and tumorigenesis in in vivo models. ## Switching the hippo pathway on and off The main signal responsible for the activation of the Hippo pathway is contact with neighboring cells. In epithelial cells, the mechanisms involved in establishing cell orientation (apical, basal and lateral polarity) are important regulators of the pathway. Additionally, cells can "sense" the characteristics of the extracellular matrix and the presence of extracellular soluble growth factors. Multimeric complexes situated in different locations of the cell membrane are responsible for maintaining adherens and tight junctions as well as cell polarity. Strict specification of apical-basolateral polarity is particularly important in epithelial cells, where it regulates essential features such as stemness, differentiation and cell function. In fact, loss of cell polarity leads to dysplasia and eventually to EMT, a hallmark of cancer. The cadherin-catenin complex at adherens junctions, the aPKC-PAR complex at thigh junctions, the apical Crumbs complex and the basolateral Scribble complex, can work as scaffolds that recruit the kinases of the multimeric core complex of the Hippo pathway, promoting the activation of MST1/2 and LATS1/2 and the subsequent inactivation of YAP/TAZ. Furthermore, α-catenin at adherens junctions can sequester phospho-YAP/14-3-3 complexes, directly preventing YAP activation . Through regulation of the Hippo-YAP pathway transcriptional targets the cell can respond and adapt to changes of cell density and polarity. The same is true for the extracellular matrix (ECM). The properties of the ECM can vary during physiological processes and disease, such as tissue remodeling and regeneration, inflammation, fibrosis and cancer. Under these circumstances YAP/TAZ transcriptional activity can be modulated. For instance, when different cell types, including human mammary epithelial cells, are grown on stiff or fibronectin rich matrixes, similar to a tumor-associated ECM, the nuclear localization of YAP/TAZ and the transcription of their target genes is promoted; while cells cultured in a soft matrix display cytosolic YAP/TAZ. These experiments were performed in 2D and 3D models including fibronectin-coated glass slides, hydrogels and cells grown on rigid or elastic pillars (microposts). Using mammary epithelial cells grown on coverslips coated with different surfaces (fibronectin, poly-D-lysine or laminin), showed that integrin receptors on the cell surface bind to fibronectin in the ECM promoting LATS1/2 inactivation through a FAK (focal adhesion kinase)-Src-PI3K-PDK1 (phosphoinositide-dependent kinase 1) kinase cascade. YAP target genes include genes encoding ECM components and ECM-modifying enzymes that alter the ECM composition. In turn, the stiffening of the ECM also affects the cancer associated fibroblasts, favoring the deposition of thick and rigid collagen fibers that further sustain proliferation of the cancer cells. Additionally, intracellular cell shape and tension impact on cytoskeleton contractility and regulate the activity of YAP/TAZ through Rho GTPases-Rho associated kinases (ROCK) independently of Hippo core kinases. Some elements of the Hippo-YAP pathway are shared with other pathways thus mediating cross-talk with inputs coming from TGF-β, Wnt and growth factor signaling pathways, and metabolism. YAP/TAZ interact with the TGF-β pathway so that responsiveness to TGF-β can be modulated by cell density. The above-mentioned Crumbs complex transmits cell density information by promoting YAP/TAZ cytoplasmic retention. This can suppress TGF-β signaling since TAZ functions as a SMAD nuclear retention factor. Loss of cell density/polarity would cause the disruption of the Crumbs complex and YAP/TAZ nuclear translocation, enhancing TGF-β signaling and predisposing the cells to TGF-β-mediated EMT. Mitogenic signaling factors, such as EGF (epidermal growth factor) and Wnt ligands (Wnts), can oppose the effects of cell-contact growth inhibition and promote YAP/TAZ transcriptional activity. The binding of EGF to its receptor EGFR or LPA (lysophosphatidic acid) to G protein-coupled receptors (GPCRs) activates the PI3K-PDK1 axis. In this context PDK1, which forms a complex with MST and LATS kinases favoring LATS1/2 activation, is recruited to the cytoplasmic membrane causing the dissociation of the complex resulting in loss of LATS1/2 activation and nuclear accumulation of YAP. Wnts bind to GPCRs and can induce YAP activation mainly through the Wnt canonical pathway, which involves destabilization of the Axin/APC/GSK3 (axin/adenomatous polyposis coli/glycogen synthase kinase 3) destruction complex and the release of β-catenin and YAP from this complex and allowing the transcription of their target genes . In turn, the Hippo pathway can inhibit Wnt signaling. Cytosolic YAP/TAZ in combination with DVL (dishevelled segment polarity protein) can regulate the stability of β-catenin in the cytosol counteracting Wnt signaling, for instance high levels of cytosolic YAP inhibit intestinal crypt proliferation. On the contrary, YAP is required for the development of APC-deficient adenomasand tumorigenesis in β-catenin driven cancers relay at least in part in the formation of YAP-β-catenin-TBX5 transcriptional complexes. Thus, interactions between the Hippo and Wnt pathways might depend on cell type, cell context and subcellular localization. Finally, it has been shown that intrinsic signals such as energy stress, glucose metabolism, aerobic glycolysis and the mevalonate pathway can regulate YAP activity. ## The hippo-yap pathway in hnscc It is of no surprise that a signaling pathway controlling cell growth and connected to cell polarity and adhesion, cytoskeleton dynamics, cell survival factors signals as well as metabolism, is almost necessarily deregulated during cancer initiation, progression and metastasis. Different components of the Hippo pathway act as oncogenes (YAP, TEADs) or tumor suppressors (LATS1/2), and alterations in these factors have been described across different cancer types. Moreover, many of the above-mentioned signals controlling Hippo-YAP activity are well-known cancer pathways. This could explain why YAP/TAZ dependent gene expression is more widely deregulated in human cancer than might be expected by the frequency of alterations in its core components. Furthermore, it has been recently shown that through binding to chromatin readers, YAP/TAZ can heighten the expression of a specific set of genes to which cancer cells are addicted to. Interestingly, YAP/TAZ signaling seems to be largely dispensable for the normal homeostasis of adult tissues, making the pathway an amenable therapeutic target in cancer. The average frequency of alterations in the Hippo pathway across human cancer is 10%. However, this figure rises to more than 90% in IDH mutant low grade glioblastoma, around 50% in MSI-POLE (microsatellite instability-DNA polymerase epsilon) subtypes in colorectal, stomach and endometrial tumors, and to 42% in HPV negative HNSCC. HNSCC arises in different locations of the upper aerodigestive tract and one might expect that the primary site where the tumor arises could have an influence on the characteristics of the tumor, including its genetic features. However, current datado not show an association between alterations in the Hippo pathway genes and different locations of HNSCC. Future research in the subject could demonstrate otherwise. Amplification in YAP and TAZ are found in 5% and 9%, respectively, of the HNSCC tumors of the TCGA Pan Cancer Atlas. Additionally, two upstream regulators of this pathway are frequently altered in HNSCC, namely FAT1 and PIK3CA. Inactivation of FAT1 (deletion, truncating mutations) or activation of PIK3CA (overexpression) are associated with YAP-dependent transcriptional activation in HNSCC. The precise molecular mechanisms that contribute to tumor development in the context of FAT1 functional loss or PIK3CA overexpression are not fully understood. It has been shown that, in HNSCC derived cell lines, FAT1 directly associates with MST1 which favors its phosphorylation and the assembly of the Hippo kinase core complex leading to the subsequent phosphorylation of LATS1/2 and YAP. Overexpression of PIK3CA is associated with poor outcome in HPV negative HNSCC; these tumors show YAP nuclear localization and a YAP-activation transcriptional signature. Although the molecular mechanism linking PI3K and YAP in HNSCC has not been identified so far, in other epithelial cell lines activated PI3K recruits PDK1 to the plasma membrane disrupting its association with the Hippo core complex kinases and promoting YAP dephosphorylation. The PI3K-PDK1 pathway integrates signals from fibronectin, LPA, GPCR receptors and EGFR. Interestingly, despite the fact that nuclear YAP localization has been described in oropharyngeal HPV positive tumors, Hippo pathway alterations and in particular FAT1 inactivation or YAP amplification are not frequent events in HPV positive HNSCC. Although further research into the role of the Hippo-YAP pathway in this tumor subtype is needed, it is tempting to speculate that other mechanisms might lead to YAP activation. For instance, PIK3CA alterations are the most common genetic event in HPV positive tumors, and it has been described that the HPV E6 oncoprotein can degrade the Hippo core complex scaffolding element Scribble. In the normal oral epithelium YAP and TAZ level is generally low except for the basal layer cells that display evident nuclear YAP staining and some TAZ staining. During hyperplasia and dysplasia cells with nuclear YAP extend beyond the basal cell population and are frequent in regions of severe dysplasia. Activation of YAP/TAZ would confer these cells a proliferative advantage. There is no evidence that YAP and TAZ are significantly mutated, amplified or overexpressed in OSCC tumors. YAP and TAZ expression was not associated with tumor stage or grade in some cohorts. This suggests that alteration in YAP/TAZ upstream regulators (i.e., FAT1 and PIK3CA in HNSCCtakes place during HNSCC tumor progression leading to the activation of these two co-transcriptional factors and their target genes. At the clinical level in HNSCC, YAP and TAZ have been proposed as poor prognosis markersand YAP activation has been associated with resistance to different anticancer therapies. Analysis of YAP/TAZ expression signatures in OSCC cell lines indicated that YAP has a more prominent role than TAZ in the regulation of transcription, at least in this type of cancer. That said, in OSCC, overexpression of TAZ has been associated with poor outcome and aggressive tumor features such as size, grade and lymph node spreading in some studieswhile other show no association with tumor grade or stage. Interestingly, the analysis of YAP/TAZ transcriptional targets in OSCCrevealed that increased expression of TEAD4, but not other canonical targets such as CTGF or CYR61, associated with increased tumor grade or stage in the TCGA cohort of HNSCC. Additionally, TEADs can favor YAP/TAZ nuclear retentionthus further enhancing YAP/TAZ-TEAD4 mediated gene transcription in these tumors. A signature based on HNSCCs with YAP amplification and overexpression, defined a YAP-activated subgroup of tumors with worse prognosis across different HNSCC cohorts. In this study, the YAP-inactivated subgroup associated with HPV positive status, which was consistent with the absence of YAP amplification in HPV positive HNSCC. YAP amplification, but not high EGFR protein levels, was identified as biomarker of resistance to Cetuximab. Increased YAP expression has been associated with resistance to cisplatinand to radiotherapy. Furthermore, TAZ depletion restores sensitivity to cisplatin in nasopharyngeal carcinoma cells. . Note that PIK3CA codes for the catalytic subunit of PI3K (phosphatidylinositide 3-kinase). In non-tumor cells, in the presence of low levels of EGFR and normal PIK3CA expression, PDK1 (phosphoinositide-dependent kinase 1) forms a complex with the Hippo signaling core complex promoting YAP phosphorylation. Similarly, FAT1 acts as a scaffold for Hippo kinases, thus favoring their activation. In a tumor cell, the absence of FAT1 or the presence of high levels of EGFR and increased PI3K activity, which recruits PDK1 to the cell membrane, dismantles the Hippo core complex leading to YAP dephosphorylation and its translocation to the nucleus. RTKs: receptor tyrosine kinases. The tumor immune microenvironment (TIME) plays an important role in many tumors including HNSCC. HNSCCs with YAP amplification and/or overexpression associate with resistance to the immunotherapy agent Pembrolizumab. Conversely, it has been reported than in other tumor types such as lung and melanoma, YAP induced PD-L1 expression suggesting that immunotherapy could be effective against these tumors. There is further evidence that YAP expression in cancer cells can influence the recruitment and characteristics of the immune cells in the through the production of cytokines. YAP activity in cancer cells induces the expression of cytokines such as IL-6 (interleukin 6), CXCL5 (C-X-C motif chemokine 5), and granulocyte-macrophage colony stimulating factors that stimulate the recruitment of myeloid-derived suppressor cells (MDSCs). MDSCs inhibit cytotoxic T cell activity, contributing to promote an immune-suppressive tumor-tolerant microenvironment, and promote tumor angiogeneisis. Immunodepletion of MDSCs reduced tumor growth in an in vivo model of oral cancer. Large numbers of Tregs (regulatory T cells) are present in the HNSCC and their abundance is associated with poor prognosis. This subset of T cells also contributes to an immunosuppressive microenvironment. In Tregs, YAP activity is required for their accumulation and suppressive function. ## Therapeutic opportunities for hnscc targeting the hippo-yap pathway Different inhibitors have been identified to directly target YAP/TAZ, their upstream regulators or their downstream effectors. The challenge is, however, to find those more suitable for the treatment of HNSCC and to identify subgroups of patients that would benefit from these therapies. Direct inhibitors of YAP/TAZ include the drug Verteporfin and a synthetic polypeptide termed "super-TDU" designed to hamper YAP-TEAD interaction. Verteporfin is an FDA-approved drug marketed under the name of Visudyne for the treatment of patients with certain serious eye conditions. It has shown good results in different cancer models including liver, pancreatic, gastric and head and neck, and is under clinical trials for prostate and breast cancer (ClinicalTrials.gov Identifier NCT03067051 and NCT02872064, respectively). However, clinical trials are based on the photodynamic properties of the drug and Verteporfin is used there as a photosensitizer. While Verteporfin can effectively disrupt YAP/TAZ interaction with TEADs, other effects for this drug have been described, such as inhibition of autophagosome formation, thus making it not so suitable for use as a specific YAP/TAZ transcriptional inhibitor in cancer. Super-TDU competes with YAP in binding to TEADs and is still under preclinical development. It has been proven to be effective in gastric and colorectal cancer models. To our knowledge, there are no reports regarding the use of this peptide in HNSCC. However, two different HNSCC cell lines (WSU-HN13and FaDuwere assayed in our laboratory and Super-TDU did not show an inhibitory effect in cell growth even at nearly micromolar concentrations (unpublished observation). A feasible anti-tumor strategy would be to target pathways upstream or downstream of YAP/TAZ relevant for each cancer type. Statins and Dasatinib (Src inhibitor) were identified as candidate drugs to inhibit YAP/TAZ activity in cancer cells. Although there are examples of the use of both, studies in HNSCC specifically addressing their effect on YAP/TAZ activity are scarce. It is worth mentioning that an inhibitory role for simvastatin has been observed in a model of OSCC; this statin was able to repress TAZ resulting in an anticancer effect. Given that the PI3K-PDK1 axis mediates YAP/TAZ activation by different stimuli (EGFR, FAK, fibronectin, GPCRs) and that alterations in the PIK3CA gene are frequent events in HNSCCs and are associated with YAP transcriptional activation and poor outcome, specific PI3K inhibitors should be evaluated. Recently, inhibitors of the bromodomain and extra-terminal domain (BET) family of proteins (BRD1-4) have shown successful results in the treatment of HNSCC, including tumor models resistant to Cetuximab. This brings HNSCC therapeutic options into the thriving field of epigenetics. BETs are chromatin readers and mainly recognize lysine acetylation in H3 and H4, thus influencing gene expression. In the nucleus, YAP-TAZ-TEAD1 complexes interact with BRD4 and drive the expression of sets of genes involved in cancer transcriptional programs. Furthermore, alterations in some components of the Hippo pathway are determinants of sensitivity to BET protein inhibitors. Some BET protein inhibitors, such as Birabresib, are under clinical trials in different hematologic and solid tumors, but not in HNSCC. The small molecule JQ-1 specifically targets BRD4 and inhibited tumor growth and metastasis in a chemical-induced orthotropic model of HNSCC and in PDX (patient derived xenografts). Thus, although BET protein inhibitors seem a promising therapeutic strategy for the treatment of HNSCC with YAP/TAZ activation, more research is needed in the field before translating these advances into the clinical setting. The identification of key players in HNSCC such as FAT1 and PI3K as regulators of the Hippo-YAP pathway, as well as the activation of YAP as a relevant oncogenic mechanism in head and neck cancer opens the way for the use of different therapeutic strategies targeting this pathway in this tumor type. In particular, small-molecule inhibitors of Hippo-YAP upstream activators or inhibitors of YAP transcriptional activity are currently available in the clinical setting or are under development. Presently, research should focus on understanding the precise mechanisms of action of these drugs in the context of HNSCC using both in vivo and in vitro models. These studies will set the bases for much needed clinical trials that contribute to broaden the therapeutic options for this type of cancer. Ideally, the application of these therapies should go hand-in-hand with the identification and validation of biomarkers for this pathway, such as the abovementioned, that allow a molecularly-based stratification of patients.

Strontium ranelate and alendronate have differing effects on distal tibia bone microstructure in women with osteoporosis The structural basis of the antifracture eYcacy of strontium ranelate and alendronate is incompletely understood. We compared the eVects of strontium ranelate and alendronate on distal tibia microstructure over 2 years using HR-pQCT. In this pre-planned, interim, intention-totreat analysis at 12 months, 88 osteoporotic postmenopausal women (mean age 63.7 § 7.4) were randomized to strontium ranelate 2 g/day or alendronate 70 mg/week in a double-placebo design. Primary endpoints were changes in microstructure. Secondary endpoints included lumbar and hip areal bone mineral density (aBMD), and bone turnover markers. This trial is registered with http://www. controlled-trials.com, number ISRCTN82719233. Baseline characteristics of the two groups were similar. Treatment with strontium ranelate was associated with increases in mean cortical thickness (CTh, 5.3%), cortical area (4.9%) and trabecular density (2.1%) (all P < 0.001, except cortical area P = 0.013). No signiWcant changes were observed with alendronate. Between-group diVerences in favor of strontium ranelate were observed for CTh, cortical area, BV/TV and trabecular density (P = 0.045, 0.041, 0.048 and 0.035, respectively). aBMD increased to a similar extent with strontium ranelate and alendronate at the spine (5.7% versus 5.1%, respectively) and total hip (3.3% versus 2.2%, respectively). No signiWcant changes were observed in remodeling markers with strontium ranelate, while suppression was observed with alendronate. Within the methodological constraints of HR-pQCT through its possible sensitivity to X-ray attenuation of diVerent minerals, strontium ranelate had greater eVects than alendronate on distal tibia cortical thickness and trabecular volumetric density. # Introduction Bone fragility increases as age advances because of the appearance of abnormalities in bone remodeling [bib_ref] Relationships between surface, volume and thickness of iliac trabecular bone in aging..., Parwtt [/bib_ref]. The negative balance between the relatively greater volume of bone resorbed by osteoclasts than that subsequently deposited by osteoblasts in the basic multicellular unit (BMU) combined with a high rate of bone remodeling produce trabecular thinning, loss of trabeculae, cortical porosity and cortical thinning [bib_ref] Bone quality-the material and structural basis of bone strength and fragility, Seeman [/bib_ref]. Several agents reduce the risk of fractures in women with postmenopausal osteoporosis [bib_ref] Treatment of postmenopausal osteoporosis, Delmas [/bib_ref]. However, the microstructural changes that reduce disease progression or partly reverse bone fragility are not completely understood, in part, because non-invasive techniques for measuring them have only recently become available [bib_ref] In vivo assessment of trabecular bone microarchitecture by high resolution peripheral quantitative..., Boutroy [/bib_ref] [bib_ref] Ridge number density: a new parameter for in vivo bone structure analysis, Laib [/bib_ref] [bib_ref] Calibration of trabecular bone structure measurements of in vivo three-dimensional peripheral quantitative..., Laib [/bib_ref]. DeWning these changes is important because only a small proportion of the fracture risk reduction with antiresorptives is accounted for by the increase in aBMD. Indeed, aBMD does not capture the contribution of the reduction in cortical and trabecular thinning, or intra-cortical porosity that may occur in response to therapy [bib_ref] Bone quality-the material and structural basis of bone strength and fragility, Seeman [/bib_ref] [bib_ref] Is a change in bone mineral density a sensitive and speciWc surrogate..., Seeman [/bib_ref]. The antiresorptive agent alendronate reduces the rate of remodeling by about 60% [bib_ref] EVect of oral alendronate on bone mineral density and the incidence of..., Liberman [/bib_ref]. Following initiation of treatment, steady state is perturbed as many cavities excavated by high remodeling before treatment partly Wlled by bone formation while 40% fewer new BMUs in their resorptive phase appear [bib_ref] Bisphosphonates: how do they work?, Papapoulos [/bib_ref]. The result of this perturbation is a net rapid increase in aBMD with alendronate [bib_ref] Mechanisms of action of bisphosphonates: similarities and diVerences and their potential inXuence..., Russel [/bib_ref]. However, no longitudinal in vivo data in humans are available to determine the microstructural changes that accompany this modiWcation. In cross-sectional studies on single bone biopsies from postmenopausal osteoporotic women, alendronate preserves existing architecture but has not been reported to improve or restore the architectural integrity of bone [bib_ref] Histomorphometric assessment of the long-term eVects of alendronate on bone quality and..., Chavassieux [/bib_ref] [bib_ref] DiVerential eVects of teriparatide and alendronate on bone remodeling in postmenopausal women..., Arlot [/bib_ref] [bib_ref] Trabecular bone microarchitecture after alendronate treatment of osteoporotic women, Recker [/bib_ref]. Bone strength has been shown to increase in monkeys after treatment with alendronate [bib_ref] The eVects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism,..., Balena [/bib_ref]. Strontium ranelate does not reduce the rate of bone remodeling as assessed by dynamic histomorphometry [bib_ref] Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated..., Arlot [/bib_ref]. It does increase in vitro pre-osteoblastic cell replication and osteoblastic diVerentiation with mineralized nodule formation [bib_ref] Osteoblasts play key roles in the mechanisms of action of strontium ranelate, Brennan [/bib_ref] [bib_ref] Dual EVect of strontium ranelate: stimulation of osteoblast diVerentiation and inhibition of..., Bonnelye [/bib_ref]. Exposure of cells to strontium ranelate in vitro increases the ratio of osteoprotegerin to receptor activator of nuclear factor kappa B ligand (OPG/ RANKL) in the medium, while treatment of cells reduces resorption in calvaria [bib_ref] The calcium-sensing receptor (CaR) is involved in strontium ranelate-induced osteoblast proliferation, Chattopadhyay [/bib_ref] [bib_ref] Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while..., Atkins [/bib_ref]. However, there is no in vivo evidence for a reduction in volume of bone resorbed by osteoclasts or an increase in mean wall thickness (a measure of the volume of bone formed by the BMU) with strontium ranelate [bib_ref] Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated..., Arlot [/bib_ref]. Nevertheless, CT analysis on unpaired biopsies from patients treated for 3 years with strontium ranelate suggests a higher cortical thickness and trabecular number than in the placebo group [bib_ref] Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated..., Arlot [/bib_ref]. Results on 4 paired iliac crest bone biopsies from postmenopausal osteoporotic women from a 3-year phase III study suggest that these changes might be due to improvement in trabecular and cortical bone microstructure [bib_ref] Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated..., Arlot [/bib_ref] , an observation requiring conWrmation in larger-scale studies. There is also evidence for improvements in bone strength with strontium ranelate [bib_ref] Strontium ranelate improves bone strength in ovariectomized rat by positively inXuencing bone..., Bain [/bib_ref]. The development of non-invasive methods for quantifying bone microstructure has made it possible to study the early changes in bone structure that may, in part, explain fracture risk reductions observed with antiosteoporotic agents [bib_ref] Ridge number density: a new parameter for in vivo bone structure analysis, Laib [/bib_ref] [bib_ref] Calibration of trabecular bone structure measurements of in vivo three-dimensional peripheral quantitative..., Laib [/bib_ref]. We compared the eVects of strontium ranelate and alendronate, both of which have demonstrated antifracture eYcacy [bib_ref] The eVects of strontium ranelate on the risk of vertebral fracture in..., Meunier [/bib_ref] [bib_ref] Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with..., Reginster [/bib_ref] [bib_ref] Randomised trial of eVects of alendronate on risk of fracture in women..., Black [/bib_ref] , on bone microstructure of the distal tibia and distal radius using HR-pQCT in a pre-speciWed interim analysis at 1 year, in a trial lasting 2 years. # Materials and methods ## Study design In this randomized, double-blind, double-dummy, two-year study, patients were randomly assigned (1:1) to strontium ranelate 2 g/day or alendronate 70 mg/week. All received calcium (500 mg) and vitamin D (400 IU) supplementation. Compliance was deWned as the number of sachets taken divided by the theoretical number of sachets to be taken. Patients were ambulatory Caucasian women, aged¸50, at least 5 years after menopause with a lumbar, femoral neck, or total hip T-score ·¡2.5. The main exclusion criteria were body mass index (BMI) >30 or <18 kg/m 2 , diseases and treatments that interfere with bone metabolism (bisphosphonate for more than 1 year, strontium ranelate or glucocorticoids in the previous year, calcitonin within previous 6 months, or hormone replacement therapy within previous 3 months), oesophageal disease, and patients at risk of venous thrombo-embolism. This trial is registered with http://www.controlled-trials.com, number ISRCTN8 2719233. The protocol was approved by local ethics committees and followed ethical principles stated in the Declaration of Helsinki, 1964, amended Edinburgh 2000. All women gave their written informed consent prior to participation. HR-pQCT techniques HR-pQCT (Xtreme CT, Scanco Medical AG, Bruttisellen, Switzerland) examinations were performed at baseline, at 3 and 6 months, and then every 6 months at the non-dominant distal tibia and distal radius. For tibia, the reference line was set at the distal joint limit and for the radius at the most proximal location of the sub-chondral plate. The region of interest (ROI) consisted of 110 slices measured proximally to a 22.5-mm point from the reference line for the tibia and at 9.5 mm for the radius. Scans were read centrally (Scanco Medical AG). The central facility was blind to treatment assignment. Scan quality was rated as follows: very good quality, exam without any movement artifacts; good quality, minor movement artifacts not aVecting the integrity of the cortical circumference; medium quality, clearly visible movement artifacts with horizontal streaks at cortical edge; poor quality, marked movement artifacts with disruption in cortical edge and image partially blurred; and very poor quality, completely blurred images. A scan was considered as assessable if it was rated very good or good quality. Daily quality controls were centrally reviewed, and cross-calibration of the devices was performed using a density phantom and an anthropomorphic phantom (a nonhuman bone specimen prepared by Dr M. A Krieg, Lausanne, Switzerland) to follow the stability of both density and structural parameters. The methods used to process and validate the HR-pQCT data have been reported elsewhere [bib_ref] Ridge number density: a new parameter for in vivo bone structure analysis, Laib [/bib_ref] [bib_ref] Calibration of trabecular bone structure measurements of in vivo three-dimensional peripheral quantitative..., Laib [/bib_ref]. In brief, cortical and trabecular compartments were separated by segmentation using a low-pass Wlter. The threshold to determine cortical bone from trabecular bone was set at 1/3 of the apparent cortical bone density value [bib_ref] In vivo resolution 3D-QCT of the human forearm, Laib [/bib_ref]. Cortical thickness (CTh) was the mean cortical volume divided by the outer bone perimeter. Trabecular bone density (Dtrab, trabecular volumetric BMD) was computed as the average mineral density within the trabecular volume of interest. Trabecular bone volume fraction (BV/TV) was derived from Dtrab (BV/TV = 100 £ Dtrab/1200). Trabecular number (TbN) was evaluated using the Ridge number [bib_ref] Ridge number density: a new parameter for in vivo bone structure analysis, Laib [/bib_ref]. TbN was the inverse of mean spacing of the mid-axes. Heterogeneity of the trabecular network (Tb.1/N.SD) was evaluated measuring the SD of the individual distribution of the trabeculae separation. Trabecular thickness (TbTh) and separation (TbSp) derived from BV/TV and TbN:TbTh = (BV/TV)/TbN and TbSp = (1 ¡ BV/TV)/TbN [bib_ref] Relationships between surface, volume and thickness of iliac trabecular bone in aging..., Parwtt [/bib_ref]. Periosteal perimeter and cross-sectional area (CSAtotal, mm²) of the tibia and radius were used to match the volumes of interest on the baseline and follow-up scans. Area of the cortical bone (CSA cort ) and the trabecular bone (CSA trab ) was calculated. For follow-up, only the bone volume common to all assessable scans was used to assess density and microstructure. Common region was 93.0% (range, 74-99; median, 94.0%). ## Other analyses Total hip, femoral neck and lumbar aBMD was measured by dual-energy X-ray absorptiometry (DXA) using Hologic or Lunar devices at baseline, at 1 and 2 years. Baseline values are means of two examinations. A cross-calibration program was performed throughout the study (Dr M-A Krieg). Bone markers were assessed at baseline, 3 and 6 months, and then every 6 months over 2 years. The bone formation marker bone alkaline phosphatase (b-ALP), and the bone resorption marker C-telopeptides crosslinks of type-1 collagen (S-CTX) were centrally assessed (Supreme, Liège, Belgium). b-ALP was assayed by immunoradiometric assay (Tandem ® -ROstase ® , Beckman Coulter, San Diego, CA, USA), and S-CTX was assayed using an enzyme-linked immunosorbent assay (Serum Cross-Laps ® ELISA, Nordic Bioscience Diagnostic, Herlev, Denmark). Adverse events were recorded at each study visit. Full blood cell count, creatinine, blood calcium and blood strontium were measured at all visits. # Statistical analysis The study was exploratory. No formal sample size calculation was performed. However, a calculation of the study power was conducted and showed that, with a risk = 0.05, the statistical power to detect a diVerence was 86% for CTh and 70% for Dtrab. An interim pre-planned analysis was performed at 1 year while maintaining the blinding for the sponsor, investigator and patients. All eYcacy analyses were performed in intention-to-treat (ITT) and included randomized patients having taken at least one dose of study medication, with a baseline and postbaseline assessable HR-pQCT scans. All analyses were performed on non-calibrated data. For the tibia, 92% of the patients had at least two scans rated as good or very good quality. For the radius, 28% of the examinations were not satisfactory. Criteria analyzed were as follows: TbN, Tb.Th, TbSp, CTh, Tb.1/N.SD, BV/TV, D cort (cortical volumetric BMD), D trab (trabecular volumetric BMD), CSA total , CSA trab and CSA cort . Relative change from baseline to last observation (end) at the 1-year visit was analyzed with a linear model with center as covariate (Wxed eVect). The robustness of this main analysis was veriWed using the same model with center as random eVect. Moreover, since the distribution of HR-pQCT data is not well deWned, a second robustness analysis was performed using a non-parametric approach without adjustment based on Hodges-Lehmann's estimator. This type of non-parametric approach avoids hypotheses on the normality of the distribution of the data usually made in the parametric model. Treatment eVect was analyzed using a Student t test or a Wilcoxon signed-ranks test, and treatment groups were then compared using Student t test based on the overall general linear model (least-squares norm) or with a Mann-Whitney Wilcoxon test when appropriate. The withingroup results are presented as means § SD or median when non-normally distributed, and between-group diVerences are presented as estimates § SE. Similar analyses were performed for aBMD and bone turnover markers. Safety was analyzed using descriptive statistics. The type I error rate was set at 5%. ## Correlation between cortical thickness and bone strontium content To evaluate a possible inXuence of bone strontium content on CTh measurement by computerized tomography, we analyzed the correlation between CTh and bone strontium content in 10 biopsies from postmenopausal women treated for 3 years with strontium ranelate 2 g/day in the SOTI and TROPOS trials [bib_ref] Histomorphometric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated..., Arlot [/bib_ref] [bib_ref] The eVects of strontium ranelate on the risk of vertebral fracture in..., Meunier [/bib_ref] [bib_ref] Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with..., Reginster [/bib_ref] [bib_ref] In osteoporotic women treated with strontium ranelate, strontium is located in bone..., Boivin [/bib_ref]. In brief, the embedded biopsy blocks were examined at 20 m isotropic resolution using a CT scanner ( CT 40, Scanco Medical AG, Bruettisellen, Switzerland). Mineralized bone was separated from bone marrow with a 3D segmentation algorithm. CTh was calculated as the average thickness of the 3D cortex from the inner to the outer cortical surfaces of the biopsy specimens. For measurement of bone strontium, a part of the embedded bone biopsies was subjected to nitric acid/perchloric acid digestion at high temperature. Total bone calcium and strontium concentrations were measured by inductively coupled plasma optical emission spectrometry using a vacuum polychromator. Total bone strontium content was calculated and expressed as the ratio Sr/Sr + Ca. In addition, we analyzed the correlation between distal tibia cortical thickness and area, and serum strontium levels, as a surrogate of bone strontium content, since there were no bone biopsies collected in the present trial. ## Role of the funding source Servier funded this study. Statistical analyses were subcontracted by Servier to Biotrial (Rennes, France). Data concerning HR-pQCT, biochemical markers and blood strontium were collected centrally by independent centers and transferred to Biotrial for statistical analysis. BMD print-outs were sent directly from the investigator centers to Biotrial and data captured. Remaining data collected in this study were held by Servier, locked, and then transferred to Biotrial. Only Biotrial had access to the decoding list. To maintain blinding, results of quantitative data analyses were given by treatment groups (mean § SD), but minimal and maximal values, and values that could lead to the identiWcation of a patient, were not provided. For qualitative data, only statistics on the whole population were provided. All authors reviewed the manuscript and jointly agreed to submit the Wnal version of the manuscript. The corresponding author had access to all the data and had the Wnal responsibility for the decision to submit the manuscript. # Results The two groups had comparable baseline characteristics [fig_ref] Table 1: Baseline characteristics Values are mean § SD BMI body mass index [/fig_ref]. Of 88 patients randomized, 75 (85%) completed the Wrst year and 85 (97%) were included in the ITT population. Compliance was ¸80% in 93% of subjects. Treatment duration and exposure were similar in the two groups. For the tibia, at 12 months, D cort increased by 1.1 § 2.7% (P = 0.005) with strontium ranelate but not with alendronate (0.5 § 2.0%, P = 0.114). D cort was 758.9 § 86.0 mg HA/cm 3 with strontium ranelate and 749.6 § 78.0 mg HA/cm 3 with alendronate [fig_ref] Table 2: Relative change [/fig_ref]. CTh increased by 5.3 § 0.2% in the strontium ranelate group (P < 0.001) but was unchanged in the alendronate group (1.3 § 6.0%, P = 0.13). The between-group diVerence in favor of strontium ranelate was 3.9 § 1.9% (P = 0.045). The increase in CTh with strontium ranelate was 2.9 § 4.9% at 3 months versus baseline (P < 0.001) and was 2.3 § 0.9% (P = 0.012) greater than in the alendronate group [fig_ref] Figure 1: Change in cortical thickness [/fig_ref]. The increase in CTh was associated with a signiWcant increase in cortical area by 4.9 § 9. Distal tibia trabecular BV/TV increased by 2.0 § 5.3% and D trab by 2.1 § 5.2% (both P = 0.002) in the strontium ranelate group [fig_ref] Figure 1: Change in cortical thickness [/fig_ref]. These changes were signiWcant from 6 months (BV/TV, 0.9 § 2.3%, P = 0.031; D trab , 0.9 § 2.2%, P = 0.033). No signiWcant changes were observed in the alendronate group. The estimated betweengroup diVerences were 1.7 § 0.8% (P = 0.048) for BV/TV and 1.8 § 0.8% (P = 0.035) for D Trab . TbN increased in the strontium ranelate and alendronate groups, (4.2 § 8.2%, P = 0.011, and 4.3 § 10.7%, P = 0.030, respectively) with a corresponding decrease in trabeculae spacing (TbSp; ¡3.7 § 8.3%, P = 0.001, and ¡3.1 § 9.8%, P = 0.031) with no signiWcant between-group diVerences. TbTh remained unchanged with strontium ranelate (¡1.6 § 8.6%, P = 0.096) but decreased with alendronate (¡2.8 § 8.3%, P = 0.024), and this was signiWcant from 3 months (¡3.4 § 7.5%, P = 0.007). The heterogeneity of the trabec-ular network decreased with strontium ranelate (¡3.6 § 8.6%, P = 0.007), signiWcantly from 6 months (¡3.2 § 8.1%, P < 0.017) but remained unchanged with alendronate (¡1.9 § 12.2, P = 0.361). There was no between-group diVerence. Trabecular area decreased at 12 months in the strontium ranelate group (¡0.34 § 0.42%, from 594 to 592 mm², P < 0.001) but not in the alendronate group (¡0.11 § 0.42%, from 542 to 541 mm², P = 0.097). The estimated between-group diVerence was ¡0.22 § 0.10% (P = 0.024). Similar results were found at 3 months. Out of 88 patients, only 63 (72%) had assessable distal radius examinations, mainly due to movement artifacts. Of these patients, the relative changes in structural parameters evaluated by HR-pQCT was ·1% for strontium ranelate and alendronate, respectively (mean relative change at M12 in CTh: +0.3 § 6.2% and ¡1.1 § 5.8%; D cort : 0.9 § 2.1% The estimated between-group diVerence was 0.95 § 0.47% (P = 0.05). There were no other signiWcant within-or between-group diVerences (data not shown). The mean bone strontium content from biopsies sampled in postmenopausal women after 3 years of treatment from the SOTI and TROPOS studies was 1.8%, four times more than at 1 year. No correlation was detected between CTh and bone strontium content (r 2 = 0.003, y = ¡30.03x + 816). Similarly, there was no correlation between distal tibia cortical thickness and area, and serum strontium levels (r 2 = 0.235; P = 0.134 and r 2 = 0.241; P = 0.124 respectively). Lumbar, total hip and femoral neck aBMD increased in both groups and did not diVer between groups. In the strontium ranelate group, b-ALP increased by a median of 0.55 ng/mL (+4.1%, P = 0.095) at 3 months and 0.6 ng/mL (+5%, P = 0.082) at 12 months. S-CTX remained unchanged (median, ¡0.04 ng/mL, ¡7%, P = 0.250). In the alendronate group, both b-ALP and S-CTX decreased (median, ¡4.7 ng/mL, ¡35%, P · 0.001, and ¡0.36 ng/mL, ¡58%, P < 0.001, respectively). Between-group diVerences at 1 year were 40 and 55%, respectively (P · 0.001 each) [fig_ref] Figure 2: Change from baseline over time [/fig_ref]. Adverse events were analyzed in pooled groups to maintain blinding. One patient withdrew because of development of a peptic ulcer and one for upper abdominal pain. Both events were considered as related to the study drug by the investigator. Four patients suVered a rash, three were considered as possibly or doubtfully related to the study drug; all were mild, one led to treatment withdrawal. Two patients had an increase in CPK judged clinically signiWcant by the investigator, one case at 6 months that spontaneously recovered on treatment and one case at 12 months. Both values were lower than three times the upper limit of the normal range. # Discussion To our knowledge, this is the Wrst head-to-head, doubleblind, randomized controlled trial comparing eVects of strontium ranelate and alendronate on bone microstructure. Strontium ranelate increased cortical thickness, cortical area and trabecular density after 1 year of treatment. Trabecular bone volume and density increased with a decrease in the heterogeneity of the trabecular network. No change was observed with alendronate. The increases in cortical thickness, cortical area, and BV/TV and decrease in trabecular bone area were greater in the strontium ranelate than in the alendronate group. Trabecular number increased in both groups. While these results suggest that strontium ranelate has a positive eVect on bone microstructure, they require cautious interpretation given changes in tissue mineralization may modify the attenuation characteristics of photons during HR-pQCT imaging [bib_ref] The mineralization of bone tissue: a forgotten dimension in osteoporosis research, Boivin [/bib_ref]. Both drugs might artifactually increase edge detection. Strontium ranelate has twice the atomic number of calcium and alendronate increases the mean degree of mineralization [bib_ref] Alendronate increases bone strength by increasing the mean degree of mineralization of..., Boivin [/bib_ref]. Cortical thickening suggests that there is an increase in periosteal and/or endocortical bone formation but neither can be conWrmed as endocortical perimeter was not available with the HR-pQCT technique used in this study, while periosteal perimeter (a measure of periosteal apposition) and total crosssectional area are used to match the volumes of interest between baseline and postbaseline exams. Moreover, cortical area may increase without change in periosteal or endocortical perimeters if there is Wlling of intracortical pores with mineralized bone. This will also result in an apparent increase in the cortical thickness given it is calculated as cortical area/perimeter [bib_ref] The eVects of geometric and threshold deWnitions on cortical bone metrics assessed..., Davis [/bib_ref]. While these possibilities cannot be excluded, bone strontium content is low after 1 year (0.43 § 0.28%, n = 8) [bib_ref] In osteoporotic women treated with strontium ranelate, strontium is located in bone..., Boivin [/bib_ref]. After 3 years treatment, with a strontium content of 1.8%, four times more than at 1 year, strontium is present in only 37% of the bone surface and mainly trabecular surfaces [bib_ref] In osteoporotic women treated with strontium ranelate, strontium is located in bone..., Boivin [/bib_ref]. Furthermore, after 3 months of treatment, when bone strontium content is plausibly lower (2.2% of the bone area with strontium and 0.14% of strontium in bone) [bib_ref] In osteoporotic women treated with strontium ranelate, strontium is located in bone..., Boivin [/bib_ref] , increases in cortical thickness, cortical area and BV/TV are already observed in the strontium ranelate group, making less likely that the changes observed are entirely due to the higher atomic number of strontium and issues concerning edge detection. Furthermore, we did not detect any positive correlation between cortical thickness and area, and serum strontium levels. We also performed a simulation to quantify the possible inXuence of strontium content on X-ray linear attenuation in the evaluation of CTh (A. Laib, Scanco Medical AG, personal communication). Calculations were based on the results of Boivin and colleagues [bib_ref] In osteoporotic women treated with strontium ranelate, strontium is located in bone..., Boivin [/bib_ref] who quantiWed, in bone biopsies of patients treated for 2 or 3 years with strontium ranelate 2 g/day, the strontium content in cortical bone (newly formed and "old" bone) and the percentage of bone area containing strontium. We also used the linear attenuation coeYcients (mu/rho) of cortical bone, of calcium and of strontium given by the National Institute of Standards and Technology (http://physics.nist.gov/PhysRefData/Xray MassCoef/cover.html). We calculated the percentage of strontium after 1 year of treatment in the cortical bone and estimated the variation of X-ray linear attenuation due to the replacement of calcium atoms by this quantity of strontium atoms. This led to an increase in linear attenuation coeYcient of 0.255%. Then, we electronically changed the pixel values of the cortex pixels to increase them by the estimated 0.255%, and processed the scans again. The resulting CTh was 0.49% higher than the initially measured CTh. This means that a small part of the increase in cortical thickness in strontium ranelate group could be related to strontium dependent overestimation of the measurement. Trabecular volumetric BMD increased in the strontium ranelate group due to an increase in trabecular number not thickness. This was associated with a corresponding decrease in trabecular spacing and an improvement of the trabecular homogeneity consistent with improved trabecular architecture. These observations may also be the result of changes in attenuation characteristics. Trabeculae that were not detectable at baseline may have become detectable due to an increase in edge detection. This possibility would also account for the increase in TbN in the alendronate group without a change in BV/TV as alendronate increases secondary mineralization (even though no increase in tissue density was detected at 1 year) by suppressing remodeling [bib_ref] Histomorphometric assessment of the long-term eVects of alendronate on bone quality and..., Chavassieux [/bib_ref] [bib_ref] Trabecular bone microarchitecture after alendronate treatment of osteoporotic women, Recker [/bib_ref]. This could also explain the apparent decrease observed in trabecular thickness since this parameter is derived from TbN. The groups were similar in terms of baseline characteristics, including HR-pQCT parameters. The increases in aBMD in the two groups were similar to those previously observed [bib_ref] The eVects of strontium ranelate on the risk of vertebral fracture in..., Meunier [/bib_ref] [bib_ref] Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with..., Reginster [/bib_ref] [bib_ref] Randomised trial of eVects of alendronate on risk of fracture in women..., Black [/bib_ref]. The results on bone markers are also in accordance with published data. The changes in b-ALP with strontium ranelate were of a similar magnitude to those observed in the SOTI study [bib_ref] The eVects of strontium ranelate on the risk of vertebral fracture in..., Meunier [/bib_ref]. The lack of signiWcance in the present study may be due to a small sample size and a great inter-patient variation for the change from baseline to end. In the alendronate group, the decrease in S-CTX by roughly 60% preceded the decrease in b-ALP, as demonstrated elsewhere [bib_ref] Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic..., Garnero [/bib_ref]. Within the constraints imposed by methodological issues described earlier, with strontium possibly inXuencing X-ray attenuation, we infer that strontium ranelate improved bone microstructure within 3 months, while alendronate preserves existing structures. The observed changes in structure and density using strontium ranelate require conWrmation with larger sample sizes and using dynamic histomorphometry. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. [fig] Figure 1: Change in cortical thickness (CTh) (a) and relative trabecular bone volume (BV/TV) (b) over time (mean § sem) in the strontium ranelate and alendronate groups ¡0.2 § 1.7%; BV/TV: ¡0.4 § 3.7% and ¡0.3 § 4.4%; D trab : ¡0.4 § 3.6% and ¡0.6 § 4.2%; TbN: ¡0.1 § 5.9% and ¡0.1 § 5.3%; TbTh: 0.1 § 6.7% and ¡0.3 § 6.1%; TbSp: 0.4 § 6.1% and 0.3 § 5.6%; CSA tot : 0.0 § 0.2% and 0.1 § 0.2; CSA cort : 0.4 § 5.7% and ¡0.8 § 6.0%; CSA trab : 0.2 § 1.0% and ¡0.4 § 0.9%). A slight increase in cortical density was signiWcant in the strontium ranelate group (P = 0.015) but not in the alendronate group (P = 0.577). [/fig] [fig] Figure 2: Change from baseline over time (median, Q1 and Q3) of b-ALP (a) and S-CTX ( [/fig] [table] Table 1: Baseline characteristics Values are mean § SD BMI body mass index; BMD bone mineral density; BV/TV bone volume fraction; CSA cross-sectional area (total, trabecular, or cortical); CTh cortical thickness; D bone density (trabecular or cortical); TbN trabecular number; TbSp trabecular separation; TbTh trabecular thickness; Tb.1/N.SD heterogeneity of the trabecular network [/table] [table] Table 2: Relative change (%) from baseline to end of microstructure parameters at the distal tibia P values <0.05 (signiWcant) are in bold SE standard error; CI conWdence interval; BV/TV bone volume fraction; CSA cross-sectional area (total, trabecular, or cortical); CTh cortical thickness; D bone density (trabecular or cortical); TbN trabecular number; TbSp trabecular separation; TbTh trabecular thickness; Tb.1/N.SD heterogeneity of the trabecular network a P < 0.05 versus baseline [/table]

Legal & ethical compliance when sharing biospecimen When obtaining samples from biobanks, resolving ethical and legal concerns is a time-consuming task where researchers need to balance the needs of privacy, trust and scientific progress. The Biobanking and Biomolecular Resources Research Infrastructure-Large Prospective Cohorts project has resolved numerous such issues through intense communication between involved researchers and experts in its mission to unite large prospective study sets in Europe. To facilitate efficient communication, it is useful for nonexperts to have an at least basic understanding of the regulatory system for managing biological samples. Laws regulating research oversight are based on national law and normally share core principles founded on international charters. In interview studies among donors, chief concerns are privacy, efficient sample utilization and access to information generated from their samples. Despite a lack of clear evidence regarding which concern takes precedence, scientific as well as public discourse has largely focused on privacy concerns and the right of donors to control the usage of their samples. It is therefore important to proactively deal with ethical and legal issues to avoid complications that delay or prevent samples from being accessed. To help biobank professionals avoid making unnecessary mistakes, we have developed this basic primer covering the relationship between ethics and law, the concept of informed consent and considerations for returning findings to donors. # Introduction The Biobanking and Biomolecular Resources Research Infrastructure-Large Prospective Cohorts project has provided valuable experience on the issues of sample access through its open calls to provide funding for accessing biobanked samples. For the cohorts who did participate in various projects, attaining ethics approval and sorting the legal issues were timeconsuming but not insurmountable tasks because of intense communication between involved researchers and experts. The risk of biobank samples being used in an inappropriate manner has received increasing attention in scientific discourse. In comparison, the threat of under-utilization of samples or an inability to return the benefits of research to donors has received relatively little attention, despite also being among the chief concerns of interviewed donors [bib_ref] The ethics of research biobanking: a critical review of the literature, Hoeyer [/bib_ref]. Furthermore, the genomic revolution means that pretty much any sample can be considered to contain potentially identifiable personal data in the form of DNA. Researchers therefore face an intricate extralegal regulatory system complete with steering documents Tomas Klingströ m is a PhD student at the Swedish University of Agricultural Sciences (SLU) and employed at SLU-Global Bioinformatics Centre. His main research deals with data integration and quality verification of the scientific process from sample procurement to data analysis working in the BBMRI-lpc, BBMRI.se and B3Africa projects. Erik Bongcam-Rudloff is a professor in Bioinformatics at the Swedish University of Agricultural Sciences (SLU) and the head of the SLU-Global Bioinformatics Centre. His main research deals with the development of bioinformatics solutions for the Life Sciences community including nematode, plant, animal and human genomics research; numerous projects on metagenomics; and the building of LIMS and Biobank systems based on open-source elements (www.b3africa.org). Jane Reichel is a professor in Administrative Law at the Faculty of Law and is tied part-time to the Centre for Research Ethics and Bioethics, both at Uppsala University. She is currently vice dean and chairman of the research committee at the Faculty of Law. Her research focuses on the globalization and Europeanization of administrative law, mainly in the areas of transparency and data protection in cross-border biomedical research. (ethics guidelines), overseeing bodies (research ethics committees) and formal procedures (informed consent) [bib_ref] Making researchers moral: why trustworthiness requires more than ethics guidelines and review, Johnsson [/bib_ref] when attempting to access samples. Although laws regulating research oversight have been implemented differently in every country, there is a similarity of core principles founded on international charters such as the Helsinki Declaration. International consortia have translated these core principles into policies, procedures, tools and governance that facilitate interoperability between biobanks across national borders in a manner acceptable to national law makers [bib_ref] Ethical aspects of human biobanks: a systematic review, Budimir [/bib_ref] , thereby enabling the scientific community to operate despite a lack of clarity and international agreements that may provide a stable and enabling environment for international collaboration [bib_ref] Biobanking: international norms, Knoppers [/bib_ref]. As biobanks mature, priorities tend to shift [bib_ref] Biobanking 3.0: evidence based and customer focused biobanking, Simeon-Dubach [/bib_ref] , and it is not uncommon that biobanks find themselves prevented from providing samples due inappropriate decisions taken several years earlier. These complications are often the result of requests with unforeseen requirements causing uncertainties if given consents are sufficient and how or if information from new research projects should be returned to the donors. The primer therefore covers how these obligations are governed under international agreements and national law, the practice of establishing this relationship by the concept of informed consent and the difficulties on deciding when and what information should be provided to sample donors. ## Hard and soft law, the key to international collaboration The national legal framework of biobanking is often substantially different even between countries of comparable jurisdictional systems [bib_ref] Biobanking and international interoperability: samples, Kiehntopf [/bib_ref]. To accommodate international collaboration, it is therefore necessary to rely on 'soft law' or extra-legal means to bridge the gap between the national legal systems, which operate on a 'one nation, one law, one project' approach [bib_ref] From single biobanks to international networks: developing e-governance, Kaye [/bib_ref]. When dealing with such matters, it is therefore important to understand and recognize how research is regulated by a combination of 'hard law' and 'soft law' where the terms can be defined as follows: Hard law: Binding legal instruments, either in the form of international law (conventions, treaties or agreements) or national law (statutory law). International law is often drafted in a more general form and subsequently implemented in national law. For the individual researcher, it is most often the national statutory law that regulates the legality of actions. Soft law: Nonbinding instruments such as guidelines and codes of conducts that may lay down suitable and commonly accepted ways to deal with a matter. Soft law in different forms varies in form from openly phrased to rather strictly defined rules, bearing close resemblance to hard law. Hard law is codified in legal text, which makes it relatively straightforward for a trained expert to access and identify the relevant laws. Soft law is on the other hand more flexible but makes it harder to find and understand the regulatory mechanisms, as it allows governmental and nongovernmental experts to update regulations and standards without requiring active engagement of law-making bodies, and often these experts may be specified in hard law as bodies tasked with providing legally binding regulations and decisions. Funding bodies are becoming an increasingly important source of soft law by enforcing contracts requiring certain guidelines or procedures to be followed by researchers to be eligible for funding. For European researchers, an important source of this kind of regulation is the European Union (EU) funding programs managed by the European Commission. It requires applicants to state in their proposal that they will conform to specific standards where failure to comply mean that the researcher will not be eligible to receive the funds provided by the grant. Similar approaches are not only used for international projects but are also a way for national agencies to harmonize activities in nations where legislation is done at a regional or state level. For example, in the United States, the National Research Council stipulates the following for the international transfer of embryonic stem cells: If a U.S.-based investigator collaborates with an investigator in another country, the ESCRO committee may determine that the procedures prescribed by the foreign institution afford protections consistent with these guidelines, and the ESCRO committee may approve the substitution of some of or all of the foreign procedures for its own.These guidelines are defined by one selected group of experts (the National Research Council) who delegate decisions to another group of experts [the Embryonic Stem Cell Research Oversight (ESCRO) Committee], which is charged with deciding if there is a comparable set of checks and balances in the partner country in the form of a, yet to be identified, third group of experts. These guidelines are a good example of how a soft law approach with several layers reduces transparency in return for increased flexibility, as guidelines, review committees and research practitioners make up an ever-changing system of stakeholders. Under such circumstances, collaboration is substantially more likely to be accepted between nations where the respective authorities have had the possibility to become familiar with each other's customs and traditions, and above all, where the legal requirements applicable to the matter have been enacted as a result of international agreements. A lack of trust, harmonization or the local preferences of the committee may therefore significantly affect the outcome of an application for the transfer of data or samples. Decisions by judicial authorities covering one of the partners in a collaboration may also have an immediate impact on international collaboration, as certain procedures are deemed to be in conflict with national law. The EU has, for example, chosen a high standard for data protection, as seen in the recent Safe Harbor-ruling from the Court of Justice of the European Union (C-362/14), where the US level of protection was found not to uphold an adequate protection. However, most modern national laws are based on an ambition to adhere to a common set of core principles derived from the declaration of human rights and international declarations such as the Declaration of Helsinki. This means that even if there is yet little legal harmonization between countries. There is a strong case for researchers to argue that institutional review boards should take into account decisions from review boards in other countries, in a soft version of a principle of mutual recognition. ## Consent as the basis of international collaboration The signed consent form provides a receipt that verify that the donor has been provided with sufficient information to make an informed consent when donating his or her samples. Modern regulations regarding informed consent were codified in an international setting by the Helsinki declaration and Nuremberg code [bib_ref] The origins of informed consent: The International Scientific Commission on medical war..., Weindling [/bib_ref] as a result of the horrors in World War II and subsequent development. Respect for the autonomy of research subjects and their right to refuse participation in research does however have a much longer history in research [bib_ref] Informed consent in human experimentation before the Nuremberg code, Vollmann [/bib_ref] even if modern researchers may find certain practices troubling or even barbaric. For example, in the mid-19th century in America, it was considered acceptable for a slave owner to obtain consent for invasive experimental surgery from slaves [bib_ref] The medical ethics of Dr J Marion Sims: a fresh look at..., Wall [/bib_ref]. While it for a modern person is hard, if not impossible to accept slavery or the concept of 'a consenting slave'. From an academic context, this intuitive protest can be interpreted as an example of how we instinctively respect that a person in a position of dependence cannot make a truly autonomous decision [bib_ref] Paternalism in the name of autonomy, Sjostrand [/bib_ref]. The concept of donors as autonomous agents is one of the key concepts of modern research, and the question of identifying what information and freedom is necessary before a person can make an autonomous decision is therefore central to all forms of biobanking and genomic research with human participants. When establishing a new biobank, it is important to rely on forward-looking consent procedures to ensure the future viability of the sample collection. A large number of different forms of consent have been proposed in scientific literature. But in practice, consent forms likely available to a biobank would need to result in a presumed, broad or specific kind of consent [fig_ref] Table 1: Forms of consent described in literature [/fig_ref]. In bioethicist literature, concepts such as 'tiered' or 'dynamic' consent are suggested as compromises between specific or broad forms of consent. In practice, these forms of consent can either be broad or specific depending on whether the components of the consent are widely or narrowly specified. It is however not always possible or feasible to obtain information from a known, informed and willing donor. In some cases, a presumed consent is necessary, and several ethicists also argue that a consent can never be truly informed unless strict requirements are met [bib_ref] Broadening consent-and diluting ethics, Hofmann [/bib_ref] [bib_ref] Banking together. A unified model of informed consent for biobanking, Salvaterra [/bib_ref] [bib_ref] Scientists' perspectives on consent in the context of biobanking research, Master [/bib_ref]. When looking at large biobank infrastructures, a broad consent is favored among the major infrastructures [bib_ref] Ethics and biobanks, Hansson [/bib_ref] [bib_ref] Broad' consent, exceptions to consent and the question of using biological samples..., Petrini [/bib_ref] [bib_ref] Active choice but not too active: public perspectives on biobank consent models, Simon [/bib_ref] even if there still is debate among ethicists on how broad a consent can be while still maintaining the autonomy of the donor [bib_ref] Biobanks, consent and claims of consensus, Master [/bib_ref]. The dominance of broad consent in infrastructures based on soft law is in this context a good example of how soft law solutions allow society to adapt more quickly to new possibilities and risks compared with hard law where important laws may be debated for years before implementation. Specific consent is by its nature reactive, as it is impossible to request specific consent for purposes not yet foreseen. As a response to this issue, proponents of specific consent have made numerous proposals where modern communication technology makes it possible to repeatedly (or dynamically) ask donors for consent [bib_ref] Ethical endgames: broad consent for narrow interests; open consent for closed minds, Karlsen [/bib_ref]. Thus, initial consent only needs to cover foreseeable research, while new projects are made possible by a renewed consent, thereby, in the opinion of its proponents, creating a balance between maximizing the value of samples and the necessary safeguards to ensure that consent is truly informed. However, research rarely takes place in clearly defined modules, and there is often a continuum where it is hard to define the acceptable threshold for clarity, which requires new consent [bib_ref] The consent problem within DNA biobanks, Shickle [/bib_ref]. In practice, this means that a biobank will require a similar independent ethics review board, regardless of if the biobank operates under a legislation requiring specific, broad or any other form of consent. Recent research further underlines the support for a broad consent among biobank experts [bib_ref] Scientists' perspectives on consent in the context of biobanking research, Master [/bib_ref] , but even a broad consent is limited in how much freedom may be given to researchers to initiate new projects. That an administrative framework remains in place for the sample collection and that the new research does not change the overall aims or governance structure are core conditions and may be regarded as a minimal set of regulations for a broad consent to remain valid [bib_ref] Broad consent versus dynamic consent in biobank research: is passive participation an..., Steinsbekk [/bib_ref]. For European needs, Carlo Petrini at the Bioethics unit of the Presidentã s office in Italy has conducted a bibliographical study of the requirements necessary to operate a biobank under a broad consent in Europe, suggesting that the following requirements must be met: - Adequate sample coding procedures are used. - Adequate procedures for personal data protection are used. - The importance of the research aim is sufficient to justify conducting the study and is evaluated on a case-by-case basis by an ethics committee. - The sensitivity of the data is evaluated on a case-by-case basis. Genetic information varies in sensitivity based on its significance, ranging from stringent protection to a lesser degree of protection. - Generic research results are always released without specifically identification of individual subjects. - 'Opt-out' consent is allowed for subsequent or secondary studies. Every subject must be guaranteed the possibility of withdrawing consent at any time. - Participants must have adequate means of involvement, such as encouraging participant consultation or communicating information through the mass media before project initiation. The multiple modes of involvement should be complementary as opposed to mutually exclusive. It is especially important that forms of direct participation also be available, for example, by having population representatives serve on the ethics committees that will decide on the approval of the research before it begins. - Measures to ensure transparency and supervision must be in place. Adequate supervisory, procedural and technical systems are necessary to guarantee information protection. Further, it is highly advisable to have external and independent supervisory bodies monitoring procedural correctness. ## The reporting of planned or incidental findings Another controversial subject with far-reaching consequences for sample availability is whether researchers should be obliged to return information on findings to the donor [bib_ref] To tell or not to tell? A systematic review of ethical reflections..., Christenhusz [/bib_ref]. There is currently no overall consensus on when to tell and when not to tell participants of incidental findings [bib_ref] Incidental findings: the time is not yet ripe for a policy for..., Viberg [/bib_ref]. Careful planning of procedures to satisfy local or national expectations is therefore necessary to ensure that donor interests are managed properly. In cases where a study is based on samples, not yet collected, researchers can, and should, plan ahead to ensure that donors are properly informed at the time of consent on reporting procedures. For studies on samples already collected or where clinically relevant findings are incidental in nature, it instead becomes necessary for study manager to base their reporting procedures on their own judgment or guidelines provided by local experts or governing boards suited to the task. Based on the conflicting opinions described by researchers conducting systematic reviews of the field, it would be foolhardy to claim that practitioners and ethicists are anywhere near a consensus in the field [bib_ref] Ethics and biobanks, Hansson [/bib_ref] [bib_ref] Broad' consent, exceptions to consent and the question of using biological samples..., Petrini [/bib_ref] [bib_ref] Active choice but not too active: public perspectives on biobank consent models, Simon [/bib_ref] [bib_ref] To tell or not to tell? A systematic review of ethical reflections..., Christenhusz [/bib_ref] ]. It may however be possible to break down disclosure into two dimensions to separate situations where researchers are closer to consensus from areas where there still is severe disagreement [fig_ref] Figure 1: A breakdown of potential situations encountered when conducting genetic analysis on collected... [/fig_ref]. Given this four-field breakdown and preceding information, ethicists are at least approaching a consensus on the lower left and upper right corners. Which mean that incidental findings with a high level of actionability and clinical validity should, if possible, be reported back to the donor [bib_ref] Changing practice: the controversy over obligations to return incidental findings in genomic..., Bradbury [/bib_ref] and findings of low validity and actionability should not be reported to the donors (2, upper right corner) ahead of [bib_ref] Changing practice: the controversy over obligations to return incidental findings in genomic..., Bradbury [/bib_ref] and (3, lower left corner). There is however no consensus on whether it is a moral necessity to actively look for such genes in genetic data, and many researchers also feel uncertain when judging if specific markers are actionable and clinically valid [bib_ref] Changing practice: the controversy over obligations to return incidental findings in genomic..., Bradbury [/bib_ref]. To support clinicians, the American College of Medical Genetics has taken initiatives to support researchers to reduce these difficulties with lists of ## Specific informed consent Allows the use of biological specimens and related data only in immediate research; forbids any future study that is not foreseen at the time of the original consent Salvaterra et al. Note: Terms used in literature are not always univocal and may also be used with different levels of specificity. In the table, the specific definitions described by the authors have been clustered into more general of consent described in accordance with this article. The more specific definitions are listed in the column 'Definition', and the terms used to name them are outlined in 'Type of consent' and 'Disagreement'. valid and actionable genetic biomarkers [bib_ref] ACMG recommendations for reporting of incidental findings in clinical exome and genome..., Green [/bib_ref] , which can be consulted by clinicians to determine if incidental findings should be reported. The procedures for how and if findings are to be reported to the donor should be outlined to the donor at least by the time of consent, thereby helping to set donor expectations and define their future relationship with their donated samples. This means that the researchers, when developing the consent form, must take care to ensure the long-term viability of the biobank and balance their obligations to donors with the scientific needs of the project. A high level of reciprocity cannot, for example, be offered in a biobank where a large portion of the research is expected to be conducted by external researchers limited to anonymized data to maintain privacy. It is therefore necessary that researchers make important decisions such as coding [bib_ref] Reporting actionable research results: shared secrets can save lives, Hunter [/bib_ref] versus anonymization before contacting potential donors for consent. Failure to do so may otherwise result in major issues in the future, as national laws on privacy or obligations outlined in the consent form may prevent the efficient usage of biospecimen. ## Concluding remarks International collaboration relies on soft law connecting national legal systems, which creates an environment that is inconsistent, unfair and often lacking in transparency. But replacing the soft law with hard law may be even worse, as a codification of overly restrictive standards into law may stifle or outright halt scientific progress in regions within the jurisdiction of such laws. Furthermore, it is unlikely that hard law solutions would be able to possess the necessary flexibility to keep up the pace with the rapid advancement of research and genomics. As a researcher, it is easy to become frustrated and avoid engaging in such a complex, and ever-changing field of work. But despite calls for harmonization, it is unlikely that issues will be solved in the immediate future. There are significantly different legal traditions [bib_ref] What are the main roadblocks to transnational biobank collaboration, and how can..., Watson [/bib_ref] [bib_ref] A call for global governance of biobanks, Chen [/bib_ref] as well as variation in public perception [bib_ref] Publics and biobanks: pan-European diversity and the challenge of responsible innovation, Gaskell [/bib_ref] [bib_ref] Demographic differences in willingness to provide broad and narrow consent for biobank..., Ewing [/bib_ref] of research. Taken together, this makes it a perhaps insurmountable task to reach harmonization of national laws regarding biological samples and data protection. The legal obligations of biobank professionals concerning consent and reciprocity are therefore likely to change over time and remain areas associated with a high risk of interfering with the individual goals and aims of researchers. In this context, adhering to best practices contributes to the long-term value of samples, as new implementations of soft law instruments and codified law are likely to take established best practices in consideration. Guidance and templates provided by international organizations such as International Society for Biological and Environmental Repositories (ISBER, www.isber.org), Global Alliance for Genomics and Health (http://genomicsandhealth.org), the Asian Network of Research Resource Centers (http://anrrc.org), the Biobanking and BioMolecular resources Research Infrastructure-European Research Infrastructure Consortium (www.bbmri-eric.eu) and the Human Heredity and Health in Africa (http://h3africa.org), here, form a platform for harmonization as well as generating the opportunities to build the mutual trust necessary to enable the transfer of samples or data. The role and function of these soft law tools must however take into account the constitutional aspect of the bioethical framework involving several human rights. Traditionally, these rights, and especially the limiting of the rights, are usually thought to be best regulated by democratically elected parliaments [bib_ref] The need for a legitimate regulatory regime in bioethics: a global and..., Reichel [/bib_ref]. These international soft law tools do thus not supersede national authorities and courts, but their status as internationally recognized authorities may provide considerable support in achieving approval from institutional review boards acting under mandate from national laws. It is therefore in the best interest of researchers to respect and promote core principles codified by international conventions and organizations. Connecting local interpretations on law to an international context also makes it easier to compare decisions and encourage the development of trust that is necessary for collaboration using sensitive genomic data. It is therefore advisable for biobank builders to adopt a system of governance where: - The ethical standards set forth by the Global Alliance for Genomics and Health are upheld. - Samples are stored and managed in accordance with the internationally recognized ISBER standards for best practice [bib_ref] Development of the ISBER best practices for repositories: collection, storage, retrieval and..., Campbell [/bib_ref]. - Sharing is handled in a manner compliant with the International Charter of principles for sharing bio-specimens [bib_ref] International charter of principles for sharing bio-specimens and data, Mascalzoni [/bib_ref]. This does not preclude researchers from having to abide by the national law of each state involved in international research collaborations and is far from an exhaustive list of tools to support international sharing of samples. But it may provide an international research project with a common foundation and framework, which make the project more easily acceptable to the national authorities charged with reviewing projects. The inherent adaptability of soft law also mean that international collaboration through soft law mechanisms may steadily improve, as experience is gained among stakeholders and thus alleviate the need for global governance via codified hard law solutions within the field. If given time to adapt, researchers and associated organizations might instead be able to contribute to a bottom-up harmonization of a soft global bioethical framework. ## Key points - To accommodate international collaboration, it is necessary to bridge the gap between national legal frameworks. This is usually done by designated experts and organizations who determine if material transfer agreements are able to protect the rights of the donors in accordance with what they could expect when giving their consent for samples to be stored for future usage. - Collaboration is substantially more likely to be accepted between nations where the respective authorities have had the possibility to become familiar with each other's customs and traditions. Identifying successful precedents by other researchers participating in collaborative projects can therefore greatly reduce the time necessary to access samples. - Different institutions define terms such as consent, informed consent and broad consent differently. This mean that an 'informed consent' at one institution may not be accepted as truly informed by another. Under such circumstances, researchers are likely to face a situation where the strictest interpretation in terms of data protection or privacy becomes the governing one. - There is a conflict between reciprocity, anonymity and the right to not know. Research must therefore be planned and conducted in accordance with what the donors could reasonably expect when donating their samples and giving their consent. [fig] Figure 1: A breakdown of potential situations encountered when conducting genetic analysis on collected samples and practical examples of cases clearly belonging to each quarter. Support for returning information to the donor is strong when a finding is both reliable (possessing a high level of clinical validity) and actionable (the donor can act on the given information) as in the example given in Square 2 and, there is little support for providing information that is neither reliable nor actionable (Square 3). Decisions are harder and in greater need of consideration when the reliability of findings is low (Square 4) or when there is little the donor can do about the situation (Square 1). [/fig] [fig] Funding: This work was financed by the BBMRI-LPC and the B3Africa projects. BBMRI-LPC is supported by the European Community's 7th Framework Programme (FP7/20072013) grant agreement no. 313010, B3Africa is supported by the European Union s Horizon 2020 research and innovation programme under grant agreement No 654404. [/fig] [table] Table 1: Forms of consent described in literature [/table]

ObjectiveThe aim of the study was to investigate whether obese children and adolescents have a disturbed body representation as compared to normal-weight participants matched for age and gender and whether their body representation changes in the course of an inpatient weightreduction program.MethodsSixty obese (OBE) and 27 normal-weight (NW) children and adolescents (age: 9-17) were assessed for body representation using a multi-method approach. Therefore, we assessed body size estimation, tactile size estimation, heartbeat detection accuracy, and attitudes towards one's own body. OBE were examined upon admission and before discharge of an inpatient weight-reduction program. NW served as cross-sectional control group.ResultsBody size estimation and heartbeat detection accuracy were similar in OBE and NW. OBE overestimated sizes in tactile size estimation and were more dissatisfied with their body as compared to NW. In OBE but not in NW, several measures of body size estimation correlated with negative body evaluation. After weight-loss treatment, OBE had improved in heartbeat detection accuracy and were less dissatisfied with their body. None of the assessed variables predicted weight-loss success.ConclusionsAlthough OBE children and adolescents generally perceived their body size and internal status of the body accurately, weight reduction improved their heartbeat detection accuracy and body dissatisfaction. # Introduction Childhood obesity is increasing worldwide, and it is associated with both psychosocial and medical complications [bib_ref] The impact of obesity on psychological well-being, Wardle [/bib_ref] [bib_ref] Trends in Obesity Prevalence Among Children and Adolescents in the United States, Ogden [/bib_ref]. Awareness of the problem and motivation are considered a key factor in changing health behavior [bib_ref] The Transtheoretic Model and Stages of Change, Prochaska [/bib_ref] [bib_ref] Is there utility in the transtheoretical model?, Armitage [/bib_ref]. In this sense, it has been suggested that a lack of awareness of the own body size or indifference towards own weight status contribute to overweight, as they hamper motivation for weight loss [bib_ref] Weight perceptions in a population sample of English adolescents: cause for celebration..., Jackson [/bib_ref] [bib_ref] Obesity and body image, Schwartz [/bib_ref] [bib_ref] Mediating Effect of Perceived Overweight on the Association between Actual Obesity and..., Assari [/bib_ref]. In addition, it has been suggested that a disturbed interoceptive processing, as indicated by poor heartbeat detection accuracy, might contribute to an excessive food intake [bib_ref] Resting-state brain connectivity after surgical and behavioral weight loss, Lepping [/bib_ref] [bib_ref] Interoceptive sensitivity, body weight and eating behavior in children: a prospective study, Koch [/bib_ref]. As yet, no study has comprehensively investigated different types of body representation in obese children and adolescents. It is still unclear whether obese children and adolescents really have a disturbed body representation and whether weight loss also involves changes in body representation that could be addressed in weight loss treatment. Body representation is not uniform, but a conglomerate of multiple body representations that are informed by different modalities [bib_ref] Touch and the body, Serino [/bib_ref] [bib_ref] Body schema and body image-pros and cons, De Vignemont [/bib_ref]. In this notion, body representation comprises not only attitudes about body weight and shape, but also a mental picture of one's own body and implicit representations informed by proprioception, somatosensation and interoception. It is assumed that different body representations are organized along a continuum between implicit and explicit representations [bib_ref] Implicit and Explicit Body Representations, Longo [/bib_ref]. Studies on childhood obesity have typically focused on explicit body representation only and mostly used cross-sectional designs. It has been shown that a significant proportion of overweight children tend to underestimate their current body size in figure rating scales [bib_ref] An exploration of body dissatisfaction and perceptions of Black and White girls..., Kelly [/bib_ref] [bib_ref] Weight status and thebody image in children: The effect of maternal immigrant..., Gualdi-Russo [/bib_ref] , though this has not been replicated in adults when using methods that have a less social focus [bib_ref] Weight status and the perception of body image in men, Gardner [/bib_ref]. Also, children and adolescents with a high body mass index (BMI; kg/m 2 ) tend to have high body dissatisfaction and low self-esteem, contradicting the idea that the problem of obesity is often ignored [bib_ref] The impact of obesity on psychological well-being, Wardle [/bib_ref] [bib_ref] An exploration of body dissatisfaction and perceptions of Black and White girls..., Kelly [/bib_ref] [bib_ref] Perceived weight, not obesity, increases risk for major depression among adolescents, Roberts [/bib_ref]. Longitudinal studies, suggested that both body dissatisfaction and body size estimation in figure rating tasks approach performance of normal-weight children when overweight is reduced [bib_ref] Prospective changes in body image dissatisfaction among adolescent bariatric patients: the importance..., Ratcliff [/bib_ref] [bib_ref] Service evaluation of the GOALS family-based childhood obesity treatment intervention during the..., Watson [/bib_ref] [bib_ref] Inpatient treatment for children with obesity: weight loss, psychological well-being, and eating..., Braet [/bib_ref]. Recently, more implicit types of body representation came into the focus of obesity research and reopened the debate again. Heartbeat detection accuracy has been observed to be diminished in adults with high BMI [bib_ref] Attenuated interoceptive sensitivity in overweight and obese individuals, Herbert [/bib_ref] and it is associated with healthier eating behavior and better physical fitness in children [bib_ref] Interoceptive sensitivity, body weight and eating behavior in children: a prospective study, Koch [/bib_ref] [bib_ref] Interaction of physical activity and interoception in children, Georgiou [/bib_ref]. Also, studies in adults indicate that participants with high BMI might have difficulties in estimating the size of an object touching their skin (tactile size estimation), possibly reflecting a disturbed sense of the own size [bib_ref] Keeping the world a constant size: object constancy in human touch, Taylor-Clarke [/bib_ref] [bib_ref] Laterality and sex differences in tactile detection and two-point thresholds modified by..., Boles [/bib_ref] [bib_ref] Tactile mental body parts representation in obesity, Scarpina [/bib_ref]. Taken together, while the observed disturbances in explicit measures of body representation could be interpreted as effect of social teasing and stigmatization, implicit measures suggested that an inaccurate representation of the own body size could still play a role in obesity. In the present study, we wanted to obtain a more comprehensive picture of a possible body image disturbance in childhood obesity than previously reported. Specifically, we aimed to find out i) whether and in which measures of body representation obese children and adolescents differ from normal-weight mates matched for age and gender and heartbeat detection ii) how the different measures of body representation are associated with each other in both groups. In addition, we followed up obese children and adolescents until discharge from a weight-loss therapy, as we wanted to investigate iii) whether weight loss induced any changes in the body representation of obese children and adolescents. Finally, we also wanted to test iv) whether any of the body representation measures would serve as a predictor for weight loss success in obese children, as suggested by health behavior theories [bib_ref] The Transtheoretic Model and Stages of Change, Prochaska [/bib_ref] [bib_ref] Is there utility in the transtheoretical model?, Armitage [/bib_ref]. # Materials and methods ## Study design and participants The study presented here was conducted as part of the DROMLIN-study (PreDictor Research in Obesity during Medical care-weight Loss in children and adolescents during an INpatient rehabilitation) [bib_ref] PreDictor Research in Obesity during Medial care-weight Loss in children and adolescents..., Sauer [/bib_ref]. The study protocol was approved by the Ethics Committee of the medical faculty for the University Tübingen, Germany. This study is registered at the German Clinical Trials Register (DRKS) with the clinical trial number DRKS00005122. Children and parents were informed about the study purpose and provided written consent prior to inclusion. In short, 60 overweight and obese children (OBE; age 9-17, 47% male) with a BMI over the 90 th percentile for their age and sex specific norms [bib_ref] Percentiles of body mass index in children and adolescents evaluated from different..., Kromeyer-Hauschild [/bib_ref] and an indication for hospitalization for weight loss intervention were included. All OBE participated in a weight loss program at the Children Rehabilitation Hospital for Respiratory Diseases, Allergies and Psychosomatics in Wangen i.A., Germany. The program comprised physical activity, cognitive behavioral therapy, and a balanced diet. A detailed description of the setting is reported elsewhere [bib_ref] PreDictor Research in Obesity during Medial care-weight Loss in children and adolescents..., Sauer [/bib_ref]. Exclusion criteria were severe psychological comorbidities, linguistic or intellectual limitations, type-1 diabetes, malignant tumors, systemic disorders, or severe cardiovascular diseases. Additionally, 27 normal weight children (NW; 11-14 years, 56% male) matched for age and gender with a BMI between the 10 th to 90 th BMI percentile from the surrounding area of the University Hospital Tübingen, Germany were recruited and served as control group. OBE were tested twice, upon admission (T1) and prior to discharge (T2). The anthropometric and body perception assessments took place in an afternoon session and the heartbeat perception assessment in the morning session. NW were tested once in a single session, and served as control group for T1. ## Assessments Anthropometry. The physical development of the children was assessed using the tanner stages [bib_ref] Variations in pattern of pubertal changes in girls, Marshall [/bib_ref] [bib_ref] Variations in the pattern of pubertal changes in boys, Marshall [/bib_ref]. The tanner scale ranges between 1 (prepubertal) and 4 (mature). In the context of anthropometric measurements, the actual body widths (spine, hip, thigh, upper arm) and body depths (abdomen, buttocks), were measured with a caliper and body circumferences (abdomen, buttocks, thigh, upper arm) with a tape measure, respectively in the morning. Participants were not informed about their body dimensions. Body size estimation. Two hours after the anthropometry, the same investigator assessed the corresponding body size estimations by instructing the participants to set their dimensions by moving sliders on a 2 m wooden slat. Then, the investigator measured the adjusted dimension without providing any feedback. At the beginning of each trial, the investigator placed both sliders in the middle of the slat. The children's cognitive ability to discriminate between physical dimensions was tested by presenting everyday objects of different size that had to be estimated: a mobile phone (9 cm), a book (24 cm), and a bottle (34 cm). After each presentation, the object was removed and the participant was asked to set its length on the wooden slat. Tactile size estimation. We conducted a tactile size estimation test similar to the one reported by Keizer et al. [bib_ref] Tactile body image disturbance in anorexia nervosa, Keizer [/bib_ref] at four different body sites (upper spine, upper arm, buttocks, thigh). The participants were blindfolded and the investigator pressed a small caliper/pair of compasses with predefined distances on different body sites. After each tactile stimulation (each distance and body site), the blindfold was removed and the participants had to reproduce the perceived distance using the wooden slat. The distances between the two points were as follows: spine-20 cm, upper arm-10 cm, buttocks-15 cm, thigh-10 cm. ## Perception indices and scores. A perception index for each body size and tactile size estimate was calculated according to the formula: perception index = (estimated / actual body size) x 100 [bib_ref] Awareness of body dimensions in anorexia nervosa: cross-sectional and longitudinal studies, Slade [/bib_ref]. Next, mean perception scores for each group were calculated as average of the single measures for everyday objects (mobile phone, book, bottle), body width (spine, hip, thigh, upper arm), body depth (abdomen, buttocks), body circumference (abdomen, buttocks, thigh, upper arm) and tactile size estimation (spine, upper arm, buttocks, thigh). Values below 100 indicate an underestimation and values above 100 indicate an overestimation in terms of percent of the actual size. Heartbeat detection. The heartbeat detection task was performed as reported previously by Pollatos and Schandry [bib_ref] Accuracy of heartbeat perception is reflected in the amplitude of the heartbeatevoked..., Pollatos [/bib_ref] in a modified version. During the procedure, a conventional ECG (3991/3-GPP BioLog, UFI Company, Morro Bay, CA) recorded the actual cardiac activity while the child was comfortably seated in a chair and was not allowed to speak and to move. A short test interval of 15 seconds was followed by four intervals of 25, 35, 45 and 55 seconds. Between the intervals were resting periods of 30 seconds. The children were instructed to count during each interval their own heartbeats by concentrating on their heart activity. The procedure was standardized by giving the instructions from a tape. A heartbeat detection index for every interval was calculated by the following formula: 1-(|recorded heartbeatscounted heartbeats|/recorded heartbeats). Next, the mean heartbeat detection score was calculated as average of the heartbeat detection indices of the four intervals 25s, 35s, 45s and 55s. The maximum score is 1, the minimum score is 0. A high index or score indicates a good concordance between the detected and actual heartbeat whereas a low score indicates a poor agreement between the detected and the actual heartbeat. Concerns about body weight and shape. Eating behavior and concerns about body weight and shape were assessed with the validated Eating Behaviour and Weight Problems Inventory for Children (EWI-C), consisting of 60 items and 10 subscales [bib_ref] Attitudes to eating and body weight of 11-to 16-year-old adolescents] Einstellungen zu..., Diehl [/bib_ref]. In this study, the subscales "figure dissatisfaction" (consisting of 5 items), and "concerns about eating" (consisting of 8 items) are reported. Percentile ranks for the values of the subscales were retrieved by sex and age specific norm tables. Values between the 16 th and 84 th percentile can be considered as normal. ## Statistical analyses The data were analyzed using SPSS version 19. Normally distributed data are presented as mean±standard deviation. Non-normally distributed data are presented as median [interquartile range] and the perception indices additionally by mean±standard deviation. Differences between OBE T1 and NW were calculated using unpaired t-tests (age, weight, height, BMI-SDS), Chi 2 test (sex) or Mann-Whitney-U-tests if data were not normally distributed (EWI-C, perception indices). Differences between OBE T1 and OBE T2 were analyzed with paired t-test (weight, BMI-SDS) or Wilcoxon signed-rank test if data were not normally distributed (EWI-C, perception indices). We used Spearman correlations to analyze associations between variables, because in all analyzed pairs, at least one variable was not normally distributed. In order to analyze the association between body representation distortion and successful weight loss in OBE, Spearman correlations between the T1 perception scores and the delta BMI-SDS were calculated. The same Spearman correlations were computed using the T1 absolute values of mis-estimation instead of the T1 perception scores. Spearman correlations were computed for correlation analyses between all perception scores and EWI-subscales. In order to control for multiple testing the p-values were false discovery rate (FDR) adjusted [bib_ref] Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple..., Benjamini [/bib_ref]. FDR-values of <0.05 and for correlation analyses <0. [bib_ref] Weight status and the perception of body image in men, Gardner [/bib_ref] were considered as statistically significant. [fig_ref] Table 1: Characteristics of the study populationdeviation and non-normally distributed data as median[interquartile range] [/fig_ref] provides an overview on the characteristics of the study population. At T2, seven children had dropped out so that the longitudinal data refers to a sample of 53 obese children. The length of intervention in OBE was 38±10 (min-max: 16-70) days. To exclude possible age effects, all analyses were repeated excluding the four youngest children (aged 9 to 10 years from the OBE group), which however, did not influence the results. Similarly, we explored whether results would change if absolute values of percentage of mis-estimation instead of perception scores were used. Again, this was not the case. # Results ## Group differences obe t1 versus nw As displayed in , both groups overestimated their body widths and body depths while they underestimated their body circumferences. However, the perception indices of body widths, body depths and body circumferences for the different body sites did not differ significantly between OBE and NW. As a result, the corresponding three aggregated perception scores "Body widths", "Body depths" and "Body circumferences" were also similar in OBE versus NW respectively. Both groups greatly overestimated the distances in the tactile size estimation task . However, the perception indices "Spine", "Buttocks" and "Thigh" of this task differed significantly between OBE and NW, with OBE overestimating the distances more than NW . Consequently, the aggregated perception score "Tactile Size Estimation" also differed between OBE and NW (U(N = 87) = 434.0, FDR = 0.007, d = 0.81; . In order to exclude that either changes in mechanoreceptor density through growth or central nervous system maturation influenced tactile size estimation performance, we explored whether performance correlated with the children's height and their tanner stages, which was not the case. The heartbeat detection indices for the different counting intervals were similar in OBE and NW in all intervals and consequently also the aggregated detection accuracy score . The results for the two subscales " and "Concerns about eating" are presented in [fig_ref] Table 1: Characteristics of the study populationdeviation and non-normally distributed data as median[interquartile range] [/fig_ref]. In both subscales OBE scored significantly higher than NW ("Concerns about eating": U(N = 87) = 40.00, p<0.001, "Figure dissatisfaction": U(N = 87) = 72.00, p<0.001), reflecting higher body dissatisfaction and eating concern in obese children. ## Correlations between body representation measures Correlations between all the perception scores and the subscales of the EWI-C were computed separately for OBE (at T1) and NW . In OBE, the perception score "body width" correlated weakly to moderately with the perception scores "body depths", "tactile size estimation and the EWI-C scale "eating concern". The perception score "body depth" correlated weakly with "tactile size estimation". A moderate correlation was observed between the two EWI-C scales "eating concern" and "figure". In NW, the perception score "body width" correlated moderately with "tactile size estimation". Further, the perception score "body width" correlated with "body depth" and "body circumference", but this finding did not withstand FDR-adjustment. In NW, the two EWI-C scales "eating concern" and "figure" correlated strongly with each other. Neither in OBE nor in NW, correlations between the heartbeat detection score and other perception scores or EWI-C scales were found. ## Changes in obe body representation between t1 and t2 In the OBE group, the weight loss treatment induced neither a significant effect with regard to any of the aggregated perception scores for "Body widths", "Body depths", Body circumferences" ## . perception indices of body perception in obese children (obe) before (t1) and after weight loss (t2) in comparison to normal-weight children (nw). ## Mean±sd Median nor for the "Tactile Size Estimation" [fig_ref] Fig 2: Perception Scores of body perception in obese children [/fig_ref]. We also analyzed the individual changes in aggregated perception scores between T1 and T2 and found no trend of improvement or worsening [fig_ref] Fig 2: Perception Scores of body perception in obese children [/fig_ref]. In contrast, the heartbeat detection accuracy improved significantly in the course of weight loss from T1 to T2 in all examined intervals and in the aggregated score (Z(N = 52) = -5.174, FDR<0.001, d = 0.67, [fig_ref] Fig 2: Perception Scores of body perception in obese children [/fig_ref]. Also, we observed that OBE improved in the EWI-C subscale "Concerns about eating" (Z(N = 53) = -2.81, p = 0.005). ## Prediction of weight loss success None of the assessed measures of body representation at T1 correlated with the weight loss success (delta BMI-SDS). # Discussion Our observations suggest that obese children and adolescents generally represent their bodies as accurate as normal weight age mates, though in OBE, body size representation was associated with eating concern. Our observation that none of the assessed variables predicted weight loss success is contradictory to ideas that a lack of awareness of their excess body size or poor interoception contributes to being overweight. However, we observed that in the obese children and adolescents, not only eating concern, but also heartbeat detection accuracy improved throughout weight loss, suggesting that the program induced improvements in interoceptive processing. We observed no uniform group differences between OBE and NW with regard to their general body size perception and heartbeat detection accuracy, but only in tactile size estimation and body dissatisfaction. In the body size estimation task, our observations do not confirm previous results from figure rating tasks suggesting that obese children underestimate their size [bib_ref] An exploration of body dissatisfaction and perceptions of Black and White girls..., Kelly [/bib_ref] [bib_ref] Weight status and thebody image in children: The effect of maternal immigrant..., Gualdi-Russo [/bib_ref] [bib_ref] Accuracy of perception of body size among overweight Latino preadolescents after a..., Gesell [/bib_ref]. Rather, our observations match findings obtained with depictive methods in adults by [bib_ref] Weight status and the perception of body image in men, Gardner [/bib_ref] that showed no difference between obese and normal-weight participants. The discrepancy may be due to the fact that figure rating tasks assess own body size perception as compared to a certain social range, whereas metric body size estimation, as used in this this study, assesses body size estimation for the actual size. Several studies have already shown that families and peers of obese children often do not perceive the child as obese, which may be beneficial for the child's quality of life [bib_ref] A little on the heavy side": a qualitative analysis of parents' and..., Eli [/bib_ref] [bib_ref] Is obesity becoming the new normal? Age, gender and racial/ethnic differences in..., Twarog [/bib_ref] [bib_ref] When ignorance is bliss: weight perception, body mass index and quality of..., Hayward [/bib_ref]. It is likely that obese children do not see themselves as different from their peers as they are, and thus underestimate in figure rating tasks while they might be accurate in tasks that do not require a social comparison. Our observation that OBE children were less accurate than NW children in tactile size estimation, is in line with previous findings in adults [bib_ref] Tactile mental body parts representation in obesity, Scarpina [/bib_ref]. Interestingly, differing from these previous studies, we found that both groups overestimated, but OBE children did so to a significantly higher degree. Tactile size estimation performance did neither correlate with height nor with tanner stages of physical development. We therefore consider it unlikely that differences in growth and maturation processes might have caused the group differences, as previous studies suggested [bib_ref] Age-related changes in tactile spatial resolution from 6 to 16 years old, Bleyenheuft [/bib_ref] [bib_ref] Tactile Spatial Acuity in Childhood: Effects of Age and Fingertip Size, Peters [/bib_ref]. Rather, our results are similar to those found in anorexia nervosa suggesting that tactile size, despite being considered to assess implicit body representation, might be influenced top-down, e.g. by body dissatisfaction [bib_ref] Tactile mental body parts representation in obesity, Scarpina [/bib_ref]. Hence, it might be the case that the overestimation of OBE children reflects their perception of being large although correlations between body dissatisfaction and tactile size estimation did not yield significance. ## Fig 1. perception scores of body perception in obese children (obe) before weight loss (t1) in comparison to normalweight children (nw Heartbeat detection accuracy is assumed to be the central construct underpinning other interoceptive measures [bib_ref] Knowing your own heart: distinguishing interoceptive accuracy from interoceptive awareness, Garfinkel [/bib_ref]. Further, it has been observed to be negatively correlated with the tendency to evaluate one's body based on appearance rather than for its effectiveness [bib_ref] Body conscious? Interoceptive awareness, measured by heartbeat perception, is negatively correlated with..., Ainley [/bib_ref]. Our observation of no group differences in heartbeat detection accuracy contradicts previous claims that a diminished perception of the inner status of the body might contribute to overweight [bib_ref] Attenuated interoceptive sensitivity in overweight and obese individuals, Herbert [/bib_ref]. Interestingly, in a large sample of children (n>1500) aged 6 to 11 years, no differences were observed between overweight and normal-weight children at a first assessment, whereas differences between the groups were evident one year later [bib_ref] Interoceptive sensitivity, body weight and eating behavior in children: a prospective study, Koch [/bib_ref]. Our observations suggest that diminished heartbeat perception is likely not a general symptom of obesity. However, heartbeat perception is involved in weight regulation, possibly as a mediator for body-related cognitions. Finally, our observation of high body dissatisfaction in OBE children confirm Wardle and Cooke [bib_ref] The impact of obesity on psychological well-being, Wardle [/bib_ref] ,who identified high body dissatisfaction in OBE children as one of the main factors of their compromised psychological well-being. At the same time, our observation suggests that it is not the case that obese children have a lack of awareness of the problem but that they are aware of and suffering from their excess weight. Group wise correlation analyses of the different measures of body representation revealed an interesting pattern: Whereas in the NW group questionnaire measures of eating concern and body dissatisfaction were independent from other body representations, high eating concern was associated with body size overestimation in the OBE group. This indicates that in obese children and adolescents, cognitions of being too fat are possibly internalized to a higher degree than previously assumed and thereby might influence body size estimation on a very basic level. Interestingly, we found that different measures of body representation do not homogeneously correlate with each other. In both the OBE and the NW group, measures related to size estimations were correlated moderately with each other, but not with heartbeat detection accuracy. This supports the notion of body representation as conglomerate of multiple rather independent representations and emphasizes the necessity of a multi-method account. Our third research question asked for the role of body representation in weight loss treatment. Although, heartbeat detection accuracy is unlikely to be involved in the etiology of overweight, our observation of improved heartbeat detection accuracy at T2 indicates that weight loss treatment affects interoception. From our data, it is unclear whether heartbeat perception accuracy is rather a marker or even a potential moderator variable for weight loss. A possible mechanism of this relationship includes physical fitness, which has been observed to be associated with heartbeat detection accuracy in high BMI children [bib_ref] Interaction of physical activity and interoception in children, Georgiou [/bib_ref]. Alternatively, it could reflect changes in body image, as weight loss might reduce tendencies to evaluate oneself based on appearance [bib_ref] Body conscious? Interoceptive awareness, measured by heartbeat perception, is negatively correlated with..., Ainley [/bib_ref]. However, the causal structure of this association is yet unknown. In line with other studies, we also observed that body dissatisfaction, as reflected by the scale "eating concern" improved throughout the weight loss treatment [bib_ref] Prospective changes in body image dissatisfaction among adolescent bariatric patients: the importance..., Ratcliff [/bib_ref] [bib_ref] Service evaluation of the GOALS family-based childhood obesity treatment intervention during the..., Watson [/bib_ref] [bib_ref] Body image change in obese and overweight persons enrolled in weight loss..., Chao [/bib_ref] [bib_ref] Body image after sleeve gastrectomy: reduced dissatisfaction and increased dynamics, Teufel [/bib_ref]. It has to be noted that body dissatisfaction remained on a level higher than in the NW group even at the end of the program. However, this suggests that positive effects on psychological wellbeing apply as soon as weight loss starts. With regard to the predictive power of body representation for weight loss success, we found that none of the investigated measures predicted weight loss success of OBE children. Opposed to widely used models of health behavior change, our results suggest that a lack of awareness and, consequently, motivation for weight loss, is not the main hurdle for weight loss in obesity [bib_ref] The Transtheoretic Model and Stages of Change, Prochaska [/bib_ref] [bib_ref] Mediating Effect of Perceived Overweight on the Association between Actual Obesity and..., Assari [/bib_ref] [bib_ref] The Association between Weight Perception and BMI among High School Students, Brener [/bib_ref]. However, we observed body dissatisfaction to be very sensitive to weight loss, suggesting that motivational variables might be relevant for therapy adherence and success. It is a limiting factor of this study i) that we were not able to analyze body representation in longer follow-up intervals and ii) that our design does not allow us to disentangle whether the observed changes in body dissatisfaction and interoception were rather consequences or even actively contributing to weight regulation. Although we have not found an immediate link between weight and body representation, it is still possible that some of the body representations investigated here are associated with weight loss, weight loss maintenance or weight gain in a long-term perspective. Studies with a longer follow-up interval and more measurement time intervals could help to clarify this question and to learn more about the mechanisms through which body representation and weight regulation interact. There are also several strengths to our study. To our knowledge, we are the first to examine body representation of obese children from a multi-modal perspective and in both a cross sectional and a longitudinal setting. That way, we were not only able to compare body representation of obese children to normal weight children, but could also identify changes that occur in the course of weight loss. We observed that obese children do not have general problems to represent their excess body size. However, correlation analyses indicate that their self-categorization as "too large" is likely influencing their body representation on a basal level. Further studies focusing on the association between perception and representation of the body might help to better explain this observation. For clinical practice, it is important that we observed counter-evidence for the idea that obese children lack awareness of their excess size or motivation to lose weight. Still, we observed that interoceptive awareness, as indicated by heartbeat detection accuracy, changes throughout weight loss therapy, suggesting that it might play a role in weight regulation. Further research is needed that tracks different types of body representation throughout development and long-term treatment of overweight. Specifically, the role of interoceptive awareness for weight loss treatment needs further exploration. Our findings also show that neither high body dissatisfaction nor accurate awareness of the own excess size translate into higher weight loss success. However, to improve psychosocial well-being of overweight children, weight loss interventions that specifically target body image may be useful. [fig] Fig 2: Perception Scores of body perception in obese children (OBE) before (T1) and after weight loss (T2). A: The perception scores of everyday objects, body width, body depth, body circumference, tactile size estimation and heartbeat detection are displayed as box-whiskers with a cross, the latter depicting the mean. Except for the heartbeat detection score, values below 100 indicate an underestimation and values above 100 indicate an overestimation in terms of percent of the actual size. For the heartbeat detection, a score of 1 indicates absolute accuracy of heartbeat detection and the minimum score of 0 indicates that no heartbeat was perceived. The mean±standard deviation of the perception scores were as follows: Everyday objects-T1: 103±08, T2: 105±08; Body width-T1: 120±15, T2: 120±14; Body depth-T1: 115±20, T2: 119±16; Body circumference-T1: 85±16, T2: 85±12; Tactile size estimation-T1: 182±41, T2: 183±35; Heartbeat detection-T1: 0.47±0.26, T2: 0.63±0.21. Due to multiple testing the p-values were false discovery rate (FDR)-adjusted. FDR values <0.05 were considered as statistically significant. *** indicates FDR<0.001. B: The change values of the perception scores in OBE are presented as box-whiskers. doi:10.1371/journal.pone.0166826.g002 [/fig] [table] Table 1: Characteristics of the study populationdeviation and non-normally distributed data as median[interquartile range]. Data were compared between OBE T1 versus OBE T2 and OBE T1 versus NW, respectively. P-values <0.05 were considered as statistically significant. Min = Minimum, Max = Maximum, n.a. = not applicable, n.s. = not significant. [/table] [bib_ref] Weight status and the perception of body image in men, Gardner [/bib_ref]

Nature’s Derivative(s) as Alternative Anti-Alzheimer’s Disease Treatments Alzheimer's disease (AD), the 'Plague of Twenty-First Century,' is a crippling neurodegenerative disease that affects a majority of the older population globally. By 2050, the incidence of AD is expected to rise to 135 million, while no treatment(s) that can reverse or control the progression of AD are currently available. The treatment(s) in use are limited in their ability to manage the symptoms or slow the progression of the disease and can lead to some severe side effects. The overall care is economically burdensome for the affected individuals as well as the caretakers or family members. Thus, there is a pressing need to identify and develop much safer alternative therapies that can better manage AD. This review discusses a multitude of such treatments borrowed from Ayurveda, traditional Chinese practices, meditation, and exercising for AD treatment. These therapies are in practice since ancient times and reported to be beneficial as anti-AD therapies. Ayurvedic drugs like turmeric, Brahmi, Ashwagandha, etc., management of stress by meditation, regular exercising, and acupuncture have been reported to be efficient in their anti-AD usage. Besides, a combination of vitamins and natural dietary intakes is likely to play a significant role in combating AD. We conclude that the use of such alternative strategies will be a stepping-stone in preventing, treating, curing, or managing the disease. # Introduction Alzheimer's disease (AD) is a neurodegenerative disease that was first described by German psychiatrist and neuropathologist, Alois Alzheimer in 1906. It results in deterioration of cognition and memory, ultimately leading to the loss of a person's ability to carry out day to day activities independently. It is the most common form of dementia, contributing to 60-70% of the cases. Generally observed in people around 65 years of age or more, the onset of onset of aging. In the initial stages, neurons of the entorhinal cortex and hippocampus, responsible for memory, are destroyed. The disease then proceeds to the later stage to affect areas accountable for reasoning, language, and social behavior of the cerebral cortex. This fatal neurodegenerative disease then affects other areas of the brain, ultimately leading to the stage where the patient becomes incapable of functioning and living independently, before death. The significant changes taking place in the brain at the molecular level are: 1. Oxidative stress. Reactive oxygen species (ROS) bring about the oxidative stress, especially in the case of age-related disorders like AD. Oxidative damage occurs early in the AD patient's brain. Its impact can be seen even before the plaque pathology is initiated as it precedes amyloid-␤ (A␤) deposition as well as abnormal formation of intracellular neurofibrillary tau protein tangles. ROS also lead to neurodegenerative processes and neuronal death, at the cellular and tissue levels. 2. Formation of A␤ plaques. Formation of A␤ proteins occurs due to breakdown of the amyloid-␤ protein precursor (A␤PP). These proteins begin to clump together to form plaques between neurons at abnormal levels. Ultimately, these clumps result in disruption of cellular function.. Formation of neurofibrillary tangles. Accumulation of abnormally high levels of hyperphosphorylated tau protein inside the neurons results in the generation of neurofibrillary tangles. Tau protein in healthy neurons is responsible for binding to and stabilizing microtubules. However, after detaching from microtubules, tau sticks to other tau molecules in case of AD and forms threads that join together to form tangles that lead to blockage of synaptic communication between the neurons. Due to the breakdown of a retrograde barrier at the axon initial segment, tau proteins present in the axonal region of neurons get missorted to the somatodendritic compartment. 4. Chronic inflammation. Microglial cells act as macrophages of the brain, clearing cell debris, including A␤ plaques, toxins, and dead neurons. Astrocytes also help in removing the plaques and cell debris from the brain. In the case of AD, these microglial cells of the brain assem-ble around the neurons but are not successful in performing their function of clearing the debris. Thus, their build-up results in the release of chemicals that lead to chronic inflammation in the brain. 5. Vascular attributions of AD. AD may also cause A␤ accumulation in arteries of the brain, hardening of arteries, i.e., atherosclerosis, ministrokes, etc., and can reduce blood flow and oxygen supply to the brain. Ultimately, this leads to a degenerative and complicated cycle that is a cause as well as a consequence of AD. 6. Brain atrophy. Neuron cells are damaged and die in case of AD. Thus, the neural network may breakdown, and brain regions begin to shrink in volume. The brain activity deterioration begins from the hippocampus in the medial temporal lobe, which is mainly related to memory and emotion. The deterioration then escalates, and the whole brain glucose metabolism is also affected, resulting in a reduction in neuronal processing of bilateral parietal and temporal lobes. The parietal lobe is responsible for sensation, self-awareness, attention, memory retrieval, and theory of mind. The temporal lobe is associated with episodic memory, emotions, and mood. Thus, the regions affected by AD are responsible for the regulation of memory, emotions, and awareness. ## Current treatments for ad At present, there are a limited number of drugs available for the treatment of AD, and most of these can only treat the symptoms. These are inept at preventing the progression of the disease further or reversing its effects. The development of symptoms like memory loss is, however, slowed, prolonging life and alleviating the quality of life of the affected individuals. Some of the compounds used in case of mild to moderate AD are donepezil, rivastigmine, and galantamine. These are classified as cholinesterase inhibitors that prevent the breakdown of acetylcholine, necessary for memory and learning. For patients with moderate to severe AD, memantine, a N-methyl D-aspartate (NMDA) antagonist, is used. A combination of memantine and donepezil is used for the same. Some other drugs are also used to treat depression, aggression, restlessness, and anxiety associated with AD. These include citalo-With the ever-increasing population and the growing life expectancy of individuals, AD incidences are also rising rapidly across the globe. As per the WHO factsheet on dementia, worldwide 50 million people are reported to have dementia, with 10 million new cases being added every year. It is also proposed that globally, one person develops dementia every 3 seconds. This rapid increase necessitates the development of alternative treatment strategies to prevent and treat AD. The currently available treatments have limited efficiency, a more significant number of side effects, and poor patient compliance. They are also burdensome for the patients and caregivers to bear as the socioeconomic costs of long-term attendance and hospital expenditure continue to rise annually. This calls for the development of much safer alternative therapies that have been practiced since ancient times and have been reported to be beneficial in their use as anti-AD therapies. A multitude of such therapies can be borrowed from Ayurveda, traditional Chinese practices, meditation, and exercise. Many of the treatments aimed at fixing or preserving the functioning of synapses and cognition (the main attributes of AD) have proven to be useful for contributing in slowing the progression of AD and complementing the mainstream treatments currently in use. Some of the alternative therapeutic and preventive strategies that can be adopted for long-term anti-AD beneficial effects are discussed in detail. ## Ayurveda Ayu meaning longevity of life (Chetananuvrutti), Ayurveda, a Sanskrit word, is the 'scripture of longevity'; it is the science of life and wellbeing that encourages a holistic view of treatment to maintain the equilibrium of the body, mind, and soul. This traditional system of medication is based on the five basic elements of the universe: space, fire, water, air, and earth. It finds its roots in India and the Indian subcontinent, dating back to the Indus Valley Civilization (about 3,000 BC). Ayurveda not only cures but acts as an excellent preventive medication system that can delay the very onset of aging and ailments associated with it. According to Ayurveda, disturbances in the Tridosha (Vata, Pitta, and Kapha) and Triguna (Sattva, Raja, and Tama) results in functional disorder of Indriya (cognitive and motor organs), Mana (psyche), and Buddhi (intellect). It leads to impaired memory or cognitive defects. Ayurvedic drugs help in maintaining and re-establishing the balance between Tridosha and Triguna. They also provide Medhya (intellect promoting) effect to improve the memory of the patients. Recently, the world's first Ayurvedic drug against cancer has been launched, which is non-chemical, non-synthetic, and has no side-effects. 'Kudos CM9 ® ' (CSIR-IICB) was developed by the Ministry of Science & Technology, CSIR-IICB (Council of Scientific & Industrial Research-Indian Institute of Chemical Biology), India. It helps in cure as well as control the progression of benign and malignant tumors. It can also be used to prevent cancer in high-risk individuals. Ayurvedic herbal medications often have a biosafety profile and generally, do not show significant side-effects. Thus, there is a surge in research interest of Ayurvedic drugs. Mechanistic studies being carried out have helped in elucidating the effect and efficacy of these drugs on central nervous system (CNS) disorders, like AD. Some Ayurvedic herbal/traditional plant-based drugs that can treat disorders of the nervous system and improve cognitive function and memory are discussed. ## Bacopa monniera (brahmi) In the traditional Indian system of medicine, Brahmi acts as Medhyarasayana (from the Sanskrit words; 'medhya,' meaning intellect or cognition, and 'rasayana,' meaning 'rejuvenation') plant-derived nerve tonics that enhance memory, intellect, and aid mental health. Brahmi extracts, functioning as a nootropic, are used for disorders like epilepsy, insomnia, anxiety, etc.since they have effects that can enhance memory, help in anti-inflammation, and act as an analgesic, antipyretic, sedative, and antiepileptic agents. Steroidal saponins, Bacosides A and B, are the active constituents of Brahmi that scavenge free radicals and inhibit lipoxygenase activity, reducing lipid peroxidation, thus protecting neurons of the hippocampus, prefrontal cortex, and striatum 282 A. Sharma and Y. Kumar / Nature's Derivative(s) as Alternative Anti-AD Treatmentsagainst cytotoxicity and DNA damage related to AD. Bacosides also chelate iron and increase glutathione peroxidase. The extracts show neuroprotective effects due to nitric oxide-mediated cerebral vasodilation. A study by Le et al. on olfactory bulbectomized mice demonstrated that Brahmi protects the cholinergic neurons in the medial septal nucleus that projects into hippocampus. A study on rat model of AD also shown the inhibition of degeneration of cholinergic neurons due to alcoholic Brahmi extracts suggesting the potential therapeutic use of the drug. These studies suggest the cholinergic effect of Brahmi extracts are similar to the current treatments like donepezil, rivastigmine, and galantamine. Studies also show that Brahmi extracts decrease A␤ deposition and stress-induced hippocampal damage as demonstrated by Mathew & Subramanianand Holcomb et al., indicating its potential therapeutic use in the case of AD. There are no proven side effects of the drug; however, excessive consumption of the same leads to conditions like diarrhea, stomach upset, and nausea. Limited intake of the drug for pregnant and breastfeeding women is recommended. ## Centella asiatica (mandukaparni) Also known as Gotu kola and Asiatic pennywort, the extracts of this herb are useful in nerve regeneration, thereby helping in the recovery of their function. Enhancing memory retentionand promoting longevity, this neurodegenerative adaptogen also helps in preventing cognitive impairments and alleviating oxidative stress associated with AD due to its antioxidant properties. It is an alternative for acetylcholinesterase inhibitors, which generally comprise the first-line of treatment for AD and can also help in lowering the phospholipase A 2 activity. A natural process that plays a role in causing neurodegeneration, i.e., mitochondrial dysfunction, can be altered by the antioxidant activity of this herb. It consists of saponins, asiaticoside derivatives at a molecular level as active compounds, which can significantly reduce cell death induced from hydrogen peroxide, lowering free radical levels and preventing A␤-mediated neural cell death in vitro. Thus, its use can be significant in A␤ toxicity and AD prevention and treatment. A study conducted on 28 healthy elderly individuals also suggested the potential of Gotu kola extracts on alleviating mood disorders as well as the age-related cognitive decline in them. It is quite safe to use; however, higher doses of it can result in drowsiness. ## Withania somenifera (ashwagandha) It very commonly is known as Indian ginseng and is known to possess multiple therapeutic properties, serving as a nerve tonic, memory enhancer, and a medicinal plant having anti-stress, immunomodulatory, and antioxidant properties. These properties are due to its constituent compounds, Withaferin A and Withanolide A, which have comparable pharmacokinetic roles except for different oral bioavailability, with that of Withaferin A being greater than Withanolide A. Its roots facilitate inhibition of the activation of the nuclear factor NF-κB, which controls the expression of genes responsible for inflammation and oxidative stress and prevents A␤ production, thus protecting from inflammation and oxidative stress. It also helps in the restoration of synaptic function, and reduction of apoptotic cell death. It aids in migration of nuclear factor related E2-related factor 2 (Nrf2) to the nucleus leading to a surge in the antioxidant enzyme expression and neuroprotective proteins like heme oxygenase-1, thus substantiating antioxidant effects beneficial in AD treatment and prevention. It also contributes to neurite outgrowth and neuroprotective effects in vitro, axonal regeneration, and synaptic reconstruction in vitro and in vivo. Crossing the BBB, it can alleviate inflammation of the brain. It has no side effects, but some people report experiencing nausea or diarrhea after consuming its root. ## Curcuma longa (turmeric) The yellow curry, Indian spice consists of a hydrophobic compound known as curcumin or diferuloylmethane (C 21 H 20 O 6 ). This polyphenolic yellow pigment has anti-inflammatory and antioxidant characteristics. The molecule can cross the BBB and bind to A␤, thus preventing its aggregation and fibril formation in vivo as well as in vitro. Turmeric acts as a therapeutic agent in case of AD as in addition to reducing amyloid deposition; it can also prevent tau phosphorylation, both of these events being significant implications of the neurodegenerative disorder. Curcumin shows anti-inflammatory characteristics by suppressing pro-inflammatory pathways by preventing the production of TNF-␣, IL-1␤, and other pro-inflammatory cytokines, like, IL-8, MIP-1␤, and MCP-1, in astrocytes and microglia. It also inhibits phospholipase A 2 and cyclooxygenase (COX-2) enzymes associated with the metabolism of neural membrane phospholipids to prostaglandins, thus reducing neuroinflammation. It reduces oxidative damage and enhances cognitive function, especially in the case of aging by modulating the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway. Nrf2 bound to Keap1 and present in the cytoplasm get released upon binding of curcumin to Keap1. It then translocates to the nucleus and binds to antioxidant responsive elements in DNA as a heterodimer, thus, targeting the genes controlling the expression of antioxidant enzymes, DNA repair enzymes, molecular chaperones, and antiinflammatory response proteins. These proteins lead to the lowering of ROS production and aid the cell's ability to fix any damage that follows. A study also established that regular curry consumers performed better on the standard test (Mini-Mental State Examination, MMSE) of cognitive function than those who have never or rarely consumed it. It also has the potential to lower cholesterol, inhibit acetylcholinesterase, mediate insulin signaling pathway, metal-chelation (binds copper) that prevents cerebral deregulation caused by bio-metal toxicity, and decrease microglia formation. It naturally has lower bioavailability, but when consumed with black pepper (Piper nigrum), its bioavailability and absorption rate increases due to the active ingredient 'piperine' of black pepper. The lower rates of AD in the Indian population has also been attributed to the extensive consumption of turmeric, hinting at the neuroprotective role of turmeric. It can be used as a food additive and is safe to consume, with no significant side effects except diarrhea, nausea, dizziness, or stomach upset when consumed excessively. ## Convolvulus pluricaulis (shankhpushpi) This plant commonly found in India is a nerve tonic that serves to improve memory and cognition. It has a calming effect, and it is used for neurological disorders, like anxiety, insomnia, stress, and mental fatigue. Its ethanolic extracts induce antioxidant effects along with enhancing learning and memory in rats. It comprises of glycosides, flavonoids, coumarins, anthocyanins, and alkaloids as its major bioactive components and has also shown to contain sitosterol glycoside, octacosanol tetracosane, hydroxycinnamic acid, and glucose. In an AD Drosophila model, its aqueous extracts extend the lifespan of the fly model by neutralizing human microtubule-associated protein tau-induced neurotoxicity, preventing early death. Shankhpushpi extracts significantly lower the tau protein in tauopathy. It also restores the declining acetylcholinesterase activity, boosts the antioxidant enzyme activities, and amends the tau-induced oxidative stress. The increase in acetylcholine content caused by its extracts is also complemented by a substantial upsurge in the total dendritic intersections, number of branch points, and dendritic processes developing from the neurons' cell bodies, in comparison with age-matched saline controls. Thus, the increase in neurite outgrowth caused by ethanolic extracts of Shankhpushpi augments memory. Addition of C. pluricaulis to regular standard food allows its action. The paste made from its small leaves along with its flowers and roots can be consumed regularly. ## Guggulu This pale yellow or brown oleo-gum resin is an exudate from cracks, fissures, or incisions on the bark of Commiphora wightii, Commiphora mukul, Commiphora abyssinica, Commiphora molmol, and Commiphora Burseraceae. It is composed of phenols, ferulic acids, and other nonphenolic aromatic acids that have the potential to scavenge superoxide radicals, thus, making it significant in combating neurodegenerative diseases that have oxidative stress as a substantial implication. High cholesterol is among one of the risk factors of AD. Guggulu is essential in modulating the activity of the cell membrane and regulating synaptic functions and neuronal plasticity. Guggulu helps in lowering cholesterol. Thus, this attribute of the drug contributes to reducing the frequency of AD development, since decreased neuronal cholesterol level leads to inhibition of A␤-forming amyloidogenic pathway. It becomes possible due to the removal of precursor protein from cholesterol. Studies have revealed that Z-guggulsterone pre-treatment of C57BL/6J mice models showing scopolamine (a muscarinic acetylcholine receptor antagonist)-induced memory impairments result in memory-improving effects due to the activation of the cAMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling pathway. Guggulipids also exhibit antioxidant effects, lower cholesterol, and show antiacetylcholine esterase activities, thus improving the condition of streptozotocin-induced memory deficit mice model of dementia as demonstrated in a study by Saxena et al. It can lower serum LDL cholesterol as well as triglyceride levels. Half-lives of Guggulu and its derivatives in circulation in body and brain are yet to be elucidated. ## Ginkgo biloba (ginkgo) Extracts of Ginkgo consist of flavonoid glycosides (containing quercetin, kaempferol, isorhamnetin, etc.), terpenoids (containing Ginkgolides A, B, C, and J and bilobalide), and organic acids. The flavonoids and terpenoids are constituting the pharmacological components, while organic acids contribute to the water solubility of the extract. It shows antioxidant activity attributed to its free radical scavenging action as shown in a study by Wei et al. in which pre-treatment of a cerebellar granule with Ginkgo reduced the oxidative damage caused by H 2 O 2 /FeSO 4. It also upregulates the protein level and activity of antioxidant enzymes like superoxide dismutase and catalase, to stabilize the cellular redox state, in addition to direct weakening of ROS. In LPS-activated microglial cells, Ginkgo extracts have been used to inhibit the release of prostaglandin E 2 (PGE 2 ), a pro-inflammatory mediator and pro-inflammatory cytokines, thus, displaying antineuroinflammatory activity. It also downregulates cytokines associated with inflammation, such as AGE-or LPS-induced nitric oxide (NO), TNF␣, IL-6, and IL-1. The anti-inflammation effects may also be attributed to their platelet-activating factor (PAF)-antagonist activity. PAF is shown to have a role in regulating cytokines in inflammatory responses. Ginkgo also shows anti-apoptotic action and can act synergistically on multiple signaling apoptotic pathways, like maintaining the integrity of the mitochondrial membrane, blocking the apoptotic caspase cascade and apoptosome formation by preventing mitochondrial release of cytochrome c, enhancing the transcription of anti-apoptotic Bcl-2-like protein, attenuating the transcription of pro-apoptotic caspase-12, inactivating pro-apoptotic c-Jun Nterminal kinase (JNK), and inhibiting the cleavage of the vital effector protease caspase-3. Therefore, it blocks apoptosis and also prevents nuclear DNA fragmentation, the molecular hallmark of apoptosis. It also plays a protective role in preventing neurotoxicity caused by A␤ aggregation by blocking A␤-induced events, such as ROS accumulation, glucose uptake, mitochondrial dysfunction, activation of AKT, JNK, and ERK 1/2 pathways, and apoptosis. Ginkgo also shows other protective effects such as ion homeostasis, modulation of phosphorylation of tau protein, and induction of growth factor synthesis. Ginkgo extracts can also upregulate expressions of nerve growth factor in mouseand glialderived neurotrophic factor and vascular endothelial growth factor in cultured rat cortical astrocytes. In a study by Ihl et al., treatment of 404 patients having AD or vascular dementia with standardized extracts of Ginkgo, popularly known as EGb 761, resulted in a considerable enhancement in cognitive function and neuropsychiatric symptoms. Of late a study by Savaskan et al., involving 1,628 patients with dementia, also showed the improvement of behavioral and psychological symptoms of dementia (except psychotic-like features) and caregiver distress caused by such symptoms by the administration of 22-24 weeks' treatment with EGb 761. Recent studies have also shown the use of EGb761 in providing an appropriate environment to induce differentiation of stem cells into nerve cells, which opens new and fascinating avenues for stem cell treatment of neurodegenerative diseases. Ginkgo extracts complexed with phosphatidylserine as a phytosome (Virtiva ® ) lead to an enhanced memory task performance due to increased bio-availability of active constituents of the extract when tested on 28 participants. ## Cinnamon zeylanicum (cinnamon) Cinnamon is composed of a flavonoid, cinnamaldehyde, that gives it the characteristic flavor and aroma. Most commonly present are E-cinnamaldehyde and o-methoxycinnamaldehyde. Its extracts are shown to inhibit tau aggregation and filament formation. It is not detrimental to normal tau functioning. This inhibitory effect is due to the proanthocyanidin trimer molecule as well as cinnamaldehyde. Cinnamaldehyde and an oxidized form of epicatechin (EC) (ECox) of the extracts can also dissolve tau tangles by binding to the two cysteine residues in tau. ECox can also prevent oxidation of tau by ROS and H 2 O 2 , preventing tau tangles. EC also has the potential to sequester toxic and reactive by-products of oxidation, like acrolein. It showed that a diet rich in proanthocyanidin, catechin, and epicatechin in monomeric, oligomeric, and polymeric forms could promote learning and memory in AD. It can also inhibit the TLR4/NOX4 signaling, which can then lessen the oxidative stress/nitrative stress in case of neuronal damage and apoptosis pathways. It also shows its neuroprotective effects by reducing the intracellular ROS production as well as expression of pro-inflammatory cytokines, IL-1␤, IL-6, TNF␣, and NF-kB to reduce inflammation, as shown in case of LPS-stimulated rats in a study by Zhao et al.and can also increase synaptic plasticity. Its polyphenolic compounds can counter A␤ aggregation; it also inhibits ␤-secretase and production of A␤in Chinese hamster ovary-APP CHO cells in vitro. Thus, cinnamon extracts can target all three AD hallmarks: inhibition of acetylcholinesterase activity, A␤ formation/aggregation, and tau phosphorylation as summarized by S. Momtaz et al.. ## Panax ginseng (ginseng) Ginsenosides, a type of terpenoid dammarane glycoside, constitute the major bioactive component of the plant extracts. Ginseng root has been observed to contain over and about 60 different ginsenosides, like Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, Rg2, and Rg3. It shows anti-inflammatory effects due to inhibition of phosphorylation of p38MAPK and STAT1 in BV2 microglial cells, thus reducing LPS-induced mediators that are pro-inflammation such as COX-2, TNF␣, and inducible nitric oxide synthase (iNOS). Studies show the use of its extracts to improve thinking as well as working memory in case of AD patients. Ginseng treatment improved Alzheimer's Disease Assessment Scale cognitive subscale (ADAS-cog) and MMSE scores in AD patients. A study also reported the potential of GP-17, a novel phytoestrogen isolated from ginseng, in the clearing of A␤ in vivo and in vitro in the AD mouse models, Swedish mutant of APP695 (APP695swe) and APP/PS1. The neuroprotective effect was due to the autophagy-based elimination of A␤ due to the activation of transcription factor EB responsible for lysosome biogenesis. It is useful in the inhibition of formation of A␤, stimulation of soluble A␤PP␣ formation, anti-inflammation, anti-apoptosis, decreasing oxidation stress, and enhancing CNS cholinergic function to counter impairments and implications associated with AD. Its combination with Ginkgo is expected to give better results. ## Cannabis sativa (cannabidiol) The main constituent of phytocannabinoids, cannabidiol (CBD), is present in Cannabis sativa. It is less toxic and lipophilic, making it easier to cross the BBB. It can show anti-inflammation effects and inhibit the neuroinflammation caused by A␤ aggregation. CBD inhibits glial fibrillary acidic protein (GFAP) mRNA and protein expression, impairing iNOS and IL-1␤ protein expression, and their related release in A␤-injected animals as shown by Esposito et al.. CBD also prevents tau hyperphosphorylation in A␤-stimulated PC12 neuronal cells due to reduction in phosphorylated glycogen synthase kinase 3␤ (p-GSK3␤) which is the active form of GSK3␤. GSK3␤, called tau protein kinase, is associated with tau protein hyperphosphorylation, which then leads to the formation of tau tangles. CBD treatment has shown to enhance memory and learning, along with decreasing IL-6 levels in AD mice models. Chronic CBD treatment administered following the inception of AD-like symptoms, cognitive insufficiencies and formation of A␤ plaques, has shown to reverse social recognition memory and object recognition deficits in double transgenic APPswe/PS1E9 mice. The study also suggested the modulation of cytokine TNF␣ levels by CBD. Sativex ® is a combination of CBD and 9tetrahydrocannabinol (THC) in the ratio of 1 : 1. It has shown to reduce neuroinflammation and iNOS levels in the cerebral cortex and alleviate the stress-related behaviors in parkin-null, human tau overexpressing (PK -/-/Tau VLW ) mice. It also stimulated autophagy and reduced cortical and hippocampal A␤, plaques, and phosphorylated tau. THC/CBD treatment increased fear-associated learning and preserved object recognition memory. It also reduces cortical soluble A␤ 42 levels and changes plaque composition. Also, it effectively lowers inflammation and alters neuroinflammatory processes in APPxPS1 transgenic mice. CBD shows anti-AD effects; antioxidant activity, anti-inflammation effects, and neuroprotective properties, and reduces A␤ generation and tau hyperphosphorylation in vitro. ## Natural dietary intakes An insightful review article bysheds light on the importance and use of natural dietary products, like fruits (berries: blueberries, mulberries, and strawberries, apple, , papaya, dates, grapes, pomegranate, walnut, etc.), vegetables (broccoli, etc.), spices (cinnamon, black pepper, saffron, turmeric, garlic, ginger, etc.), drinks (tea, coffee, wine, fruit juice, etc.), marine products, and Mediterranean diet, in showing positive effects for AD prevention and treatment. These, thus, have the potential for being developed into therapeutic anti-AD drugs. A recent study byshows that polyphenols have a high therapeutic value and are present in plants. Quercetin derived from fruits and vegetables, such as onions, apples, berries, peanuts, soybeans, potatoes, etc.; resveratrol from grapes' skin and seeds; curcumin from turmeric; cinnamic acid and its derivatives from cinnamon, citrus fruits, tea, cocoa, etc.; caffeine from Coffea arabica and caffeic acid abundantly found in all plants and dietary products, are some of the critical polyphenolic compounds that have been discussed. They inhibit cholinesterase activity, thus, qualifying as potential drugs for AD treatment. However, their exact mechanism of action has not been elucidated. N-acetylcysteine (NAC), abundantly present in onions, is a glutathione (GSH) precursor which is an antioxidant. It is included in the World Health Organization's List of 40 Essential Medicines, being a well-established drug since the 1960 s. GSH levels are depleted in the hippocampal regions in AD, implying that NAC supplementation can be beneficial for cognitive enhancement. A study by Moreira et al. provides evidence that NAC is efficient in enhancing the mitochondrial related oxidative stress in case of AD. It also showed that the combination of lipoic acid and NAC was more potent to use. ## Vitamins Lower levels of vitamin B are often associated with decreasing cognitive ability, commonly due to the increased level of homocysteine. Vitamin B6, B9 (folate), and B12 (cyanocobalamin) are involved in homocysteine metabolism, which can thus be boosted with the help of vitamin B supplementation, resulting in reduced risk of dementia. Vitamin A and C have antioxidant activity, thus their use in enhancing cognition and preventing dementia. Vitamin A (retinol) is essential for memory, learning, and cognition. Its levels in the brain decline with age and are very low in AD patients. Performed retinoids or provitamin carotenoids provide vitamin A, with ␤-carotene most commonly found in food. In Physicians' Health Study II (PHSII), a randomized trial of ␤-carotene supplementation, it was found that long-term use conferred cognitive benefits. Retinoic acid was also reported to protect neurons of the hippocampus by preventing A␤-induced cell death. A study demonstrated that low vitamin D dietary intake was associated with the poor cognitive performance. Similar results were obtained from another study from the Third National Health and Nutrition Examination Survey (NHANES III). Vitamin E is a potent antioxidant and antiinflammatory agent. It also has neuroprotective roles. Lower levels of vitamin E have been reported in AD patients. It has shown positive results on cognition. However, further research is required to support the use of vitamin E as a therapeutic agent for AD. ## Medical food cocktail According to a study by Parachikova et al., administration of a cocktail that comprised of curcumin, piperine, epigallocatechin gallate, ␣-lipoic acid, Nacetylcysteine, vitamin B and C, and folate for six months in transgenic mice model of AD resulted in improvement in memory and learning. It also helped in reducing the soluble A␤ concentrations, including A␤ oligomers. Thus, combinations of different medicinal compounds must be extensively studied to derive different formulations that show greater efficacy in their preventive and therapeutic anti-AD usage. ## Stress, meditation, and ad Stress, an effect of a threat to homeostasis, is a situation wherein individuals encounter aversive stimuli leading to a negative physiological change. Stress and cortisone (a hormone released by the adrenal gland in response to stress) can activate glucocorticoid receptors, leading to a reduction of hippocampal plasticity. It affects memory (especially spatial memory) and learning, potentially leading to cognitive deterioration and age-linked neurodegener-ative diseases. Excess cortisol production also leads to a decreased volume of right hippocampi and orbitofrontal cortex. It showed that stress diminishes telomerase levels, thus affecting the telomere length and the genetic health of an individual. The accelerated shortening of telomeres is associated with faster aging, inflammation, and AD. Managing stress allows alleviating the progression and implications of AD since stress is considered to be a factor that accelerates AD pathology. A review by Jesse Russell-Williams et al. analyzed ten different studies, comprising of six studies on mindfulness (mindfulness-based stress reduction, MBSR), three studies on Kirtan Kriya (KK) meditation, and one study on mindfulnessbased Alzheimer's stimulation (MBAS). The analysis highlights that the investigation by Quintana-Hernandez et al., spanning over two years, shows that mindfulness can be used as a non-pharmacological method to delay cognitive deterioration in case of mild to moderate AD patients, a study based on MBSR and KK. Newberg et al. reported that practicing KK for eight weeks in case of subjective memory loss due to AD or mild cognitive impairment resulted in a significant increase in cerebral blood flow (CBF) within the frontal lobe and right superior parietal lobe. A study by Paller et al. also showed that mindfulness augments attention as assessed by P3-related activity. The pre and postanalysis following eight weeks of MBSR also showed benefits like better subjective sleep quality, improved ratings of quality-of-life, and fewer depressive symptoms. Studies also showed that eight weeks of KK or mindfulness sessions helped in alleviating worryand soaring positive mood and energy. A majority of patients found the experience to have positive impacts since the sessions were relaxing, calming, peaceful, and uplifting. Thus, mindfulness can act as an efficient disease management method, and different kinds of meditation (KK, MBAS, and MBSR) can work as suitable complementary treatments. Synaptic changes contribute to AD pathogenesis, affecting cognition. Practicing KK has shown to stabilize brain synapses by increasing neurotransmitter levels, including acetylcholine, norepinephrine, glutamate, and possibly GABA. KK also activates the posterior cingulate gyrus (PCG)which is a metabolically active region and is vital for memory and emotional functions. Reduced cerebral blood flow and hypometabolism in PCG are considered to be early signs of AD. Thus, regular activation of PCG by KK can significantly contribute to decreasing AD risks. A study analyzed the effects of KK on caregivers and reported that practicing KK for eight weeks helps in lowering depressive symptoms, boosting mental health, memory, and well-being. It improved the telomerase activity as well, suggesting an alleviation of stressinduced cellular aging. Practicing KK also enhances sleep, poor sleep being a risk factor in the case of AD. KK is thus proved to be an effective, safe, and free of side-effects. It is a self-directed program that is easy to learn and is affordable. It can be used to complement pharmacological treatments for increased preventive or therapeutic results. A study by Luders et al. highlights that the brain age of people who have regularly been meditating for an extended period is found to be is 7.5 years lesser than that of the non-practitioners. The brain age was calculated using a machine learning algorithm, and scores obtained in years as BrainAGE index. Not just were the brains of practitioners of meditation found to be younger, but with every passing year, meditators' age reduced by one month and 22 days than their chronical age. Thus, there was a positive impact of meditation on the preservation of the brain and its activity. Therefore, by reducing the age-related atrophy, meditation slows the rate of brain aging. ## Exercise and ad Higher levels of daily physical activity and exercise, complemented by bright light and proper nutrients, aid in lowering the chances of AD development. Regular walking shows improvement in cognition. In people aged 60 years or more, regular aerobic exercises enhance the production of gray and white matters in the cortical regions. Physical activities, such as treadmill, ergo cycle, stair-climbing, etc., done for 40 min over 12 weeks consecutively, contribute to neuroprotective mechanisms of reduction of A␤ plaques, and increase blood flow to the cerebral region that improves neurogenesis and synaptogenesis, that will enhance memory and cognitive functions. The increased blood flow results in developing vascular reserve and maintenance of neuronal plasticity due to the activation of NO/endothelial NO synthase, thus lowered cerebrovascular and endothelial dysfunction pathophysiology is seen. Exercise upregulates the production of neurotrophins which are responsible for neurogenesis, improved memory, and brain plasticity. Regular physical exercising helps in reducing the rate of production of ROS, which is associated with the onset and progression of AD. Exercising helps escalate the activity and the level of antioxidant enzymes in different regions of the brain. Thus, reducing oxidative stress. Regular exercising causes a reduction in the levels of lipid peroxidation and protein oxidation. It can also limit ROS production by decreasing the ROS generating source itself or attenuating ROS generating capacity. Therefore, it acts as a preventive tool to combat AD. Physical activity not only thwarts the effects of oxidative stress but also helps in lowering cholesterol and insulin resistance, subsequently leading to increased vascularization and improved energy metabolism, such as glucose metabolism. A study by Nagarah et al. has shown that BDNF is critical for the upkeep of adult cortical neurons of the entorhinal cortex, whose early dysfunction furthers the initial short-term memory loss in AD. Thus, administration of BDNF helps in reversal of synapse deterioration, normalization of aberrant gene expression as well as the restoration of memory and learning. Moderate to high-intensity physical exercise intensifies BDNF production, thus, bringing about its positive impacts. In older healthy subjects and AD patients following one year of moderately intense aerobic exercise, i.e., 40-min session, three days per week, showed an increase of more than 2% in hippocampal volume and plasma concentration of BDNF. The environmental conditions also play a crucial role in BDNF enhancement as mice trained in enriched environment showed more significant environment-related cognitive improvement in BDNF, hippocampal neurotrophin, and activation of hippocampal neurogenesis. By enriching environmental conditions, BDNF production can thus be enhanced. Decrease of neurofibrillary degeneration and neuroinflammation attributed to physical training, along with prevention of loss of choline acetyltransferase expression. Neurotrophins are proteins that mediate neuronal survival and differentiation during development, maintain neuronal viability in adults by protecting and restoring neurons in case of injury or aging. They also function as activity-dependent modulators of synaptic plasticityin which BDNF is known to be an essential mediator in memory centers of the brain. Neurotrophins also have the potential to regulate genes coding for structural proteins, enzymes, or neurotransmitters, thus, modifying neuronal morphology and function. With the right standardization and universal protocols, this can be used to prescribe appropriate levels of physical exercises for brain health and to alleviate AD conditions. As per a recent finding, the beneficial effects of exercising on AD models are mediated by irisin, a hormone initially discovered as an exerciseinduced myokine that leads to thermogenesis and adipocyte browning, and is cleaved from a transmembrane precursor protein, fibronectin type III domain-containing protein 5 (FNDC5). Under the control of peroxisome proliferator-activated receptor-␥ coactivator 1␣, FNDC5 is expressed in muscle. FNDC5/irisin stimulates the expression of BDNF in the hippocampus. In the case of AD, FNDC5/irisin levels are lowered in the hippocampi and cerebrospinal fluid, thus leading to impairment of cognition and novel object recognition memory. Regular physical exercise boosts the molecular levels of FNDC5/irisin, which thus mediates protection and repair of synapse function and memory impairment, and prevents cognitive decline in AD. ## Acupuncture Acupuncture is a traditional Chinese practice that is said to be more than 3,000 years old. It is gaining global recognition as a complementary medicine system for a variety of conditions. It involves the application of heat or pressure or insertion of sharp, thin needles into the specific points on the body to stimulate nerve receptors via the connective tissue surrounding the site of application. It is aided by mechanical, electrical, or other physical manipulations. Acupuncture with deeper needling is shown to induce stronger, wider-ranging de qi sensations and enhance nodal centrality, chiefly in the abnormal brain regions in case of mild cognitive impairment. It was reported by a small-scale functional magnetic resonance imaging study that examined the effect of de qi sensations induced by different needling depths on the reorganizations of whole-brain networks. Some studies support the benefits of acupuncture in case of AD. Eighty-seven patients with mild-to-moderate AD were involved in a clinical trial in which acupuncture treatment, when used as a monotherapy for 12 weeks, resulted in substantial lowering in ADAS-cog scores from baseline compared with the use of donepezil. The study emphasized that acupuncture is safe and effective in improving cognitive function. showed that manual acupuncture could significantly improve spatial learning, relearning, and memory abilities in SAMP8 mice models of AD. It could be due to the increase in CBF in prefrontal lobe and hippocampus due to the acupuncture treatment since a reduction in CBF considered a sensitive biomarker to early perfusion deficiencies in AD. ## Rheumatoid arthritis and ad Rheumatoid arthritis (RA), an autoimmune disease, is marked by synovial inflammation, cartilage and bone destruction, and autoantibody production, causing progressive disability. RA has also been linked to cardiovascular disease, diabetes mellitus, and depression. RA also causes inflammation of the heart, lungs, and blood vessels. Thus, inflammation is a common implication observed in case of both RA and AD. Common inflammatory biomarkers found in the two provide evidence for the same. These include IL-6, pentraxin 3, resistin, etc. There is epidemiological evidence for reduced AD incidence in the case of RA due to the use of nonsteroidal anti-inflammatory drugs (NSAIDs). A study by Judge et al., based on observational studies and experimental data, highlights the inverse relationship between the manifestation of RA and AD. It shows that the use of classical disease-modifying antirheumatic drugs (cDMARDs) decreased AD risk, with methotrexate (MTX) being the most effective cDMARDs in the study. Thus, the protective effect of cDMARDs was independent of that of NSAIDs. This study brings forth the potential therapeutic use of cDMARDs in treatment of AD. MTX shows limited penetrance potential of the BBB. However, even moderate penetrance shows therapeutic effects. At higher doses, it is used to treat brain tumors. According to a study, administration of GM-CSF, which gets upregulated in RA, helped in reducing A␤ load and alleviating cognitive impairments in mice models of AD. The study highlighted the use of the recombinant human form of GM-CSF (Leukine ® ), approved by the FDA, as a therapeutic agent for AD. ## Diabetes and ad Insulin signaling is damaged in the brain in the case of AD. A review article by Kamal et al. highlights the similarities between AD and type 2 diabetes mellitus (T2DM) including impaired brain insulin signaling, abnormal brain glucose metabolism, amyloidogenesis, mitochondrial dysfunction, inflammation, and oxidative stress. It emphasizes that the interrelation between the two is complex and that with T2DM, AD incidences also increase. Thus, the mechanisms and factors that are common can be potential therapeutic targets. The neuroprotective effect of an antidiabetic drug, triple receptor agonist (TA), that can activate glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon receptors, was tested in the APPSWE/PS1dE9 mouse model of AD. TA treatment leads to memory and learning enhancement and reduction of amyloid plaques in the brain. It did not just ease the chronic inflammation by activating microglia and astrocytes but also reduced oxidative stress in hippocampus and cortex. TA also helped in reducing the levels of the mitochondrial proapoptotic signaling molecule BAX, increasing the anti-apoptotic signaling molecule Bcl-2, and enhancing the levels of BDNF, growth factor that protects synaptic function. The synaptic loss got prevented, indicated by a rise in levels of synaptophysin. It also enhanced the neurogenesis in the dentate gyrus. Thus, TA is a potential drug for AD. # Conclusion As the global population continues to increase, the number of people affected by age-related disorders such as AD is going to increase manifold. It is predicted among the aging population, that the number of AD cases will escalate to 135 million by 2050. Thus, alternative preventive and therapeutic strategies are attracting mounting attention for advanced research and development of appropriate medicine systems. It is attributed to their efficacy and safe usage as they are shown to have little or no side effects and potential to provide overall wellbeing. AD is not just crippling for the health of the patient but also imposes an economic burden on the patients and caregivers as it demands long-term medication, healthcare, and caregiving services, thus, necessitating the need to promptly formulate and adapt economically viable long-term medication and assistance systems to address the concerning rise of AD incidences in the coming future. Ayurveda drugs including turmeric, Brahmi, etc., natural dietary products, vitamins, meditation and management of stress, regular exercising, and traditional Chinese practices are significant in contributing toward the development of such alternative strategies. The anti-AD treatment systems discussed to provide an economical alternative to strive to combat the disease, derived from nature or naturally existing resources. Besides, various drugs in use for RA as well as diabetes also have therapeutic potential for anti-AD treatment. To efficiently establish the effectiveness of the alternative therapeutic and preventive strategies suggested, research studies with more extensive sampling size and appropriate control groups are required. It would be correct to conclude that the alternative strategies discussed have proven to be efficient in preventing, treating, curing, or managing AD. They have also proven to be useful for the wellbeing of the caregivers. The drugs and treatment options currently available do not fully cure or reverse the effects of the disease. These strategies can thus be used in combinations to complement the mainstream AD treatments presently being used for better management of the disease condition and increased cognition and memory of the patients.

Chrysopogon zizanioides aqueous extract mediated synthesis, characterization of crystalline silver and gold nanoparticles for biomedical applications The exploitation of various plant materials for the biosynthesis of nanoparticles is considered a green technology as it does not involve any harmful chemicals. The aim of this study was to develop a simple biological method for the synthesis of silver and gold nanoparticles using Chrysopogon zizanioides. To exploit various plant materials for the biosynthesis of nanoparticles was considered a green technology. An aqueous leaf extract of C. zizanioides was used to synthesize silver and gold nanoparticles by the bioreduction of silver nitrate (AgNO 3 ) and chloroauric acid (HAuCl 4 ) respectively. Water-soluble organics present in the plant materials were mainly responsible for reducing silver or gold ions to nanosized Ag or Au particles. The synthesized silver and gold nanoparticles were characterized by ultraviolet (UV)-visible spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) analysis. The kinetics decline reactions of aqueous silver/gold ion with the C. zizanioides crude extract were determined by UV-visible spectroscopy. SEM analysis showed that aqueous gold ions, when exposed to the extract were reduced and resulted in the biosynthesis of gold nanoparticles in the size range 20-50 nm. This eco-friendly approach for the synthesis of nanoparticles is simple, can be scaled up for large-scale production with powerful bioactivity as demonstrated by the synthesized silver nanoparticles. The synthesized nanoparticles can have clinical use as antibacterial, antioxidant, as well as cytotoxic agents and can be used for biomedical applications. # Introduction The synthesis of metal and semiconductor nanoparticles is an important topic of research because of their potential applications in catalysis, biosensing, recording media, and optoelectronics. [bib_ref] Gold Nanotriangles biologically synthesized using tamarind leaf extract and potential application in..., Ankamwar [/bib_ref] The chemical methods follow electrochemical, thermal, laser, microwave, polyol, radiolytic, sonochemical, and various other techniques. [bib_ref] Comparative evaluation of antibacterial activity of silver nanoparticles synthesized using Rhizophora apiculata..., Antony [/bib_ref] Currently, there is a growing need to develop an environmentally benign nanoparticle synthesis that does not use toxic chemicals in the synthesis protocols to avoid adverse effects in medical applications. The properties of noble metal nanoparticles such as silver and gold have previously been changed with many stabilizing and capping agents for various applications. The biological means of synthesizing nanoparticles provides an edge over chemical means as it is cost-effective, does not involve physical barriers for lessening agents, and expels the toxic effects of the chemicals used for the synthesis. There are several plants that have been identified to synthesize nanoparticles and the rate of synthesis of This article was published in the following Dove Press journal: International Journal of Nanomedicine 2 July 2013 Number of times this article has been viewed nanoparticles by plant extracts is comparable to those of chemical methods and faster than green synthesis by microorganisms. [bib_ref] Extracellular biosynthesis of gold nanoparticles using sugar beet pulp, Castro [/bib_ref] [bib_ref] Preparation and physicochemical characterization of ag nanoparticles biosynthesized by Lippia citriodora (Lemon..., Cruz [/bib_ref] Following our previous studies for the green synthesis of nanoparticles using Memecylon edule 5 and M. umbellatum [bib_ref] One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold..., Arunachalam [/bib_ref] aqueous extract, attempts were made to synthesize silver nanoparticles (SNPs) and gold nanoparticles (GNPs) by green technology from medicinal plants. [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] Until today, no reports have been available on the synthesis of SNPs and GNPs using aqueous leaf extracts of Chrysopogon zizanioides, a tropical medicinal plant originating from India and belonging to the Poaceae family. [bib_ref] Bacterial communities within the rhizosphere and roots of vetiver (Chrysopogon zizanioides (L)..., Monteiro [/bib_ref] C. zizanioides (vetiver) is a perennial grass commercially used to extract fragrance oils and its medicinal properties. The name "vetiver" is drawn from the Tamil word "vettiver" and the South India peninsula is considered to be the center of origin from where vetiver is said to have spread over the world because of its value as a producer of aromatic oil. Vetiver is known to have been in use in India both for its fragrant oil and as a traditional medicine since antiquity, 7 and its hedges have been applied for contour protection in India for centuries.Silver has been used in the form of metallic silver, silver nitrate, or silver sulfadiazine to treat burns, wounds, and severe bacterial infections. [bib_ref] Enhanced antimicrobial activity of silver nanoparticles synthesized by Cryphonectria sp evaluated singly..., Dar [/bib_ref] [bib_ref] Starch-directed green synthesis, characterization and morphology of silver nanoparticles, Khan [/bib_ref] The synthesized silver ions have been used in many kinds of formulations. Recently, it was shown that hybrids of SNPs with amphiphilic hyperbranched macromolecules display effective antimicrobial surface coatings. The most important applications of SNPs and GNPs are in the medical industry, such as topical ointments to prevent infection in burns and open wounds. [bib_ref] Enhanced antimicrobial activity of silver nanoparticles synthesized by Cryphonectria sp evaluated singly..., Dar [/bib_ref] Similarly, GNPs have been considered as an important area of research because of their unique and tunable surface plasmon resonance (SPR) and their applications in biomedical science including drug delivery, tissue or tumor imaging, photo thermal therapy, and immune chromatographic identification of pathogens in clinical specimens. [bib_ref] Biosynthesis of Au nanoparticles using olive leaf extract: 1st Nano Updates, Khalil [/bib_ref] The GNPs are used for developing biosensors, DNA labelling, and vapor sensing. [bib_ref] Plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial..., Mubarakali [/bib_ref] In this study, we show that an aqueous leaf extract of C. zizanioides, placed in a concentrated aqueous solution of AgNO 3 and HAuCl 4 , resulted in reduction of the silver and gold ions to form SNPs and GNPs. These green-synthesized SNPs (GSNPs) and green-synthesized GNPs (GGNPs) of C. zizanioides were examined by ultraviolet-visible (UV-vis) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDAX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) analysis for size and shape. # Materials and methods ## Collection of plants The plants C. zizanioides were collected from Villupuram, Tamil Nadu, India, the herbarium was prepared for authentication, and taxonomic identification was done by Dr Jayaraman, Madras Christian College, Tambaram, Chennai, Tamil Nadu. The voucher specimen was numbered and kept in our research laboratory for further reference. ## Preparation of aqueous extract The leaves of C. zizanioides were first washed with distilled water to remove the dirt and further washed with mild soap solution and rinsed thrice with distilled water. The leaves were blotted with tissue paper and shade dried at room temperature for at least 2 weeks. After complete drying, the leaves were cut into small pieces and powdered in a mixer and sieved using a 20 µ mesh sieve to get a uniform size range for use in further studies. The 20.0 g of the sieved leaf powder was added to 100 mL of sterile distilled water in a 500 mL Erlenmeyer flask and boiled for 5 minutes. The flasks were kept under continuous dark conditions at 30°C. The extract was filtered and stored in an airtight container and protected from sunlight for further use. [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] [bib_ref] One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold..., Arunachalam [/bib_ref] Phytochemical activity The qualitative phytochemical analyses of C. zizanioides extracts were performed following the methods of Parekh and Chanda 14 to determine the presence of alkaloids (Mayer's, Wagner, Dragendorff's), flavonoids (alkaline reagent, Shinoda), phenolics (lead acetate, alkaline reagent test), triterpenes (liberman-burchard test), saponins (foam test), and tannins (gelatine). [bib_ref] Wound healing and antigenotoxic activities of Aegle marmelos with relation to its..., Arunachalam [/bib_ref] The results were qualitatively expressed as positive (+) or negative (−). [bib_ref] Evaluation of phytochemical constituents and antioxidant activities of successive solvent extracts of..., Guruvaiah [/bib_ref] The chemicals used for the study were purchased from Sigma-Aldrich (Chennai, India). . ## Synthesis of snps and gnps ## Uv-vis absorbance spectroscopy analysis The bioreduction (by AgNO 3 or HAuCl 4 ) of nanoparticles was recorded periodically using a UV-Vis 3000+ double beam submit your manuscript | www.dovepress.com Dovepress Dovepress spectrophotometer (LabIndia, Maharashtra, India), which had slit widths of 0.5, 1.0, 2.0, and 5.0 nm. The samples were diluted with 2 mL of deionized water and measured by UV-Vis spectrum at regular time intervals.The deionized water was used as a blank for background correction of all UV-VIS spectra. All samples were loaded into a 1 cm path length quartz cuvette for sampling. The UV-Vis spectrometric readings were scanned from 200 to 800 nm and recorded at a scanning speed of 0.5 nm interval. The UV-VIS spectra were fit with Gaussian curves correcting for a cubic background for full-width at half maximum (FWHM) and wavelength of maximum absorbance measurements. The Gaussian fits to the UV-VIS spectra all had goodness of fit values (χ 2 ∼ 1), showing accurate curve analysis. [bib_ref] Green synthesis of robust, biocompatible silver nanoparticles using garlic extract, White [/bib_ref] ## Sem analysis of snps and gnps The prepared SNPs and GNPs were characterized using high resolution SEM analysis (JSM-5600 LV; JEOL, Tokyo, Japan). The samples were prepared by a simple drop coating of suspended silver or gold solution on to an electrically heated clean glass and allowing the solvent (water) to evaporate. The samples were left to dry at room temperature. [bib_ref] Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver..., Sathishkumar [/bib_ref] [bib_ref] Green-Green synthesis of silver-protein (core-shell) nanoparticles using Piper betle L leaf extract..., Usha [/bib_ref] [bib_ref] effective chemical route for the synthesis of silver nanoplates in tannic acid..., Yi [/bib_ref] ## Ftir spectroscopy analysis of dried biomass after bioreduction To identify the biomolecules present in the leaf extract of C. zizanioides and the biomolecules within the SNPs and GNPs after synthesis, a carefully weighed quantity of the synthesized nanoparticles were subjected to FTIR analysis (PerkinElmer RX1; PerkinElmer, Waltham, MA, USA). [bib_ref] The green synthesis, characterization and evaluation of the biological activities of silver..., Dipankar [/bib_ref] The bioreduced chlorauric and silver solutions were centrifuged at 10,000 rpm for 15 minutes, and the pellets were washed three times with 20 mL of deionized water. [bib_ref] Time-dependent effect in green synthesis of silver nanoparticles, Darroudi [/bib_ref] The resulting purified suspensions were dried and ground with KBr pellets and analyzed by FTIR. The FTIR were recorded in the range of 400-4000 cm −1 . To obtain a good signal and noise ratio, 512 scans were recorded. [bib_ref] Biosynthesis of silver and gold nanoparticles using Chenopodium album leaf extract, Dwivedi [/bib_ref] EDAX spectrum measurements EDAX analysis to confirm elemental silver was carried out for the detection of elemental silver. The samples were dried at room temperature and then analyzed for sample composition of the synthesized nanoparticles. The elemental composition of the synthesized nanoparticles by C. zizanioides were dried, drop coated on to carbon film, and tested using EDAX analysis (S-3400 N; Hitachi, Tokyo, Japan). [bib_ref] Coriander leaf mediated biosynthesis of gold nanoparticles, Narayanan [/bib_ref] [bib_ref] Synthesis and characterization of nanogold composite using Cylindrocladium floridanum and its heterogeneous..., Narayanan [/bib_ref] ## Xrd measurement To characterize the purified SNPs and GNPs, XRD measurements were conducted using an XRD-6000 X-ray diffractometer (Shimadzu, Kyoto, Japan) operated at a voltage of 40 kV and 30 mA with Cu Kα radiation in θ-2θ configurations. The crystallite domain size was calculated from the width of the XRD peaks by assuming that they were free from nonuniform strains using the following Scherer formula, 29 [formula] D = 0 94 . cos λ β θ (1) [/formula] where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the X-ray wavelength, β is the FWHM, and θ is the diffraction angle. [bib_ref] Green biosynthesis of silver nanoparticles using Curcuma longa tuber powder, Shameli [/bib_ref] [bib_ref] Green synthesis of biogenic metal nanoparticles by terrestrial and aquatic phototrophic and..., Narayanan [/bib_ref] [bib_ref] Synthesis of silver nanoparticles in montmorillonite and their antibacterial behavior, Shameli [/bib_ref] To expel the added instrumental broadening, the FWHM was corrected using the FWHM from a large-grained Si sample. [formula] β corrected sample si FWHM WHM = − ( ) / 2 2 1 2 F (2) [/formula] This modified formula is valid only when the crystallite size is smaller than 100 nm. 24 # Results ## Phytochemical screening The aqueous extract of C. zizanioides was evaluated for the presence of phyto constituents by qualitative chemical tests. The distinct phytochemicals that were present are shown in [fig_ref] Table 1: Preliminary phytochemical investigation of aqueous extract of C [/fig_ref]. The results infer the presence of terpinoids, alkaloids, flavonoids, triterpines, and tannins in the aqueous extract of C. zizanioides. ## Synthesis of snps and gnps The GSNPs and GGNPs displayed yellowish-brown and ruby red color, respectively, in water; these colors arose because of exciting surface plasmon vibrations in the metal nanoparticles. The color change is attributed to the collective oscillation of free conduction electrons induced by an interacting electromagnetic field in metallic nanoparticles, which is called SPR. [bib_ref] Rapid green synthesis of gold nanoparticles using Rosa hybrida petal extract at..., Noruzi [/bib_ref] In the present study, the SNPs were synthesized rapidly within 20 minutes of the incubation period in the aqueous silver nitrate solution, which turned to a brown color in 30 minutes of adding leaf extract . The intensity of the brown color increased in direct proportion to the incubation period because of the excited SPR effect and reduced AgNO 3 . [bib_ref] Plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial..., Mubarakali [/bib_ref] The control AgNO 3 solution (without leaf extract) showed no change of color with time and our results are comparable with the previous study done using Dillenia indica fruit extract 33 and with our own results done using M. edule, [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] Memecylon umbellatum [bib_ref] One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold..., Arunachalam [/bib_ref] and indigofera aspalathoides. [bib_ref] Green synthesis of crystalline silver nanoparticles using indigofera aspalathoides -medicinal plant extract..., Arunachalam [/bib_ref] The color of the reaction mixture on formation of GNPs changed to ruby red color from a colorless/straw color . This color change from colorless/straw to ruby red was noticed within the first 2 hours of reaction time. This visibly confirmed the presence of GNPs in the solution and that AuCl [bib_ref] Preparation and physicochemical characterization of ag nanoparticles biosynthesized by Lippia citriodora (Lemon..., Cruz [/bib_ref] − ions had been reduced to Au ions. ## Uv-vis spectrophotometer The silver ions immediately declined within 20 minutes, which may have been due to the presence of water soluble phytochemicals like alkaloids, phytosterols, tannins, flavonoids, and triterpines in the C. zizanioides plant extract. The reduction of silver and gold ions occurred rapidly and more than 90% of the reduction of silver and gold ions was completed within 8 hours (at 1 and 5 mL of plant extract, respectively) after adding the aqueous plant extract to the metal ion solutions. The comparatively slower reduction rate of silver ions relative to that of gold ions was most likely because of differences in the reduction potentials of the two metal ions, the redox potential being considerably lower for gold ions. The characteristic absorption peak at 420 nm in UV-Vis spectrum confirmed the formation of SNPs. SPR patterns, which detail the characteristics of metal nanoparticles, strongly depend on particle size, stabilizing molecules or the surface of adsorbed particles, and the dielectric constant of the medium. The nanoparticles showed an absorption peak around 420 nm after 1 hour of reaction, which is a characteristic SPR band of SNPs, possibly because of exciting longitudinal plasmon vibrations in the SNPs in the solution. [bib_ref] A novel 'green' synthesis of colloidal silver nanoparticles (SNP) using Dillenia indica..., Singh [/bib_ref] [bib_ref] Green synthesis and characterization of gelatin-based and sugar-reduced silver nanoparticles, Darroudi [/bib_ref] [bib_ref] Egg white-mediated green synthesis of silver nanoparticles with excellent biocompatibility and enhanced..., Lu [/bib_ref] The increase in the intensity of the ruby red color clearly suggests the formation of GNPs in the reaction mixture. The GGNPs were ruby red in the aqueous solution because of the exciting surface plasmon vibration of the GNPs at 540 nm [fig_ref] Figure 3: Time dependent absorption spectra of gold nanoparticles after the bioreduction with aqueous... [/fig_ref]. The kinetics of biosynthesis hastens with time and the intensity of the reaction mixture color increases rapidly. The process of biosynthesis is carried out at surrounding environmental conditions and the total reaction is completed within 8 hours. [bib_ref] Biofunctionalized silver nanoparticles: advances and prospects, Ravindran [/bib_ref] ## Sem images of snps The SEM images clearly suggest that there was a thin layer of other material on the surface of the SNPs because of the capping silver ions. The SEM analysis of the bioreduced SNPs confirmed that the size of the metal particles was in the nano range and were roughly cubic in shape. The size of the SNPs was in the range of 85-110 nm after 24 hours and the representative SEM image is shown in [fig_ref] Figure 4 200: nm EHT = 3 [/fig_ref]. Most of the nanoparticles was roughly cubic with flat edges. The size of the particles agreed with the noted SPR band. Some nanoparticles had isotropic nanostructures with irregular contours as shown in [fig_ref] Figure 4 200: nm EHT = 3 [/fig_ref] ; also most of the SNPs in the SEM images were in physical contact, but they were separated by a uniform interparticle distance. From our previous reports, it has been observed that the cubic shape of nanoparticles is synthesized after bioreduction. [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] [bib_ref] Green synthesis of silver nanoparticles from aqueous Aegle marmelos leaf extract, Rao [/bib_ref] SEM of gNPs A scanning electron microscope was employed to analyze the structure of the nanoparticles that were formed, as shown in [fig_ref] Figure 5: Scanning electron microscopy image of green gold nanoparticles synthesized by reducing aqueous... [/fig_ref]. The particles that formed were cubic in shape. The cubic shaped nanoparticles that formed were shown to have high surface area and were in the range of 123-138 nm in size, with an average size of 130 nm. The particles were monodispersed, with only a few particles of different size. A high magnification SEM image recorded from our previous studies, showed that the biologically synthesized GNPs at the end of the reaction with M. edule leaf extracts were predominantly cubic in morphology. [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] [bib_ref] One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold..., Arunachalam [/bib_ref] [bib_ref] Green synthesis of crystalline silver nanoparticles using indigofera aspalathoides -medicinal plant extract..., Arunachalam [/bib_ref] Low quantities of the extract can reduce the chloroaurate ions, but do not protect most of the quasi-spherical nanoparticles from aggregating because of the lack of biomolecules to act as protecting agents, which were clearly viewed from the SEM images. ## Edax for snps The analysis through EDAX spectrometers confirmed the presence of the elementary silver signal of the SNPs, as shown in . The vertical axis displays the number of X-ray counts, while the horizontal axis displays energy in keV. The identification lines for the major emission energies for silver (Ag) are displayed and they agree with the peaks in the spectrum, thus giving confidence that silver has been correctly identified. The EDAX spectrum clearly confirms that 93.8% was silver. The weak signals that arose at 0.5 keV correspond to proteins or enzymes that are bound to the silver nanoparticle. There was also a strong signal at 0.24 keV for C atom, which is due to the functional compounds present in aqueous plant extract. Individual cubic shaped SNPs using C. zizanioides are formed in the range of 2-4 keV. Similar signal energy peaks were also observed by other researchers. [bib_ref] Synthesis of silver nanoparticles using plants extract and analysis of their antimicrobial..., Logeswari [/bib_ref] [bib_ref] Facile green synthesis of gold nanostructures by NADPH-dependent enzyme from the extract..., Narayanan [/bib_ref] As reported from our earlier studies the EDAX pattern clearly shows the SNPs are crystalline in nature and showed strong signal energy peaks for silver atoms in the range of 2-4 keV with weaker signals for carbon, oxygen, and chloride, which were prevenient biomolecules of M. umbellatum, 13 M. edule. 5 ## Edax for ggnps The GGNPs were further confirmed using EDAX spectrometry for the presence of gold with no other contaminants. The optical adsorption peak from was observed at nearly 4.60 keV, which is typical for the adsorption of gold nanocrystallites because of SPR. The current profile of EDAX of GGNPs of C. zizanioides showed strong gold atom signals around 4.60, 7.90, 9.65, and 13.63 keV. Similar peaks for GNPs synthesized from Trachy spermumammi and Papaver somniferum were observed by Vijayaraghavan et al, [bib_ref] One step green synthesis of silver nano/microparticles using extracts of Trachyspermum ammi..., Vijayaraghavan [/bib_ref] for GNPs synthesized from Jatropha curcas by From our previous results, similar peaks for GNPs synthesis from M. edule and M. umbellatum were observed. [bib_ref] Memecylon edule leaf extract mediated green synthesis of silver and gold nanoparticles, Elavazhagan [/bib_ref] [bib_ref] One-step green synthesis and characterization of leaf extract-mediated biocompatible silver and gold..., Arunachalam [/bib_ref] FTIR of SNPs and gNPs FTIR analysis was used to identify the possible biomolecules responsible for the reduction and capping on nanoparticle surfaces. In the present study, FTIR spectra of both the aqueous extract of C. zizanioides and synthesized SNPs and GNPs were recorded. FTIR measurements were carried out to identify the potential biomolecules in the C. zizanioides aqueous plant extract responsible for reducing the chloroaurate ions. It was noted the capping reagent responsible for the stability of the bioreduced GNPs involved the secondary amines. The size distribution and characterization of the GSNPs was further corroborated by FTIR, as shown in . The interaction of nanoparticles with phytochemicals of C. zizanioides showed intensive peaks at 2884, 1600, 1507, 1387, 1074, and 1335 cm −1 . Relative shifts in position and intensity distribution were confirmed with FTIR. This clearly shows that the oxidized polyphenols capped the surface of the nanoparticles and kept them stable for longer periods. The FTIR for dry gold nanopowder of C. zizanioides showed strong bands at 2832, 1731, 1612, and 1403 cm −1 . Similar peaks were also observed by other researchers. [bib_ref] Time-dependent effect in green synthesis of silver nanoparticles, Darroudi [/bib_ref] [bib_ref] Green synthesis and characterization of gelatin-based and sugar-reduced silver nanoparticles, Darroudi [/bib_ref] [bib_ref] Synthesis of silver nanoparticles using Dioscorea bulbifera tuber extract and evaluation of..., Ghosh [/bib_ref] ## Xrd analysis of gsnps and ggnps The X-ray structural diffraction pattern of the GSNPs and GGNPs produced using the leaf extracts was proved and confirmed by the characteristic peaks observed in the XRD images for silver and gold . The XRD submit your manuscript | www.dovepress.com Dovepress Dovepress pattern recorded for both SNPs and GNPs showed four intense peaks in the whole spectrum of 2θ values ranging from 20 to 80. The XRD analysis showed distinct diffraction peaks at 24.08°, 32.4°, 38.5°, 45.9°, 55.2°, 58.6°, 64.02°, and 76°, which indexed the planes 1 1 1, 2 0 0, and 2 2 0 of the cubic face-centered silver; whereas any peaks originating because of potential silver oxide interference could not be observed and it could not be confirmed that the entire silver nitrate was converted to SNPs. The average grain size of the SNPs formed in the bioreduction was determined using the Scherrer equation [formula] d = ×°( . )/ cos 0 9 180 λ β θπ(3) [/formula] and estimated as 105 nm. The XRD pattern clearly explains the crystalline structure of the SNPs formed by green biosynthesis. In comparing the XRD patterns of GSNPs and GGNPs, the content of the crystalline potassium chloride was significantly higher in the SNPs. The average crystallite size of the nanoparticles was estimated by the Scherrer equation and was found to be 105 nm. The GGNPs had phase peak positions corresponding to a highly crystalline potassium chloride phase. The XRD analysis showed higher distinct diffraction peaks at 38° and 44°. Also, no other gold containing compounds other than the metallic gold could be recognized from the XRD pattern. The crystallite sizes of the GGNPs were estimated by the Scherrer equation as 136 nm. The elemental peaks found in the EDAX study agreed with the XRD results. 43 # Conclusion Our results described a simple and eco-friendly timedependent method to biosynthesize green crystalline SNPs and GNPs in metal solution using medicinal plant extracts which does not need special physical conditions. Our research explained that C. zizanioides can be an excellent bioreductant and is an easily available, less expensive plant source for the synthesis of SNPs and GNPs. The C. zizanioides aqueous leaf extract is environmentally friendly and therefore this protocol could be used for the rapid production of SNPs. The size of GSNPs and GGNPs can be easily adjusted by varying the concentration of the leaf extract. The successful synthesis of SNPs and GNPs by reducing silver and gold ions using an aqueous extract of C. zizanioides leaves showed the reduction rate of silver ions was much faster than that of the gold ions. The water soluble compounds, like alkaloids and phytosterols, present in the C. zizanioides were mostly responsible for reducing silver and gold ions to nanosized silver and gold particles. The synthesized and well-studied green nanoparticles can be used for promising potential applications, including water purification, recording media, biosensing devices, nanoelectronics, and catalysis, as reported by Shukla et al. [bib_ref] Black pepper assisted biomimetic synthesis of silver nanoparticles, Shukla [/bib_ref] In one of our recent publications, we tissue engineered plant extracts by electrospinning, which makes it possible to combine the advantages of using these plant extracts in the form of nanofibrous mats to serve as skin graft substitutes or as nanofibrous wound dressings for the treatment of burns and wounds. [bib_ref] Tissue engineered plant extracts as nanofibrous wound dressing, Jin [/bib_ref] The synthesized SNPs and GNPs were well-capped and showed strong antibacterial activity (results not shown) which is very important for the aspects of its biomedical applications, such as a hydrogel dressing without any preservatives, which would be most efficient for cuts, new burns, and dry wounds. [bib_ref] Green synthesis of crystalline silver nanoparticles using indigofera aspalathoides -medicinal plant extract..., Arunachalam [/bib_ref] Other major applications that could be worthwhile are drug delivery, gene delivery, and biosensor applications where there is a direct contact of these nanoparticles with blood. [bib_ref] Green synthesis of gold nanoparticles with Zingiber off icinale extract: characterization and..., Kumar [/bib_ref] This eco-friendly method for SNP and GNP biosynthesis does not use any chemicals and thus has the potential to be exploited in biomedical applications and will play an important role in future optoelectronic and biomedical applications. In our recent studies, we have conferred the ability of the silver nano particles for preventing biofilm in urinary catheters. [bib_ref] Screening and characterisation of silver nanoparticles for the prevention of biofilm in..., Meenakumari [/bib_ref] [fig] Figure 1, Figure 2: Synthesis of silver and gold nanoparticles. Notes: (A) AgNO 3 ; (B) synthesized silver nanoparticles in brown color solution after 24 hours; (C) hAuCl 4 solution; (D) synthesized gold nanoparticles in ruby red color after 24 hours. Time dependent absorption spectra of silver nanoparticles after the bioreduction of silver in the aqueous extract of Chrysopogon zizanioides. [/fig] [fig] Figure 3: Time dependent absorption spectra of gold nanoparticles after the bioreduction with aqueous extract of Chrysopogon zizanioides. [/fig] [fig] Figure 4 200: nm EHT = 3.00 kV WD = 3.9 mm Signal A = InLens Date: 9 Aug 2012 Time: 13:19:49 Mag = 50.00 K X Scanning electron microscopy image of green silver nanoparticles synthesized by reduction of aqueous AgNO 3 ions using Chrysopogon zizanioides extract. Abbreviations: EHT, extra high tension; Mag, magnification; WD, working distance. submit your manuscript | www.dovepress.com Dovepress Dovepress [/fig] [fig] Figure 5: Scanning electron microscopy image of green gold nanoparticles synthesized by reducing aqueous AuCl 4 − ions using Chrysopogon zizanioides extract. Abbreviations: Mag, magnification; WD, working distance; ETD, Everhart-Thornley detector; Vac, vacuum; HV, high voltage; Det, detector. [/fig] [fig] Figure 6, Figure 7, Figure 8: Energy dispersive X-ray spectrum of silver (Ag) nanoparticles. submit your manuscript | www.dovepress.Energy dispersive X-ray spectrum of gold nanoparticles. Abbreviation: Au, gold. Fourier transform infrared spectroscopy spectrum of silver and gold nanoparticles. [/fig] [fig] Figure 10 XFigure 9: -ray diffraction spectrum of green-synthesized gold nanoparticles. X-ray diffraction spectrum of green-synthesized silver nanoparticles. submit your manuscript | www.dovepress.com Dovepress Dovepress [/fig] [table] Table 1: Preliminary phytochemical investigation of aqueous extract of C. zizanioides [/table]

Neoatherosclerosis　― Long-Term Assessment of Bioresorbable Vascular Scaffold ― only device available for clinical use in Japan. In 2017, the Absorb BVS was withdrawn from the world market because of unfavorable mid-term results, contrary to expectations. The concept of a resorbable stent, however, is still very attractive to cardiologists, especially PCI operators, and we have an obligation to learn from failed devices and to elucidate what will happen in the coronary artery after BVS implantation, in order to identify future bioresorbable materials and improve treatment for patients with atherosclerotic coronary artery disease. The goal of this review is therefore to summarize clinical data available for Absorb BVS to understand the characteristics of the pathophysiology of the scaffolded vessel, based mainly on our previous study, 7 including individual cases involving Absorb BVS from Shonan Kamakura General Hospital.Absorb BVS Clinical DataAbsorb BVS was a drug-eluting BRS that consisted of 157-μm-thick struts with a poly-L-lactide backbone and a poly-D,L-lactide coating with the antiproliferative drug everolimus. 8 To date, the Absorb BVS is the most investigated BRS. Several multicenter, prospective, clinical trials compared the Absorb BVS with the Cobalt-chromium everolimus-eluting stent (CoCr-EES) Xience (Abbott Although metallic stents improved the safety and efficacy of percutaneous coronary intervention (PCI), even the latest generation of drug-eluting stents (DES) is still limited by several factors. The limitations of DES are mainly related to the permanent metallic caging in vessel, chronic inflammatory response to the polymer and adverse effects of antiproliferative drug on endothelial tissue, leading to impaired physiological vasomotor response and late stent-related adverse events such as stent thrombosis and neoatherosclerosis. Bioresorbable vascular scaffold (Absorb BVS; Abbott Vascular) was designed to overcome these drawbacks of DES by disappearing from the vessel wall. Absorb BVS, however, was withdrawn from the world market because of increased incidence of scaffold thrombosis compared with DES. Importantly, only very limited long-term post-BVS implantation data are available, especially with regard to neoatherosclerosis, which can lead to very late adverse events even after resorption of the scaffold. Therefore, the goal of this review was to highlight the mid to long term clinical outcomes published to date, and to describe the features of the intimal healing process and neoatherosclerosis in the 5 years following Absorb BVS implantation, mainly based on our previous study. This may provide important information on the pathophysiology of the scaffolded vessel for clinicians, and promote identification of future bioresorbable materials for PCI that will minimize the stimulus for neoatherosclerosis. S ince the introduction of metallic stents into clinical use, these devices have substantially improved the quality of coronary revascularization and have continued evolving. [bib_ref] A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment..., Fischman [/bib_ref] Drug-eluting stents (DES) led to a significant reduction in the incidence of restenosis in comparison with bare-metal stents, and improved the overall safety and efficacy of percutaneous coronary intervention (PCI). 2,3 Permanent metallic stents in a coronary artery, however, play a role in the occurrence of neoatherosclerosis, that is, a de novo atherosclerotic plaque overlying the stent, and could strongly contribute to late catch-up phenomenon and very late stent thrombosis. [bib_ref] From metallic cages to transient bioresorbable scaffolds: Change in paradigm of coronary..., Serruys [/bib_ref] Neoatherosclerosis is histologically characterized by an accumulation of lipid-laden foamy macrophages with or without necrotic core formation and/or calcification in the neointima and is also an accelerated process compared with atherosclerosis in native vessels. 5, [bib_ref] Pathophysiology of native coronary, vein graft, and in-stent atherosclerosis, Yahagi [/bib_ref] For the purpose of overcoming these drawbacks of metallic DES, bioresorbable scaffolds (BRS) were introduced into clinical practice. They are antiproliferative DES-like devices that are designed to provide mechanical scaffolding during the first year following implantation, and are completely absorbed and disappear from the vessel after several years. The bioresorbable vascular scaffold (Absorb BVS Rev.1.1; Abbott Vascular) is the most comprehensively characterized, and was the 544 MORIYAMA N et al. coronary artery, scaffold thrombosis is mainly caused by problems during implantation, such as suboptimal vessel sizing to scaffold, over-or under-expansion and malapposition of Absorb BVS. 20- [bib_ref] What determines long-term outcomes using fully bioresorbable scaffolds: The device, the operator..., Yamaji [/bib_ref] These factors have been reported as the risk factors for scaffold discontinuity, late malapposition and uncovered struts leading to late or very late scaffold thrombosis. [bib_ref] Two-year clinical, angiographic, and serial optical coherence tomographic follow-up after implantation of..., Onuma [/bib_ref] With the aim of overcoming early and late events deemed to be related to procedural issues, the concept of PSP (optimal pre-dilatation, vessel and device sizing, and aggressive post-dilatation) was advocated and has been evaluated. [bib_ref] Clinical outcomes prior to and following complete everolimus-eluting bioresorbable scaffold resorption: Five-year..., Kereiakes [/bib_ref] To date, the results of PSP have been controversial. 16,21 Therefore, the question of whether technical features can have positive effects on BRS device outcomes remains inconclusive. In contrast, long-term 5-year follow-up data on Absorb BVS are still scarce. Our group reported on the occurrence and progression of in-scaffold neoatherosclerosis with luminal narrowing in the 5 years after Absorb BVS 1.1 implantation. 7 Neoatherosclerosis, by its nature, can lead to very late adverse events and may affect late prognosis in patients who have undergone Absorb BVS implantation. Therefore, from the viewpoint of neoatherosclerosis, it is worth re-reviewing the Absorb BVS data at this stage. ## Neoatherosclerosis of absorb bvs Neoatherosclerosis has recently been defined as a novel disease manifestation of atherosclerosis in the coronary artery in the neointima following stent implantation. [bib_ref] Pathophysiology of native coronary, vein graft, and in-stent atherosclerosis, Yahagi [/bib_ref] Histologically, neoatherosclerosis is characterized by an accumulation of lipid-laden foamy macrophages with or without necrotic core formation and/or calcification in the Vascular). 9-14 Although the rates of cardiac death and clinically driven target lesion revascularization (TLR) for the Absorb BVS were similar to those for the CoCr-EES during 1-year follow-up, a numerically higher incidence of definite or probable stent thrombosis was reported from the ABSORB trials, EVERBIO II and TROFI II trials. 10- [bib_ref] A bioresorbable everolimus-eluting scaffold versus a metallic everolimus-eluting stent for ischaemic heart..., Serruys [/bib_ref] In a meta-analysis of these trials, involving 3,738 patients, patients who received the Absorb BVS had a significantly higher risk of scaffold thrombosis than those with CoCr-EES (1.3% vs. 0.5%; P=0.05). [bib_ref] Everolimus-eluting bioresorbable vascular scaffolds versus everolimus-eluting metallic stents: A meta-analysis of randomised..., Cassese [/bib_ref] In addition, the AIDA trial noted a significantly higher rate of scaffold thrombosis (definite or probable) for the Absorb BVS than for the CoCr-EES through 2 years of follow-up (cumulative event rates, 3.5% vs. 0.9%; hazard ratio [HR], 3.87; 95% CI: 1.78-8.42, P<0.001). [bib_ref] Bioresorbable scaffolds versus metallic stents in routine PCI, Wykrzykowska [/bib_ref] Furthermore, in a larger-scale metaanalysis the incidence of device-oriented outcomes (cardiac death, target vessel-related myocardial infarction [MI] and ischemia-driven TLR) and of scaffold thrombosis were significantly higher for the Absorb BVS than for the CoCr-EES. [bib_ref] 2-year outcomes with the Absorb bioresorbable scaffold for Neoatherosclerosis After BVS Implantation..., Ali [/bib_ref] [bib_ref] Clinical outcomes prior to and following complete everolimus-eluting bioresorbable scaffold resorption: Five-year..., Kereiakes [/bib_ref] Several studies have determined the potential risk factors of scaffold thrombosis using intra-coronary imaging, especially optical coherence tomography (OCT). In the early to mid-phase following Absorb BVS implantation, in other words, while the scaffold is still visible inside the treated calcification: 28% vs. 94%, P<0.01; lipid-laden plaque: 17% vs. 61%, P<0.01; TCFA: 0% vs. 22%, P<0.02, respectively) on OCT follow-up. [bib_ref] Neoatherosclerosis 5 years after bioresorbable vascular scaffold implantation, Moriyama [/bib_ref] Here, we discuss each of the major findings of neoatherosclerosis and vasomotor response following Absorb BVS implantation. Pathologically, calcification occurs during cell death or when localized to peri-strut regions involving fibrin, especially after DES implantation. 5,33 Progression of calcification is one of the main findings suggestive of neoatherosclerosis following metallic stent implantation. Morphological characteristics vary widely from microcalcification to fragmented or sheet calcification. [bib_ref] Neoatherosclerosis: Overview of histopathologic findings and implications for intravascular imaging assessment, Otsuka [/bib_ref] In our study, calcification was the most frequent component of neoatherosclerosis in the in-scaffold segment rather than in the out-scaffold segment 5 mm adjacent to the edge of the neointima. [bib_ref] Neoatherosclerosis from a pathologist's point of view, Romero [/bib_ref] OCT enables the best characterization of neointimal tissue within stents due to its superior axial resolution . For quantitative OCT analysis, neoatherosclerosis is defined as lipid-laden plaque including thin-cap fibroatheroma (TCFA) with or without intimal rupture and/or thrombus, and/or calcific plaque with or without neovascularization and/or macrophages. [bib_ref] Mechanisms of very late drug-eluting stent thrombosis assessed by optical coherence tomography, Taniwaki [/bib_ref] Typical features of neoatherosclerosis at 5 years following Absorb BVS are shown in [fig_ref] Figure 1: Typical optical coherence tomography findings of neoatherosclerosis at 5 years after Absorb... [/fig_ref]. Data regarding neoatherosclerosis after Absorb BVS implantation are still very limited . 7,26-32 To the best of our knowledge, our previous study first and comprehensively described the incidence of neoatherosclerosis at 5 years following Absorb BVS rev.1.1 implantation on serial imaging. [bib_ref] Neoatherosclerosis 5 years after bioresorbable vascular scaffold implantation, Moriyama [/bib_ref] Neoatherosclerosis was more prevalent at 5 years than at 1 year (1 year vs. 5 years, [fig_ref] Figure 3: In-scaffold thin-cap fibroatheroma [/fig_ref]. [bib_ref] Neoatherosclerosis 5 years after bioresorbable vascular scaffold implantation, Moriyama [/bib_ref] A previous study reported on the time course of neoatherosclerosis after DES implantation. [bib_ref] Optical coherence tomographic observation of in-stent neoatherosclerosis in lesions with more than..., Lee [/bib_ref] At 48-month followup, 75% of all lesions with 50% diameter stenosis had lipid plaque and neovascularization. [bib_ref] Optical coherence tomographic observation of in-stent neoatherosclerosis in lesions with more than..., Lee [/bib_ref] In-stent lipid-laden neoatherosclerosis is frequently observed in patients with very late stent thrombosis (VLST) following revascularization using metallic stent. Especially, in-stent plaque rupture was identified as the dominant pathological mechanism causing VLST in 30% of patients. [bib_ref] Optical coherence tomographic observation of in-stent neoatherosclerosis in lesions with more than..., Lee [/bib_ref] In a previous analysis of patients with VLST who had undergone OCT, Taniwaki et al reported that neoatherosclerosis (observed in 27.6% of patients) was a causative factor in the occurrence of VLST following DES implantation. [bib_ref] Mechanisms of very late drug-eluting stent thrombosis assessed by optical coherence tomography, Taniwaki [/bib_ref] In another OCT analysis, neoatherosclerosis was also found to be the most frequent cause of VLST (34.7%), followed by malapposition (33.7%) and uncovered strut (24.5%). [bib_ref] Characteristics of earlier versus delayed presentation of very late drug-eluting stent thrombosis:..., Lee [/bib_ref] In terms of VLST following BVS implantation, Yamaji et al reported on the frequency of the underlying mechanism of VLST in the INVEST registry. [bib_ref] Mechanisms of very late bioresorbable scaffold thrombosis: The INVEST Registry, Yamaji [/bib_ref] Neoatherosclerosis was observed as the underlying mechanism of VLST in 18.4% of lesions at 26.9±11.3 months after BVS implantation. In a median 4.7-year follow-up, scaffold discontinuity was the most common mechanism underlying VLST (42%), followed by strut malapposition (18%). [bib_ref] Mechanisms of very late bioresorbable scaffold thrombosis: The INVEST Registry, Yamaji [/bib_ref] Theoretically, scaffold is completely absorbed by 5 years, with the exception of a rare case of persistent scaffold at >5 years. 38 Therefore, BVS. 7 On cross-sectional (CS) quantitative analysis (CS at 1 year, n=4,777; at 5 years, n=4,236), the proportion of calcification significantly increased from 0.4% (CS, n=16) at 1 year to 23.1% (CS, n=980) at 5 years (P<0.01) in the in-scaffold segments, along with a significant increase in its arc and area (arc, 42.2±15.6° vs. 68.4±29.5°, P<0.01; area, 0.24±0.18 mm 2 vs. 0.55±0.27 mm 2 , P<0.01). A typical case of calcium proliferation at 5 years is presented in [fig_ref] Figure 2: In-scaffold calcium growth at 5 years, in a 73-year-old woman with angina... [/fig_ref]. Moreover, serial coronary computed tomography analysis confirmed significant growth of calcification in the scaffolded vessel. The vasomotor response to nitroglycerine (NTG) in the in-scaffold segment was impaired compared with that in the out-scaffold segment, 7 indirectly supporting the existence of neoatherosclerosis, especially in advanced coronary calcification, as previously reported. [bib_ref] Neoatherosclerosis 5 years after bioresorbable vascular scaffold implantation, Moriyama [/bib_ref] Histological studies have clearly shown growth of calcification in preclinical animal model from 3 months to 42 months after implantation of the Absorb BVS. [bib_ref] Fully bioresorbable vascular scaffolds: Lessons learned and future directions, Jinnouchi [/bib_ref] This could support the results of our reports. 7 In contrast, Zeng et al noted contradictory results, in which calcium growth was similar between the in-and out-scaffold segments from baseline to 5 years on intravascular ultrasound echogenicity analysis (∆calcium area: in-scaffold, ∆=0.21 mm 2 ; out-scaffold, ∆=0.22 mm 2 ; P=0.881). [bib_ref] Serial assessment of tissue precursors and progression of coronary calcification analyzed by..., Zeng [/bib_ref] Also, they stated that new calcified plaque should not be confused with neoatherosclerosis. [bib_ref] Serial assessment of tissue precursors and progression of coronary calcification analyzed by..., Zeng [/bib_ref] Moreover, the phenomenon of "recapping of the underlying plaque" transforms the unstable plaque phenotype to a stable one. First, these articles by us and Zeng et al have limitations because of the technology used to assess calcification. This might explain the discrepancies between these reports. Second, we confirmed that calcification exists together with vulnerable plaque, such as lipid including TCFA and neovascularization. Hence, this finding may be associated with the failure to recap the underlying plaque ## 548 MORIYAMA N et al. # Disclosures The authors declare no conflicts of interest. after resorption of the scaffold, only neoatherosclerosis would be a potential mechanism of VLST after BVS. Further studies with longer follow-up are warranted to investigate this major concern. After metallic stent implantation, vasomotor response at the stented segment is obviously absent. Absorb BVS was developed to overcome this absence of vasomotion at the stented segment. The ability of the artery to respond to vasodilator stimuli may play an important role in physiological recovery. Small studies of patients with Absorb BVS, however, have produced mixed results. 39, [bib_ref] Vasomotor response to nitroglycerine over 5 years follow-up after everolimus-eluting bioresorbable scaffold..., Dudek [/bib_ref] Gomez-Lara et al presented data on the 3-year coronary artery assessment of the infarcted-related artery vasomotion treated with Absorb BVS or CoCr-EES. [bib_ref] Long-term coronary functional assessment of the infarct-related artery treated with everolimuseluting bioresorbable..., Gomez-Lara [/bib_ref] Vasomotion was assessed using acetylcholine (endothelium-dependent reaction) or NTG (endothelium-independent reaction) injection. At 3 years, 60% of patients had paradoxical vasoconstriction in response to acetylcholine, and this was significantly more often observed in the Absorb BVS group than in the CoCr-EES group (77.8% vs. 25.0%, P=0.008). NTG-mediated vasodilatation was either unchanged or preserved with the Absorb BVS. [bib_ref] Long-term coronary functional assessment of the infarct-related artery treated with everolimuseluting bioresorbable..., Gomez-Lara [/bib_ref] This suggests that partial resorption of the scaffold could allow more artery motion with the Absorb BVS than with the metallic stent. In terms of 5-year assessment, two reports investigated vasomotor response after NTG injection. 7, [bib_ref] Vasomotor response to nitroglycerine over 5 years follow-up after everolimus-eluting bioresorbable scaffold..., Dudek [/bib_ref] Dudek et al confirmed a numerical increase in vasomotor response to NTG from 2 to 5 years after Absorb BVS on quantitative coronary angiography (QCA) according to mean lumen diameter (mean inscaffold lumen diameter: 2 years, 0.03±0.09 mm; 3 years, 0.05±0.12 mm; 5 years, 0.07±0.08 mm, P=0.40). The degree of vasomotor response in the in-scaffold segment, however, was lower compared with the adjacent segments, [bib_ref] Vasomotor response to nitroglycerine over 5 years follow-up after everolimus-eluting bioresorbable scaffold..., Dudek [/bib_ref] and our data also support this: we confirmed that a significantly smaller change was observed for the in-scaffold than for the out-scaffold segment after NTG injection at 5 years after Absorb BVS implantation on QCA according to mean lumen diameter (∆ in-scaffold, +0.009±0.012 mm vs. ∆ out-scaffold, +0.14±0.14 mm, P<0.01), which could further support the existence of neoatherosclerosis. 7 Moreover, we encountered one case of in-scaffold vasospastic angina (VSA) deemed to be based on neoatherosclerosis at 5 year after BVS implantation [fig_ref] Figure 4: In-scaffold vasospastic angina at 5 years, in a 78-year-old man who had... [/fig_ref] ; Shonan Kamakura General Hospital, N.M., K.S., Y.T., and S.S., unpublished data 2019). Although the pathogenesis of VSA has not been fully elucidated, endothelial dysfunction based on atherosclerosis has been proposed as one of the major mechanisms of VSA. [bib_ref] Acetylcholine-provoked coronary spasm at site of significant organic stenosis predicts poor prognosis..., Ishii [/bib_ref] Hence, vasomotor response in the late phase after BVS implantation should be investigated as with neoatherosclerosis in the future study of BRS. Here, we have summarized the clinical and imaging data for the Absorb BVS [fig_ref] Figure 5: Summary of clinical and imaging data following Absorb bioresorbable vascular scaffold [/fig_ref]. We found that in-scaffold neoatherosclerosis mainly consists of lipid including TCFA, calcification, and neovascularization at 5 years after BVS resorption. The neointima of the BVS was involved in vulnerable plaque in the late phase. [bib_ref] Neoatherosclerosis 5 years after bioresorbable vascular scaffold implantation, Moriyama [/bib_ref] The development of neoatherosclerosis is a conceptually unanticipated finding after BVS deployment. Further study is needed to identify biomaterial for PCI that will minimize the stimulus for neoatherosclerosis with advances in systemic pharmacotherapy to prevent atherosclerosis. Moreover, careful long-term follow-up beyond 5 years after BVS implantation is needed, and larger studies are warranted to assess the association between neoatherosclerosis and long-term clinical events after BVS implantation. [fig] Figure 1: Typical optical coherence tomography findings of neoatherosclerosis at 5 years after Absorb bioresorbable vascular scaffold. (A) Lipid-laden intima is defined as a diffusely bordered, signal-poor region with overlying signal-rich bands in the intima. (B) Plaque rupture. (C) Thin-cap fibroatheroma-containing intima: fibrous cap thickness ≤65 μm at the thinnest segment and an angle of lipid tissue ≥180° with thrombus. (D,E) Calcification as a well-delineated, signal-poor region with sharp borders (white arrow). (F) Neovascularization as the presence of signal-poor holes or tubular structures with a diameter of 50-300 μm that are not connected to the vessel lumen (box). (G) Macrophage infiltration as a bright spot with a high signal variance from surrounding tissue (white arrowhead). Neoatherosclerosis After BVS Implantation [/fig] [fig] Figure 2: In-scaffold calcium growth at 5 years, in a 73-year-old woman with angina pectoris. Two Absorb bioresorbable vascular scaffolds rev.1.1 3.0×18 mm were implanted at the distal right coronary artery with sufficient scaffold expansion. At 1-year follow-up, optical coherence tomography (OCT) showed good expansion of scaffold without any malapposition. At 5 years, scaffolds were not visible on OCT. Calcium growth (white arrowhead) was observed in neointimal tissue (≤200 μm from the end-luminal border). CAG, coronary angiography; F/U, follow-up; PCI, percutaneous coronary intervention.546MORIYAMA N et al. [/fig] [fig] Figure 3: In-scaffold thin-cap fibroatheroma (TCFA) at 5 years, in a 65-year-old man with angina pectoris. Absorb bioresorbable vascular scaffold (BVS) rev.1.1 3.0×28 mm was implanted at the proximal left anterior descending coronary artery with sufficient expansion of scaffold. At 1-year follow-up, optical coherence tomography (OCT) showed sufficient expansion of BVS and intimalization over scaffold. At 5 years, although the scaffolds were completely absorbed, TCFA was identified with moderate luminal narrowing on OCT. CAG, coronary angiography; F/U, follow-up; PCI, percutaneous coronary intervention. [/fig] [fig] Figure 4: In-scaffold vasospastic angina at 5 years, in a 78-year-old man who had undergone percutaneous coronary intervention with bioresorbable vascular scaffold (BVS) 3.0×18 mm in significant organic stenosis of the proximal left anterior descending coronary artery. (A-C) Coronary angiography (CAG) and optical coherence tomography (OCT) showed excellent results. At 5 years after the index procedure, he was referred from the other hospital because of frequent atypical chest pressure. CAG indicated significant stenosis in the in-scaffold segment (red arrowhead). After nitroglycerine (NTG) injection, the lesion was fully expanded with resolution of symptoms. (E) On detailed observation, OCT confirmed neoatherosclerosis including lipid-plaque with luminal narrowing. (D,F) Proximal and distal edge of BVS. This patient was diagnosed with vasospastic angina, and an oral calcium channel blocker improved the chest symptom. Neoatherosclerosis After BVS Implantation after Absorb BVS rev.1.1 implantation. On CS-quantitative analysis, the proportion of lipidladen plaque significantly increased from 0.9% (CS, n=36 out of 4,777) at 1 year to 11.6% (CS, n=492 out of 4,236) at 5 years (P<0.01) in the in-scaffold segments, along with a significant increase in its arc and a decrease of cap thickness (arc, 38.7±16.8° vs. 129.1±58.4°, P<0.01; cap thickness, 172.1±24.1 μm vs. 91.8±46.9 μm, P<0.01; [/fig] [fig] Figure 5: Summary of clinical and imaging data following Absorb bioresorbable vascular scaffold (BVS) implantation. CoCr-EES, Cobalt-chromium everolimus-eluting stent; PSP, pre-dilatation, sizing and post-dilatation; TLR, target lesion revascularization. [/fig] [table] Table: Reports of Neoatherosclerosis Following Absorb BVS Implantation [/table]

Association of homocysteine with carotid-femoral pulse wave velocity in a southern Chinese population This study aimed to investigate whether plasma homocysteine levels were associated with carotid-femoral pulse wave velocity (cfPWV), a golden standard of arterial stiffness, in a population from southern China. A crosssectional study was conducted on 713 patients admitted to the First Affiliated Hospital of Fujian Medical University from February 2016 to August 2017. They were divided into four groups based on gender-specific quartile of homocysteine levels. Age, cfPWV, uric acid levels, and percentage of hypertension increased with ascending quartiles. The duration of hypertension and systolic blood pressure were higher in the highest quartile than in the lowest quartile. Pearson's correlation analysis and multivariate regression showed a correlation of homocysteine levels with cfPWV. A nearly twofold increased risk of cfPWV ≥10 m/s was observed in the highest quartile compared with the lowest quartile (in the highest quartile: odds ratio = 2.917, 95% confidence interval: 1.635-5.202, P < 0.001). After stratification, this correlation was present in both sexes, in patients aged over 65 years, and those with hypertension. The plasma homocysteine levels were independently associated with cfPWV in the population from southern China, especially in the elderly and those with hypertension. # Introduction Arterial stiffness is characterized by loss of elastin fibers and accumulation of stiffer collagen fibers in the media of large arteries, reflecting the result of arterial wall remodeling. Generally, arterial stiffness progresses with aging and now considered as a hallmark of vascular aging. Patients with arterial stiffness are at high risk of cardiovascular disease and declined cognition. Arterial stiffness may be assessed by different methods, of which the measurement of carotid-femoral pulse wave velocity (cfPWV) is proved to be simple, validated, and reproducible. cfPWV is well recognized as a golden standard for assessing arterial stiffness. Increased pulse wave velocity (PWV) has been shown to be associated with traditional cardiovascular risk factors, such as aging, hypertension, and diabetes. Furthermore, arterial stiffness progresses more rapidly with increasing numbers of traditional risk factors. Currently, some risk factors not traditionally recognized have also been linked to arterial stiffness. Homocysteine, a sulfur-containing amino acid and an intermediate product of methionine metabolism, is a nontraditional risk factor for cardiovascular disease. Deficiency of metabolic enzymes and co-factors of homocysteine, such as folic acid and vitamin B, may lead to increased homocysteine levels in humans and mice. In the last decades, the relationship between homocysteine levels and arterial stiffness has been repeatedly investigated; however, the results are controversial. This inconsistency may be due to the differences in studied populations and methods used for assessing arterial stiffness. A positive association of homocysteine with PWV has been reported in a population from Beijing in the northern part of China. However, the existence of this relationship in the general population of China has not been verified. This study aimed to investigate the relationship between plasma homocysteine levels and cfPWV in a population from southern China. # Results The clinical characteristics of all 713 patients based on the gender-specific quartile of homocysteine levels are shown in. The mean age of patients was 59 ± 12 years, 438 (61.4%) were male, and 275 (38.6%) were female. The plasma homocysteine level ranged from 0.5 to 50 μmol/L, and the median was 11.2 μmol/L in male patients and 8.70 μmol/L in female patients. From the lowest to the highest quartile of homocysteine levels, the median and range were as follows: for male patients, the lowest quartile was 8.33 (0.72-9.35) μmol/L, second quartile 10.23 μmol/L, third quartile 12.08 μmol/L, and highest quartile 15.99 (13.73-50.00) μmol/L; for female patients, the lowest quartile was 6.19 (0.50-7.34) μmol/L, second quartile 8.00 μmol/L, third quartile 9.61 (8.70-11.12) μmol/L, and highest quartile 13.00 μmol/L. Of all the patients, 489 (68.6%) were diagnosed with hypertension and 166 (23.3%) with diabetes. The patients in a higher quartile of homocysteine levels were older. Hypertension was frequently observed and uric acid levels increased in the higher quartile. The duration of hypertension [6 (5-13) vs 1 (0-10) years], systolic blood pressure [(138.0 ± 21.4) vs (129.9 ± 16.8) mm Hg] were higher in the highest quartile than in the lowest quartile (P < 0.016). With ascending quartiles of homocysteine levels, a trend of increasing cfPWV was observed (8.73 ± 1.61, 8.88 ± 1.49, 9.33 ± 1.69, and 10.40 ± 4.82 m/s, P < 0.001). No significant betweengroup differences were found in diastolic blood pressure, fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), estimated glomerular filtration rate (eGFR), percentage of diabetes, and use of medication. Percentages of cfPWV ≥10 m/s in different quartiles of homocysteine levels are demonstrated in. cfPWV ≥10 m/s was more frequently present in the higher quartile. A striking increase in the percentage of cfPWV ≥10 m/s was found from the third quartile to the highest quartile (17.2%, 18.9%, 28.7%, and 49.1%, χtrend = 54.097, P < 0.001). When analyzed as a continuous variable, the logtransformed homocysteine level correlated with cfPWV (r = 0.275, P < 0.001)and, even after adjusting for age, systolic blood pressure, heart rate, Lg triglyceride, and uric acid level (β = 0.147, adjusted r = 0.175, P = 0.001). Standardized regression equation: cfPWV = -3.638 + 0.429 × Age + 0.283 × Systolic blood pressure + 0.169 × Heart rate + 0.147 × Lg homocysteine + 0.124 × Lg triglyceride + 0.041 × Uric acid; adjusted R 2 = 0.459. Increasing odds ratios (ORs) for cfPWV ≥10 m/s were displayed from the lowest quartile to the highest quartile of homocysteine levels. The graded association remained statistically significant after adjusting for demographic data, anthropometric data, laboratory indicators, and use of medication. A nearly twofold increased risk of cfPWV ≥10 m/s was observed in the highest quartile compared with the lowest quartile (highest quartile: OR = 2.917, 95% CI: 1.635-5.202, P < 0.001). Before further stratification, interaction analyses were performed. Age and presence of hypertension were found to significantly modify the association of the quartile of homocysteine levels with cfPWV ≥10 m/s. After gender stratification, a significant association between the quartile of homocysteine levels and cfPWV ≥10 m/s was observed in both male and female patients. This association appeared to be more prominent in male patients. After age stratification, a significant association was present only in patients older than 65 years, but not those younger than 65 years. The elderly in the highest quartile of homocysteine levels had a 6.6-fold increased risk of cfPWV ≥10 m/s compared with those in the lowest quartile (highest quartile: OR = 7.605, 95% CI: 2.684-21.550, P < 0.001). After stratification of hypertension, a significant association persisted in patients with hypertension. Patients in the highest quartile had an almost 2.3-fold increased risk of cfPWV ≥10 m/s compared with those in the lowest quartile (highest quartile: OR = 3.266, 95% CI: 1.671-6.383, P = 0.001). However, the association became weak in patients without hypertension. # Discussion In this study, the homocysteine levels were shown to be independently associated with cfPWV, the golden standard of arterial stiffness. Furthermore, this association was pronounced mainly in the elderly and patients with hypertension. cfPWV has been recognized as an independent predictor of cardiovascular morbidity and mortality. Increasing evidence indicates that cfPWV may be used as a parameter for better individualized treatment. Age, blood pressure, and blood pressure-related parameters are associated with PWV, as confirmed in a previous study. In addition, some nontraditional cardiovascular risk factors, such as uric acid levels, albuminuria, and homocysteine levels, have also been reported to contribute to increased PWV. In the last decades, homocysteine was found to be associated with PWV in western populations. Recently, the association was also shown in a population from Beijing in the northern part of China. However, PWV and homocysteine levels were known to vary with ethnicity and lifestyle. Dietary profile and habit in southern Chinese are different from those in northern Chinese. Generally, the dietary folic acid level is higher in southern Chinese than in northern ChineseTherefore, it may be speculated that homocysteine levels are lower in southern Chinese, as verified in the present study. The median of plasma homocysteine levels was 11.2 μmol/L in male patients and 8.70 μmol/L in female patients, which was lower than that in the population from Beijing. However, for these lower levels of plasma homocysteine, the relationship between homocysteine levels and PWV remained unchanged. Increased homocysteine level, termed hyperhomocysteinemia, is defined as plasma homo-cysteine levels greater than 15 μmol/L. As a matter of fact, when plasma homocysteine levels are higher than 10 μmol/L, a dose-response relationship exists between homocysteine levels and cardiovascular disease. Also, a 1.6-fold increase in the risk of cardiovascular disease has been reported for every 5 μmol/L increment of the plasma homocysteine level. The present study identified a positive association between homocysteine levels and cfPWV, and a marked increase in the percentage of cfPWV ≥10 m/s was observed at the homocysteine level of around 12 μmo/L in male patients and 9.5 μmo/L in female patients. This level was below the threshold of hyperhomocysteinemia. In this study, patients in the higher quartile of homocysteine levels were found to have more concomitant risk factors for cardiovascular disease. However, after adjusting for these traditional cardiovascular risk factors, the association remained unchanged. Hence, it was suggested that the association of plasma homocysteine levels with arterial stiffness was independent. Previous studies examined the relationship between plasma homocysteine levels and cardiovascular disease. However, the conclusions remain controversial. Bortolotto et al. reported that cfPWV was higher in patients with higher homocysteine levels. In contrast, Nakhai-Pour et al. found no independent association of homocysteine levels with PWV. Even in two samples from the Framingham Heart Study, Levy et al. and Lieb et al. drew different conclusions about the relationship between these two parameters. This inconsistency might be due to the different characteristics of studied patients. The present study showed a positive association of plasma homocysteine levels with cfPWV only in patients older than 65 years and those with hypertension, which was consistent with some previous studies reporting a positive association only among patients with greater risks of cardiovascular disease, such as hypertension, diabetes, or chronic kidney disease, or elderly patients. However, the mechanism underlying the interaction between homocysteine levels and other cardiovascular risk factors has not been fully clarified. Nevertheless, all these cardiovascular risk factors, including age, may lead to endothelial dysfunction, and the damage may make the arterial wall more susceptible to the deleterious effect of homocysteine. This may explain a positive association of plasma homocysteine levels with cfPWV only in patients older than 65 years and those with hypertension. Although the positive association of plasma homocysteine levels with arterial stiffness was revealed, available evidence regarding the benefit of homocysteine-lowering therapies, such as supplementation of folic acid or vitamin B12, is inconsistent. The B-Vitamins for the Prevention of Osteoporotic Fractures study demonstrated that a 2-year supplementation of vitamin B12 and folic acid in elderly patients with hyperhomocysteinemia had no significant benefit on arterial stiffness. In contrast, a Chinese study found that the combined use of folic acid and antihypertensive agents, compared with antihypertensive agents alone, might significantly reduce the risk of first stroke in patients with hypertension. The benefit of homocysteine-lowering therapies against cardiovascular disease needs to be confirmed. The present study did not involve the use of homocysteinelowering therapy; however, arterial stiffening was known to have very limited reversibility. Therefore, it is not surprising that short-term homocysteine-lowering therapies yielded null results in the elderly. The greatest benefit of homocysteine-lowering therapies can be achieved in patients with no sign of arterial stiffness, but with multiple concomitant cardiovascular risk factors. A wide age range and a higher proportion of hypertension were unique aspects of this study, facilitating the stratified analysis. However, several potential concerns and limitations are worth mentioning. First, the sample size was relatively small. Second, no causal relationship could be determined because of the cross-sectional design of the study. Third, the population comprised inpatients, not community-based residents, thus decreasing the generalizability of the results. Finally, all patients were from Fujian province, and hence the conclusions drawn from this study could not be generalized to other populations. A prospective, long-term interventional study is needed to investigate the preventive effect of homocysteine-lowering therapy on the progression of arterial stiffness. In conclusion, an independent relationship existed between homocysteine levels and cfPWV. The association was more pronounced in patients older than 65 years and those with hypertension. This study provided evidence for the association of plasma homocysteine levels with arterial stiffness in a population from southern China. # Materials and methods ## Patients This was a single-center, cross-sectional, observational study. # Ethics statement The procedures were in accordance with the Helsinki Declaration, and the protocol was approved by the Ethical Committee of the First Affiliated Hospital of Fujian Medical University. Informed consent was obtained from all patients. A specific protocol was established before data collection, and trained physicians were responsible for the procedure. Patients were recruited from the inpatients consecutively admitted to the general medicine department of the First Affiliated Hospital of Fujian Medical University from February 1, 2016, to August 31, 2017. The reason for admission was to evaluate the patients as follows: routine health examination, or screening of hypertension-related or diabetes-related target organ damage, or treatment of coronary heart disease, peptic ulcer, Alzheimer disease, renal cyst, and so on. A total of 837 patients were included, of whom, 124 patients were excluded due to myocardial diseases, valvular heart disease, atrial fibrillation, serious arrhythmias, a history of acute myocardial infarction, stroke within the last 6 months, peripheral artery diseases, carotid artery occlusion or carotid sinus syndrome, serum creatinine (Scr) >2.5 mg/dL, chronic liver failure, autoimmune diseases, active malignancy, acute infection diseases, connective tissue diseases, use of folic acid and vitamin B, or pregnancy. Finally, 713 patients were enrolled in this study. ## Clinical data As described in a previous study, all patients were interviewed on admission to the hospital, and information was collected regarding age, gender, cigarette smoking and drinking habits, family history, medical history of hypertension and diabetes, use of medications, and so on. Current smoking was defined as consuming at least one cigarette per day for at least 6 months. According to the European Society of Cardiology (ESC)/European Society of Hypertension (ESH) guidelines for managing arterial hypertension, hypertension was defined as systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg and/or taking antihypertensive medications. Diabetes was defined as taking antihyperglycemic medications or establishing a new diagnosis of diabetes. The diagnosis of diabetes was based on the criteria recommended by the Chinese Diabetes Society in 2017. Height and body weight were recorded for patients in light clothes and without shoes. Height was recorded to the nearest 0.5 cm and body weight to 0.1 kg. Body mass index was calculated using the following formula: body mass index = body weight (kg)/height 2 (m 2 ). After patients rested for at least 5 min in the sitting position, the heart rate was taken and the blood pressure was measured using an automated sphygmomanometer (HBP-1300, Omron, Kyoto, Japan). The mean of three measurements was used for the subsequent analysis. The venous blood sample was obtained after overnight fasting. Plasma homocysteine levels were estimated by chemiluminescence microparticle immunoassay according to the manufacturer's protocol (Abbott GmbH & Co.KG, Wiesbaden-Delkenheim, Germany). The intravariable coefficient was 3%, and the inter-variable coefficient was 5%. The patients were divided into four groups based on the gender-specific quartile of homocysteine levels. The levels of fasting plasma glucose, serum creatinine (Scr), total cholesterol, HDL-C, LDL-C, and uric acid were determined using an autoanalyzer (ADVIA 2400, Siemens, Germany. Briefly, smoking, caffeine, and alcohol intake were prohibited 12 h before the measurement. The patients were asked to lie down in a supine position in a quiet room with a stable room temperature. Measurements were performed by a well-trained and experienced technician. The direct straight distance between the right common carotid artery and the right common femoral artery was measured using a tape, and the tape-measured distance multiplied by 0.8 was used as the pulse wave traveled distance. Then, two TY-360 pressure-sensitive transducers (Fukuda Denshi Co, Tokyo, Japan) were positioned at these two measurement sites, and two different pulse waves were obtained. This performance was repeated for at least 10 cardiac cycles. cfPWV was calculated according to the following formula: cfPWV (m/s) = carotid-femoral pulse wave travel distance (m)/travel time (s). The mean value of the two measurements was taken. If the difference between the two measurements was greater than 0.5 m/s, a third measurement was performed, and the median value of the three measurements was taken. Further, ≥10 m/s was used as the cutoff value of increased cfPWV. # Statistical analysis Continuous variables were expressed as mean ± standard deviation or median, and categorical variables were presented as absolute numbers or percentages. Homocysteine and triglyceride levels were logarithmically (log) transformed for further analysis because of skewed distribution. Variables were compared by one-way analysis of variance or chi-square test. The correlation between variables was assessed by Pearson's correlation analysis and stepwise multivariate linear regression. Multivariate logistic regression was performed to identify the association of the quartile of homocysteine levels with cfPWV ≥10 m/s, and the corresponding OR was calculated for increased cfPWV. Statistical significance was accepted at P < 0.05 for individual data. For multiple comparisons, the threshold levels of significance were adjusted by simple Bonferroni's correction. All the statistical analyses were performed using SPSS statistical software, version 19.0 (IL, USA).

Multiparametric ultrasound findings in acute kidney failure due to rare renal cortical necrosis Renal cortical necrosis (RCN) is a rare cause of acute kidney failure and is usually diagnosed on the basis of characteristic enhancement patterns on cross-sectional imaging. Contrast-enhanced ultrasound (CEUS) offers benefits in patients with kidney failure in the clinical setting including the use of a nonnephrotoxic intravascular contrast agent and the fact that it can be performed at the bedside in critical cases. Therefore, the aim of this study is to investigate whether CEUS can reliably identify typical imaging features of RCN. We retrospectively analyzed 12 patients with RCN examined in our department and confirmation of the diagnosis by either histopathology, other contrast-enhanced cross-sectional imaging tests, and/or CEUS follow-up. Assessed parameters in conventional US were reduced echogenicity, loss of corticomedullary differentiation, length and width of kidney, hypoechoic rim, resistance index and in CEUS delayed wash-in of contrast agent (> 20 s), reverse rim sign, maximum nonenhancing rim and additional renal infarction. Furthermore, imaging features in RCN were compared with the findings in renal vein thrombosis (RVT), among them echogenicity, corticomedullar differentiation, hypoechoic rim, RI value, delayed cortical enhancement, total loss of cortical perfusion and enhancement of renal medulla. All 12 patients showed the reverse rim sign, while a hypoechogenic subcapsular rim was only visible in four patients on B-mode ultrasound. A resistance index (RI) was available in 10 cases and was always less than 1. RI was a strong differentiator in separating RVT from RCN (RI > 1 or not measurable due to hypoperfusion as differentiator, p = 0.001). CEUS showed total loss of medullary enhancement in all cases of RVT. With its higher temporal resolution, CEUS allows dynamic assessment of renal macro-and microcirculation and identification of the typical imaging findings of RCN with use of a nonnephrotoxic contrast agent. # Material and methods Study cohort. All patients gave written informed consent to anonymized use of their data prior to imaging. This is part of the routine clinical procedure at our department. This retrospective analysis is registered at the local ethical committee of our institution (Ethikkommission der Charité-Universitätsmedizin Berlin, EA1/320/20). All study data were collected in compliance with the principles expressed in the 2002 Declaration of Helsinki. Figures were arranged considering anonymization in order to avoid their affiliation to patient's identity. The retrospective analysis included patients who underwent CEUS in our department from 2009 to 2019. Inclusion criteria were: (1) CEUS examination with documentation of sufficient image data and detailed written report of findings, (2) diagnosis of renal cortical necrosis in CEUS, [bib_ref] AJKD atlas of renal pathology: cortical necrosis, Fogo [/bib_ref] available clinical data, (4) patient age ≥ 18 years, and (5) diagnosis confirmed by biopsy and histopathology or imaging follow-up by either CEUS, ceCT, or ceMRI (in case of cross-sectional imaging one month before or after index examination and in case of CEUS follow-up within 2 months after index examination). Exclusion criteria were: (1) substandard image quality,no clinical data available, (3) no imaging follow-up and missing histopathological confirmation, and (4) proven renal vein thrombosis at the time of index examination. Using the radiology information system (RIS), we collected the following clinical information: renal or systemic primary disease, oliguria, fever, hypertonia, anemia, secondary hyperparathyroidism, diabetes mellitus, and creatinine value determined closest to the time of the index CEUS examination. Furthermore, the included cases were assessed regarding occurrence of acute RCN in the postpartum period or after renal transplant. ## Us and ceus examination protocol. Gray-scale B-mode imaging of the kidney or kidney transplant was performed using a convex array transducer to assess renal size, echogenicity, and homogeneity. Standardized CCDS was performed to assess venous outflow and arterial circulation and to calculate resistance indices (RI) for different segmental arteries at the pyeloparenchymal border (generally as a mean value of three measured RI in the upper, middle and lower third of the kidney). Power Doppler imaging was used to identify focal perfusion loss. CEUS examinations were performed as part of clinical routine using high-end ultrasound systems (Aplio 500/i900, Canon Medical Systems Corporation, Tochigi, Japan; Acuson Sequoia/S3000, Siemens Healthineers, Mountain View, CA, USA; GE Logiq E9, GE Healthcare, Chicago, Illinois, USA) with state-of-the-art CEUSspecific protocols available at the time of examination. All convex transducers employed in study patients were required to be for abdominal use with a frequency range of 1-6 MHz. The very-low-mechanical-index (< 0.1) technique was used to avoid early microbubble destruction. A bolus of 1.2-1.6 mL of ultrasound contrast agent (SonoVue, Bracco Imaging, Milan, Italy) was injected up to three times, if necessary, for example to assess arterial inflow in both kidneys. Following contrast agent injection, the kidney or renal transplant was scanned for at least five minutes to capture the wash-in phase (cine loop of 30 to 45 s) and late contrast phase between two and 5 min after injection of contrast agent. Baseline B-mode US, CCDS, and CEUS were reviewed by a highly Cross-sectional imaging. Additional multiphase ceCT examinations (arterial and venous/delayed phase) were performed in a 64-128 detector CT scanner using a standard protocol. The contrast agent was bolusinjected into an antecubital vein at a flow rate of 3.0-4.5 mL/s. Contrast media with an iodine concentration of 350-400 mg/mL were administered and their amount adopted to patient's body weight, followed by a 50 mL saline flush. The acquisition direction was craniocaudal. CeMRI was performed at 1.5 T or 3.0 T using phased-array body coils. The imaging protocols comprised T2-weighted (w) standard 2D sequences with and without fat saturation (FS) and T1-w unenhanced 2D sequences with and without FS (including in-phase/opposed-phase technique). T1-w 3D sequences with FS were acquired in breath-hold technique before and during arterial and venous/delayed phases following intravenous administration of Gadolinium-containing contrast agents (body weight adapted; manual or automatic injection at approximately 1-2 mL/s flow rate followed by 40 mL saline flush). Cross-sectional imaging datasets were used for comparison if the examination was performed within one month before or after CEUS examination with renal cortical necrosis diagnosis. Cross-sectional imaging findings were also reviewed by a highly experienced, board-certified radiologist with more than 20 years of experience in radiological imaging in consensus with a second experienced radiologist. Reference standard. Biopsies were taken on clinical indication only. All patients presented with clinically relevant native kidney or allograft post-transplant dysfunction manifesting as an otherwise unexplained increase in serum creatinine (≥ 1.3 mg/dL), proteinuria (≥ 1 g/day), or primary nonfunction in the early phase after transplantation. Pathologic examinations of biopsy samples taken in patients with renal allografts were performed by experienced nephropathologists. The diagnosis of antibody-mediated rejection was based on the presence of circulating donor-specific antibodies and significant allograft pathology according to the definitions of the up-to-date Banff classification Differentiation from renal vein thrombosis. The differentiation of RCN from renal vein thrombosis (RVT) was evaluated by comparing the following sonographic criteria between these patients with confirmed RCN and RVT (confirmed by surgery and/or cross sectional imaging and/or angiography): reduced echogenicity, loss of corticomedullary differentiation, hypoechoic rim, RI > 1 or not measurable due to hypoperfusion of interlobar arteries, delayed wash-in of contrast agent (> 20 s), total loss of cortical perfusion in CEUS, and enhancement of renal medulla. The begin of arterial phase in CEUS was described in the EFSUMB guideline for CEUS in non-hepatic applications by Sidhu et al. to start between 10 and 20 s (except lungs and liver) and so a cut-off value of 20 s was used to assess the arterial-wash-in of contrast agent as delayed [bib_ref] The EFSUMB guidelines and recommendations for the clinical practice of contrast-enhanced ultrasound..., Sidhu [/bib_ref]. The same specific protocols for renal CEUS available at the time of examination as used for RCN were utilized for CEUS in suspicion of RVT. Patients with RVT of native or transplant kidneys were identified by a systematic search for CEUS reports on examinations performed between 2009 and 2019. For inclusion, confirmation of the diagnosis of RVT by either contrast-enhanced cross-sectional imaging, intraoperative finding, or angiography was necessary. Statistical analysis. Continuous variables are reported as median and interquartile range (IQR), and categorical variables are reported as proportion of absolute number (n/N) and percentage. Categorical variables were compared using the chi 2 test and two-sided Fisher's exact test, and both values are given to evaluate possible uncertainties due to the small study cohort. A two-sided significance level of α = 0.05 was defined appropriate to indicate statistical significance. All statistical analyses were performed using the SPSS software (IBM Corp., released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp.). # Results Study cohort. Overall, our systematic query retrieved a total of 18 patients with suspected RCN. After application of the inclusion and exclusion criteria, the retrospective study cohort consisted of 12 patients ≥ 18 years who underwent CEUS of a native kidney (n = 6) or renal transplant (n = 6) between 2009 and 2019. The reasons for exclusion in the six excluded patients were: RCN was diagnosed as differential diagnosis only, the presumed RCN turned out to be caused by a macrovascular pathology (e.g. RVT) and insufficient data storage (e.g. regarding stored cine loops and images)-respectively in two cases each. Five of the six patients in the native kidney subcohort were postpartum women. The standardized renal CEUS protocol in our institution included B-mode ultrasound, CCDS, and CEUS. Baseline characteristics of the study patients are demonstrated in [fig_ref] Table 1: Baseline characteristic, comorbidities, and clinical findings in all patients [/fig_ref]. Chronic kidney disease was known in seven patients, six of them in the renal transplant cohort: one chronic glomerulonephritis, one status after rapidly progressive glomerulonephritis (RPGN) with vasculitis, two patients with diabetic nephropathy, one terminal renal failure due to shrunken kidneys (atrophic kidneys of unknown etiology), one congenital vesicoureteral reflux, and one autosomal-dominant polycystic kidney disease (ADPKD). Median serum creatinine was 6.83 mg/dL (IQR, 5.81-8.65 mg/dL). Overall, four patients underwent renal transplant nephrectomy due to cortical necrosis. US and CEUS findings. Imaging findings are summarized in An exemplary case illustrating the described imaging findings is presented in [fig_ref] Figure 1: Example of RCN of the native kidney in a 28-year-old woman suffering... [/fig_ref]. Furthermore, [fig_ref] Figure 2: Extent of RCN in CEUS [/fig_ref] shows examples of different extents of RCN in CEUS. Histopathological findings. Overall, a histopathological report was available in seven of the twelve patients (58.3%) included in this retrospective analysis. Six histopathological reports were on renal transplants, and three of them diagnosed cellular rejection. In the other three cases, the diagnoses were: pyelonephritis with ischemic alterations of the cortex, arterial alterations with thrombotic occlusions, and vasculitis. For the only native kidney, for which a histopathological examination was available, the diagnosis was cortical infarction with complete loss of viable cortical tissue. A total of five CD4 immunohistological examinations [bib_ref] A 2018 reference guide to the Banff classification of renal allograft pathology, Roufosse [/bib_ref] were performed and were negative in all cases. www.nature.com/scientificreports/ Cross-sectional imaging. Overall, nine patients underwent additional multiphase ceCT and two patients additional ceMRI, among them one patient who underwent ceCT and ceMRI [fig_ref] Figure 3: Comparison of modalities in the examination of a kidney transplant with suspected... [/fig_ref]. In two patients, RCN detected by CEUS (one native kidney, one renal transplant) was not detected with ceCT. In these two cases, the maximum necrotic rim measured in CEUS was 2 mm and 3 mm. In the two patients who underwent both CEUS and ceMRI, the two modalities showed consistent findings. Overall, classical reverse rim sign was visible in four of nine patients in case of ceCT and in one of two patients in case of ceMRI. The cortical rim sign occurred only in two patients, respectively one in ceCT and ceMRI. Differentiation from renal vein thrombosis. Overall, five patients with RVT in CEUS and surgical, angiographic, or cross-sectional imaging confirmation of the diagnosis were included for assessing the ability of CEUS to differentiate RCN from RVT, which is also characterized by a loss of cortical perfusion, however, with preserved thin subcapsular enhancement (rim sign of vascular compromise) [bib_ref] Classic signs in uroradiology, Dyer [/bib_ref]. As apparent from the data compiled in [fig_ref] Table 3: Comparison of B-mode US and CEUS findings in RCN and RVT [/fig_ref] , RI in RVT was always > 1 or not measurable due to significant hypoperfusion of interlobar arteries. Among the eleven RI values available in RCN, only one was not measurable due to hypoperfusion (9.1%) showing therefore strong statistical significance as distinctive feature between RCN and RVT (p = 0.001 according Fisher's exact test). In the differentiation of RCN from RVT, total loss of cortical perfusion was statistically significant in the chi 2 test but not in the two-sided Fisher's exact test, whereas enhancing renal medulla (100% in case of RCN, 0% in case of RVT) was found to be statistically significantly different in both tests (p < 0.001) compared with RCN. [fig_ref] Figure 4: Delimitation of RVT compared to RCN [/fig_ref] provides examples illustrating the different appearance of RVT in CEUS compared to RCN. # Discussion The main findings of our analysis can be summarized as follows: (1) RI tends to be normal in patients with RCN, (2) real-time CEUS reliably shows the reverse rim sign in RCN, and (3) the combination of CEUS and RI enables differentiation of RCN and RVT in patients with inconclusive CCDS findings. Imaging findings must always be interpreted in the clinical context and in conjunction with the patient's history: eleven of the twelve patients with RCN in our study had a renal transplant or were postpartum women. All women with postpartum acute renal failure suffered from massive bleeding, which is characteristic of RCN 1 . In 2015, Prakash et al. described RCN to be a disappearing entity in developing countries 11 , which makes it even more important to keep this rare clinical picture with its typical imaging signs in mind for correct diagnosis. The importance of the clinical background is further corroborated by the fact that RCN needs to be differentiated from RVT: our results show the RI to be a very strong distinctive feature to separate RCN and RVT. Nevertheless, in inconclusive cases, the use of a contrast agent can be expedient. Our results show-at least using the two-sided Fisher's exact test-that total loss of cortical perfusion is not clearly statistically significantly different between the two entities, which is probably driven by the small study cohort. Though, the small number of included cases in the comparative group diagnosing a RVT with CEUS is simply explained by the importance of RI in the diagnosis of RVT making the application of contrast agent in most cases needless. Our results indicate this relationship as well since all included patients in the cohort of RVT had a RI value > 1 or the RI was not measurable due to hypoperfusion [fig_ref] Table 3: Comparison of B-mode US and CEUS findings in RCN and RVT [/fig_ref]. Next to imaging features, clinical information according the patient are relevant: RVT can occur in acute cases in the post-transplant period and should therefore be considered in these cases [bib_ref] Risk factors for graft loss due to acute vascular complications in adult..., Adani [/bib_ref]. Furthermore, CEUS needs specialized examiners, who are not always available-therefore, a rational use of CEUS in patients with suspected RVT needs to take into account that emergency surgery should not be delayed highlighting the value of CCDS and RI assessment. Nevertheless-showing high importance in macrovascular problems as RVT-a normal RI value should be considered in consensus with contrast-enhanced imaging when suspected RCN, since our results show B-mode sonography assessing reduced echogenicity (50%), loss of corticomedullary differentiation (50%) and hypoechoic rim (33.33%) not to be reproductible in all included patients . External validation of the imaging pattern of RCN in CEUS is very rare. In the largest cohort of five patients with renal transplant reported so far, the investigators described similar results as in our study including an [bib_ref] The usefulness of contrast-enhanced ultrasound in the assessment of early kidney transplant..., Álvarez Rodríguez [/bib_ref] [bib_ref] Contrast enhanced ultrasound (CEUS) in the diagnosis of post-partum bilateral renal cortical..., Mckay [/bib_ref]. These studies and our results consistently show that the reverse rim sign 10 is adequately detected by CEUS. The reverse rim sign reflects loss of subcapsular enhancement [fig_ref] Figure 3: Comparison of modalities in the examination of a kidney transplant with suspected... [/fig_ref] with preserved medullary enhancement and must be differentiated from the thin cortical rim sign [fig_ref] Figure 3: Comparison of modalities in the examination of a kidney transplant with suspected... [/fig_ref] reflecting preserved supply by capsular arteries. Therefore, CEUS reliably reproduces the classical radiologic signs of RCN which are classically assessed in ceCT [bib_ref] Classic signs in uroradiology, Dyer [/bib_ref]. Evidence concerning MRI is much thinner, but was described to be comparable to , what could be confirmed in the two patients in our study who underwent ceMRI as visualized in [fig_ref] Figure 3: Comparison of modalities in the examination of a kidney transplant with suspected... [/fig_ref]. [bib_ref] Use of intravenous gadolinium-based contrast media in patients with kidney disease: consensus..., Weinreb [/bib_ref]. Therefore, they recommend to balance rarely occurring NSF and potentially delayed diagnosis against each other [bib_ref] Use of intravenous gadolinium-based contrast media in patients with kidney disease: consensus..., Weinreb [/bib_ref]. In our analysis, enhancement of the renal medulla was absent in all patients with RVT while it was present in all patients with RCN. Thus, the combination of RI and presence versus absence of medullary enhancement may distinguish between RCN and RVT. Since the absent medullar perfusion as a qualitative parameter showed high statistical significance of p < 0.001 [fig_ref] Table 3: Comparison of B-mode US and CEUS findings in RCN and RVT [/fig_ref] , it suggests that quantitative evaluation of renal perfusion might not be necessary in clinical practice to differentiate RCN from RVT. Our study patients had conspicuously high mean serum creatinine levels of 6.83 mg/dL (IQR, 5.81-8.65 mg/ dL) as indicators of renal failure. Therefore, CEUS is a suitable imaging method in patients with acute loss of kidney function since, unlike iodine-based contrast agents used in ceCT, ultrasound contrast agent is not nephrotoxic [bib_ref] The safety of Sonovue in abdominal applications: retrospective analysis of 23188 investigations, Piscaglia [/bib_ref] [bib_ref] Safety of sulfur hexafluoride microbubbles in sonography of abdominal and superficial organs:..., Tang [/bib_ref] [bib_ref] Contrast-induced nephropathy in CT: incidence, risk factors and strategies for prevention, Tao [/bib_ref]. Therefore, CEUS is also suitable for repeat follow-up examinations without a risk of nephrogenic side effects or possible adverse effects of cumulated radiation exposure. In our study, only 33.33% of the included patients with RCN showed the hypoechoic rim in B-mode ultrasound-although high-end ultrasound machines were used-underlining the need for contrast medium administration in order to diagnose RCN. In the CEUS follow-up of two patients in our cohort, regression of necrotic areas was observed, indicating that these were cases of incomplete/patchy RCN with the potential of recovery [bib_ref] Spectrum of renal cortical necrosis in acute renal failure in eastern India, Prakash [/bib_ref] [bib_ref] Acute renal cortical necrosis in pregnancy: clinical course and changing prognosis, Beji [/bib_ref]. Although partial recovery of renal function has been discussed for such cases before 4 , there was no restoration of renal function in these two patients of our cohort. A similar course was described by Wieler and Hansmann in a case report of bilateral RCN in a 31-year-old patient with recovery of renal perfusion in CEUS follow-up while retention parameters continued to be elevated [bib_ref] Renale kortikale Nekrose: Verlaufskontrollen mittels kontrastmittelunterstütztem Ultraschall (CEUS), Wieler [/bib_ref]. Furthermore, studies investigating focal renal lesions in both native kidneys and renal transplants found CEUS to have higher spatial and temporal resolution than ceCT and ceMRI [bib_ref] Contrast-enhanced ultrasound in renal imaging and intervention, Olson [/bib_ref] [bib_ref] Role of US contrast agents in the assessment of indeterminate solid and..., Harvey [/bib_ref]. Since higher resolution also helps in detecting diffuse pathology-especially in renal transplants due to the lower penetration depth in US-CEUS has an important role in these patients. Another important issue are the contrast agents used with different imaging modalities. The microbubbles used for CEUS are strictly intravascular, allowing evaluation whole organ perfusion including microcirculation [bib_ref] Ultrasound contrast agents as markers of vascularity and microcirculation, Greis [/bib_ref]. This constitutes an advantage for CEUS over both CT and MRI since iodinated and gadolinium-based contrast media are not purely intravascular 29 -which could be especially important when assessing necrotic parenchyma. # Limitations We retrospectively analyzed CEUS findings obtained in a small cohort of patients. Since we use a standardized CEUS protocol for clinical examinations in our department, a prospective study design would not have made much of a difference. Furthermore, a retrospective study design is also reasonable given the rareness of the entity investigated. Finally, we used strict inclusion criteria and required a reference standard-either ceCT, ceMRI, CEUS follow-up, or biopsy-for confirmation of the diagnosis. # Conclusion Real-time CEUS is a suitable imaging modality for identifying the reverse rim sign in patients with suspected partial RCN, especially when RI values and B-mode US findings are inconclusive. The use of a nonnephrotoxic contrast agent with visualization of renal microvascularization allows evaluation of the entire kidney and use of CEUS for repeat short-term follow-up examinations without a risk of adverse effects on renal function. In combination with RI, CEUS has the potential to differentiate RCN from RVT, which is characterized by complete loss of medullary enhancement. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. [fig] Figure 1: Example of RCN of the native kidney in a 28-year-old woman suffering from massive postpartum bleeding with acute kidney failure and HELLP syndrome. (a) B-mode image of the right kidney showing a hypoechoic rim of 3-4 mm. (b) CEUS of the right kidney showing a subcapsular loss of contrast enhancement of 3-5 mm. (c) Triplex sonography of the right kidney with a PW spectrum of an interlobar artery showing a normal resistance index of 0.65. (d) Coronal venous-phase CT scan obtained 14 days before CEUS examination showing a recess of contrast agent measuring up to 6 mm in both kidneys, confirming the diagnosis of RCN. CEUS denotes contrast-enhanced ultrasound, RCN, renal cortical necrosis, HELLP syndrome denotes hemolysis, elevated liver enzymes, low platelets, PW, pulsed-wave Doppler. [/fig] [fig] Figure 2: Extent of RCN in CEUS. (a) CEUS (0:56 min) of the right kidney in a female patient showing an uninterrupted nonenhancing circular subcapsular rim while B-mode US shows no subcapsular alteration. Note simultaneous enhancement of the medullary pyramids. This pattern is known as the reverse rim sign. (b) CEUS (1:20 min) and US image of the left kidney of the same patient consistent with bilateral RCN. (c) CEUS (2:15 min) of the right kidney in a different patient showing a subcapsular non-enhancing rim of variable width, especially at the upper pole. (d) Same patient as in (c). The 3D rendering of the right kidney during CEUS proves the variation in the with of the nonenhancing subcapsular rim. cortical band and RI not > 1 in all patients 15 . This typical enhancement pattern was also described by Álvarez Rodríguez et al. in two patients and by McKay in a case of bilateral RCN [/fig] [fig] Figure 3: Comparison of modalities in the examination of a kidney transplant with suspected RCN. Upper row: Case showing partial RCN in a renal transplant in a 51-year-old man with end-stage renal failure due to shrunken native kidneys. (a) CEUS (0:59 min) and B-mode US of the renal transplant showing typical reverse rim sign with partial loss of enhancement in subcapsular cortex. (b,c) CeCT (b) and ceMRI (c) showing the hypoenhanced rim of the renal cortex in the transverse plane. Lower row: A case of RCN in a 54-year-old woman with renal transplant. (d) CEUS (1:13 min) using a convex multifrequency probe clearly identifies the reverse rim sign as a nonenhanced rim in the superficial cortex and lower pole (arrows). (e) Increased spatial resolution by use of a linear probe, which is important for second look if CEUS with convex probe shows inconclusive findings. (f) Parametric arrival time imaging of CEUS depicts arterial inflow in interlobar arteries and cortex within 4 s(red and green). Arrows indicate the so-called cortical rim sign with preserved blood supply by capsular arteries (arrows). (g) Coronal T2w MR image confirms the cortical structure defect and moreover shows cortical perfusion deficit at the upper pole of the renal transplant. RCN denotes renal cortical necrosis; CEUS, contrast-enhanced ultrasound; ceCT, contrast-enhanced computed tomography; ceMRI, contrast-enhanced magnetic resonance imaging. [/fig] [fig] Figure 4: Delimitation of RVT compared to RCN. CEUS examination of a 55-year-old man with renal vein thrombosis. (a) The B-mode image shows signs of poor corticomedullary differentiation. (b) The pulsed wave (PW) spectrum shows triphasic flow with a resistance index of 1.1, suggesting disturbed venous outflow. (c) CEUS (after 50 s) shows no contrast enhancement (indicating loss of microcirculation) of the renal cortex and medullary pyramids. In the cine loop, a pulse-synchronous "pushing" of the microbubbles in the interlobar arteries is visible without cortical enhancement. RVT denotes renal vein thrombosis; CEUS, contrast-enhanced ultrasound, PW, pulsed wave Doppler sonography, RCN, renal cortical necrosis. permissions information is available at www.nature.com/reprints. [/fig] [table] Table 1: Baseline characteristic, comorbidities, and clinical findings in all patients. Continuous variables are given as median (IQR), categorical variables as absolute/total numbers (n/N) and percentages in brackets. RCN denotes renal cortical necrosis, IQR interquartile range. [/table] [table] Table 3: Comparison of B-mode US and CEUS findings in RCN and RVT. Furthermore, there is an ongoing debate about administration of Gadolinium containing contrast agents in patients with kidney disease which is suspected to cause nephrogenic systemic fibrosis (NSF)-which Weinreb et al. summarized to be very low [/table]

Intensive Care Weaning (iCareWean) protocol on weaning from mechanical ventilation: a single-blinded multicentre randomised control trial comparing an open-loop decision support system and routine care, in the general intensive care unit ## Mathematical models included in the cdss To do so an arterial blood gas (ABG) is required on system start up. In some patients, the system also requires modification of FIO2 to two levels for 2-5 minutes at each level to tune the pulmonary gas exchange model to the patient, a procedure previously called the automatic lung parameter estimator (ALPE) technique. The respiratory mechanics model is tuned to dynamic compliance. The model of acid-base chemistry of the blood is tuned to measured values of arterial pH, PCO2, PO2, and SO2, and haemoglobin concentration, with the acid-base chemistry of the cerebrospinal fluid (CSF) regulated to arterial bicarbonate values to account for chemical changes in respiratory drive. The respiratory drive model is tuned to describe the relationship between alveolar ventilation (VA) and arterial acid-base and oxygenation status. The ventilation model is tuned to the measured serial dead space (Vds) to allow calculation of alveolar ventilation. A series of algorithms are present in the CDSS to re-tune the models as new measurements present, or if the patient condition changes. These models are used to simulate the effect of changes in ventilator settings, with the system generating advice based upon simulated values.illustrates the mathematical models built to describe the effects of PEEP. These are formulated as empirical linear models, with the system only advising on PEEP changes of a maximum of 3 cmH2O in any step and the slope of these relationships modified automatically on measuring the response to PEEP. These models are integrated with those into enable simulation of the patient specific effects of PEEP and other ventilator settings simultaneously, enabling the system to advise on changing settings toward the best compromise for the individual patient given the model simulations. For patients with acute respiratory failure, PEEP is usually set to improve gas exchange and lung mechanics, and the sub-figures A, B and C ofillustrate the baseline expected response to changes in PEEP of pulmonary shunt, respiratory system compliance and high ventilation/perfusion (V/Q). Changes in shunt, compliance and high V/Q automatically result in model-simulated changes in oxygenation, ventilation volumes and pressures, and carbon dioxide partial pressures through the models illustrated in. For shunt and compliance, the initial slopes of the models are dependent on the initial levels of shunt and compliance, with a greater improvement expected for a greater severity of respiratory abnormality. The slopes of these models are adapted automatically according to patient response to changes in PEEP, from measurements of oxygenation (SpO2), respiratory volumes and pressures, and end tidal CO2 values. Any increase in end tidal CO2 values that are simulated to result in severe acidosis result in the system requesting an arterial blood gas. The decision to increase PEEP is then based on the potential benefit of improved oxygenation and respiratory volumes and pressures, given the tuning of the models to the patient's state. For patients responding poorly to increases in PEEP, the absolute slope of shunt and compliance models would be substantially reduced following measurement of the response to PEEP changes. The decision to reduce PEEP at high SpO2 levels depends upon the FIO2 level. FIO2 will always be optimized first, such that the competing goals of sufficient oxygenation and oxygen toxicity are balanced. Following this, the system will calculate the likely effects on oxygenation and respiratory volumes and pressures on reducing PEEP from the models illustrated on subfigures A, B and C of, combined with the models of E1. For patients recovering from respiratory injury, without substantial pulmonary shunt BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open doi: 10.1136/bmjopen-2020-042145 :e042145. 10 2020; BMJ Open , et al. Vizcaychipi MP or low respiratory system compliance, the system will calculate that the potential negative effects of reducing PEEP will be outweighed by the positive effects of reducing plateau pressure. For patients on pressure support ventilation without substantial problems related to gas exchange or respiratory system mechanics, PEEP is often used to support the respiratory muscles, preventing abnormal respiratory muscle activity as indicated by abnormal breathing patterns., D and E represent the empirical linear models included in the system to account for changes in tidal volume (VT) due to PEEP.illustrates two potential abnormal situations; the first is a very high VT at a low PEEP value, and the second a very low VT at a high PEEP, possibly representing the situation where the diaphragm may be over distended due to high PEEP. In these situations, the initial slope of the PEEP versus VT relationship is negative.illustrates the situation of a low tidal volume at a low value of PEEP, possible due to under-support of the respiratory muscles. In this situation, the initial slope of the PEEP versus VT relationship is positive. As with other PEEP models, all slopes are adapted following measurement of the response to PEEP changes. In addition to these examples, it is important to note that on occasion the system will present advice to switch ventilator mode. This occurs if the patient is in a controlled ventilator mode and the system identifies a total respiratory frequency or a total minute ventilation that is significantly greater than that set, where the system provides a message to the user suggesting changing to support mode. Similarly, if the patient is in support mode and the model simulated pH is very low then the system provides a message asking the user to consider either control mode or reduction of anesthesia. In modes containing mandatory breaths initiated by the ventilator or by patient effort, and where different settings are available for patient initiated and ventilator initiated breaths, the system provides advice on these depending upon the pattern of patient ventilation, and whether the patient is currently in a period with substantial spontaneous breathing activity. The system functions on a variety of manufactures ventilators and, as illustrated in these examples, provides advice for a variety of modes. Current exceptions to these include APRV, and modes with automatic control of ventilator settings such as ASV© or SmartCare©. Data is collected automatically by the Beacon Caresystem, and includes tidal volumes, both set if the mode dictates, and also measured. The regulation of Vt and it relationship with mode can therefore be explored for both of the study arms. The system records all ventilator settings via connection to the ventilator, all direct measurements of flow and gas concentrations, its own SpO2 measurement, and the timing and nature of all advice provided. Theillustrates the same screen as E3, but with the corners of the hexagon activated to show the current, simulated and advised values of variables simulated by the physiological models. In this patient, the current FIO2 setting of 41 %, results in a SaO2 value of 97.9 % and a current simulated SvO2 of 89.7 %. The system illustrates that the current balance between over and under oxygenation may be inappropriate with the blue symbol on the hexagon pointing slightly toward oxygen toxicity. The system therefore suggests reducing FIO2. The system also illustrates an increased risk of respiratory muscle atrophy due to a low respiratory frequency, leading the system to suggest a reduction in PS from 14 to 12 cmH2O, simulating that this may result in increased Rf.illustrates an advice including PEEP for a patient in pressure control ventilation. The vertical axis representing the balance between respiratory muscle atrophy and stress is disabled in controlled ventilation modes for cases where the patient has no spontaneous breathing activity. In this patient, the system indicated that the current balance between over and under ventilation might be inappropriate, with the blue symbol on the hexagon pointing toward lung trauma and with the patient being slightly alkalotic. The system suggested reducing PEEP and PC from 6 to 5 cmH2O and 12 to 11 cmH2O, respectively, and at the same time increasing Rf from 18 to 19 bpm. The simulated effect of PEEP was a negligible increase in pulmonary shunt, with simulated patient response to the combined advice being to reduce inspiratory pressure, reduce pH and maintain appropriate oxygenation. illustrates the result of following the advice illustrated in , and the subsequent advice provided by the system. The combined effect of PEEP and PC reduction and increase in Rf was as expected for both oxygenation and respiratory mechanics but less pronounced on pH. The advised level of PEEP was maintained in the next advice, but with an advice to decrease FIO2 expecting a resulting safe oxygenation. Simulations in the details boxes at the hexagon corners take into account differences between set and measured Rf, where measured Rf was 17 bpm despite set Rf of 19 bpm. -User interface of the system, with results according to the advice in, and the next advice., the system provides a counter on the screen which counts down from 30 minutes, and as such indicates that the patient has been within ventilator settings consistent with an SBT and stable with regard other respiratory measurements, for this duration. Cut-off values for these other variables are illustrated in figure E10-User interface illustrating variables used to time the SBT counter of the system. At any point when the counter is running, the user can view the screen illustrated in. This indicates which variables are within threshold, their values, and for how long this has been the case.

A Cross-Sectional Study of Sub-Basal Corneal Nerve Reduction Following Neurotoxic Chemotherapy M. A cross-sectional study of sub-basal corneal nerve reduction following neurotoxic chemotherapy. Trans Vis Sci Tech. 2021;10(1):24, https://doi.Purpose: Sub-basal corneal nerves have been shown to change during neurotoxic chemotherapy treatment. This cross-sectional study investigated corneal nerve morphology in patients who have completed neurotoxic chemotherapy well after treatment cessation and its association with peripheral nerve function.Methods: Central corneal nerve fiber length (CNFL) and inferior whorl length (IWL), average nerve fiber length (ANFL), corneal nerve fiber density (CNFD) and corneal nerve branch density (CNBD), and nerve fiber area (CNFA) were examined using in vivo corneal confocal microscopy in patients with cancer who had completed treatment with either paclitaxel or oxaliplatin between 3 and 24 months prior to assessment in comparison with 2 separate groups of healthy controls. Neurological assessments were conducted including clinician-and patient-reported outcomes, and neurological grading scales.Results: Both paclitaxel-(n = 40) and oxaliplatin-treated (n = 30) groups had reduced IWL and ANFL compared to the respective healthy control groups (n = 15 in each group) (paclitaxel: IWL = P = 0.02, ANFL = P = 0.009; and oxaliplatin: IWL = P = 0.008, ANFL P = 0.02). CNFL and CNFD reduction were observed only in the paclitaxel-treated group compared with healthy controls (P = 0.008 and P = 0.02, respectively), whereas CNFA was reduced in the oxaliplatin-treated group (P = 0.04). IWL reduction correlated with worse fine hand dexterity in chemotherapy-treated patients (r = −0.33, P = 0.007).Conclusions:There is evidence of corneal nerve loss in patients with cancer who have been treated with paclitaxel and oxaliplatin well after treatment cessation associated with worse upper limb function.Translational Relevance: Sub-basal corneal nerve reduction is evident even after cessation of neurotoxic treatment. In vivo corneal confocal microscopy may be useful in the monitoring of nerve function in patients receiving chemotherapy. # Introduction Chemotherapy-induced peripheral neuropathy (CIPN) is a distal, predominantly sensory neuropathy, which can affect both large and small nerve fiber function. [bib_ref] Neurophysiological, nerve imaging and other techniques to assess chemotherapy-induced peripheral neurotoxicity in..., Argyriou [/bib_ref] [bib_ref] Quantification of small fiber neuropathy in chemotherapy treated patients, Timmins [/bib_ref] With increasing survival rates due to improved diagnosis and chemotherapeutic treatment of cancer, the side effect burden of CIPN has also increased. [bib_ref] Incidence and disease burden of chemotherapyinduced peripheral neuropathy in a populationbased cohort, Shah [/bib_ref] Notably, it is one of the most common dose-limiting factors, with debilitating neuropathic symptoms, such as pain and numbness necessitating treatment discontinuation. [bib_ref] Incidence and disease burden of chemotherapyinduced peripheral neuropathy in a populationbased cohort, Shah [/bib_ref] It also has the potential to cause long-term sensory and functional impairments, which can negatively affect quality of life.The two drugs that are most commonly associated with the development of peripheral neuropathy are oxaliplatin, widely used in upper gastrointestinal and colorectal cancers, and paclitaxel, used for the treatment of breast, gynecological, and lung cancers. Chronic neuropathy has been reported to be present in up to 84% of oxaliplatin-treated patients at 24 months follow-up. [bib_ref] Long-term course of oxaliplatin-induced polyneuropathy: a prospective 2-year follow-up study, Briani [/bib_ref] Paclitaxel-induced peripheral neuropathy is also common with 71% of treated patients reporting neuropathic symptoms. [bib_ref] Early, progressive, and sustained dysfunction of sensory axons underlies paclitaxel-induced neuropathy, Park [/bib_ref] These symptoms can persist well after treatment, with 54% of patients reporting persistent neuropathy at 18 months post-treatment cessation. [bib_ref] Clinical and genetic predictors of paclitaxel neurotoxicity based on patient-versus clinician-reported incidence..., Park [/bib_ref] Despite developments in the use of validated clinician-and patient-reported outcome measures, there is no test available that provides early detection of neuropathy onset in patients receiving chemotherapy. Nerve conduction studies remain the gold standard for neuropathy diagnosis but are slow to demonstrate changes in nerve function and often correlate poorly with clinical features.In vivo corneal confocal microscopy has been proposed as an instrument for detecting peripheral neuropathy due to its noninvasive nature and rapid method of imaging small nerves particularly in patients with diabetes. [bib_ref] Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal..., Ferdousi [/bib_ref] [bib_ref] Early detection of nerve fiber loss by corneal confocal microscopy and skin..., Ziegler [/bib_ref] [bib_ref] Corneal confocal microscopy identifies small-fiber neuropathy in subjects with impaired glucose tolerance..., Azmi [/bib_ref] [bib_ref] Small-fibre neuropathy in men with type 1 diabetes and erectile dysfunction: A..., Azmi [/bib_ref] Most research involving corneal assessment in CIPN were longitudinal studies during treatment without long-term followup to assess any persistent loss of peripheral and corneal nerves. [bib_ref] Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal..., Ferdousi [/bib_ref] [bib_ref] Corneal confocal microscopy in patients with oxaliplatin-induced peripheral neuropathy, Campagnolo [/bib_ref] As investigations into corneal nerve morphology in patients with cancer treated with neurotoxic chemotherapy with high prevalence of persistent peripheral neuropathy well after treatment cessation is limited, [bib_ref] Oxaliplatin-and docetaxel-induced polyneuropathy: Clinical and neurophysiological characteristics, Bennedsgaard [/bib_ref] the current study aimed to observe corneal nerve morphology in patients with cancer who have received and completed treatment with paclitaxel or oxaliplatin. # Methods ## Study design This cross-sectional study was approved by the South Eastern Sydney Area Health Service Human Research Ethics Committee. All methods were conducted in accordance with relevant guidelines and regulations. Participants provided written informed consent after explanation of the nature and possible consequences of the study in accordance with the tenets of the Declaration of Helsinki. ## Patient selection Seventy patients with cancer who had completed treatment with chemotherapy between 3 to 24 months prior to assessment (n = 40 treated with paclitaxel: 36 breast and 4 gynecological cancers; n = 30 treated with oxaliplatin: 25 colorectal and 5 upper intestinal tract cancers) were recruited by convenience sampling from the Department of Medical Oncology, Prince of Wales Hospital (Sydney, Australia). Sample size calculation using G * Power 3.1.9.4 (Heinrich Heine University, Dusseldorf, Germany) was based on the difference between the mean values for corneal nerve fiber length (CNFL) of patients with cancer completing chemotherapy (18.08 ± 3.62 mm/mm 2 ) compared with controls (26.82 ± 4.27 mm/mm 2 ), [bib_ref] Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal..., Ferdousi [/bib_ref] and showed that 12 participants would be the minimum sample size required to show a significant difference with 90% power and alpha of 0.05. Two separate groups of age-, sex-, and body mass index (BMI)-matched healthy controls, each compared with paclitaxel-or oxaliplatin-treated groups, were recruited through contact in the local health district. Participants were excluded if they had a history of any other medical disorders known to cause neuropathy, including impaired glucose tolerance, diabetes, and chronic kidney disease, as well as those who were pregnant or lactating, had a history of ocular trauma, ocular surgery, or refractive surgery, had any active ocular disease, such as significant corneal epitheliopathy from dry eye disease (grade >2 fluorescein staining on the Efron scale), [bib_ref] Validation of grading scales for contact lens complications, Efron [/bib_ref] iritis, corneal edema, herpetic ulcers, corneal dystrophies, or glaucoma requiring treatment with intraocular pressure lowering eye drop medication, soft and rigid contact lens wear, or had known allergies to anesthetic eyedrops. Slit lamp biomicroscopy was performed to exclude clinical manifestations of these conditions. ## Assessment of neurotoxicity Clinical assessment was conducted using the clinician-reported outcome, National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE) version 3 sensory subscale for peripheral neuropathy with the following grading system: grade 1, asymptomatic loss of tendon reflex or paresthesia (including tingling) but not interfering with function; grade 2, sensory alterations or paresthesia interfering with function but not interfering with activities of daily living; grade 3, sensory alterations or paresthesia interfering with activities of daily living; and grade 4, disabling. [bib_ref] CTCAE v3.0: development of a comprehensive grading system for the adverse effects..., Trotti [/bib_ref] The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life -Chemotherapy-induced Peripheral Neuropathy questionnaire (QLQ-CIPN20) was incorporated as a patient-reported outcome, which assesses sensory, motor, and autonomic symptoms, in addition to functional activities of daily living. [bib_ref] The development of an EORTC quality of life questionnaire to assess chemotherapy-induced..., Postma [/bib_ref] The Total Neuropathy Scale (TNS -Johns Hopkins University) is a composite scale developed to grade neuropathy according to symptoms, signs, and nerve conduction studies. [bib_ref] Grading of chemotherapy-induced peripheral neurotoxicity using the Total Neuropathy Scale, Cavaletti [/bib_ref] [bib_ref] Total neuropathy score: Validation and reliability study, Cornblath [/bib_ref] The reduced TNS (TNSr) was used to grade symptomatology, clinical, and neurophysiological measures across eight domains (sensory symptoms, weakness symptoms, pin prick sensibility, vibration threshold, strength, deep tendon reflexes, sensory nerve, and motor nerve conduction studies), with grades ranging from 0 to 4, where 0 signifies no deficit and 4 represents more severe changes. [bib_ref] Grading of chemotherapy-induced peripheral neurotoxicity using the Total Neuropathy Scale, Cavaletti [/bib_ref] The total score ranges from 0 to 32. Nerve conduction studies were undertaken in the sural sensory and tibial motor nerves, as per standard protocols.Functional assessment of upper limb dexterity and hand-eye coordination was conducted via timed completion of a grooved pegboard test using identical keyholeshaped pegs with their dominant hand. [bib_ref] Gender-and age-specific changes in motor speed and eye-hand coordination in adults: normative..., Ruff [/bib_ref] The task was completed twice, and the average time of completion was reported. ## In vivo corneal confocal microscopy In vivo corneal confocal microscopy using a laser scanning confocal microscope (Heidelberg Retinal Tomograph III with Rostock Corneal module; Heidelberg Engineering GmbH, Heidelberg Germany) was performed on participants after the cornea has been anesthetized with sterile 0.4% benoxinate hydrochloride (oxybuprocaine hydrochloride). A sterile probe was then placed onto the cornea to capture images. Only the right eye was imaged for all the participants, given the symmetry between the two eyes in previous studies. [bib_ref] Interocular comparison by in vivo confocal microscopy of the 2-dimensional architecture of..., Misra [/bib_ref] [bib_ref] Standardized baseline human corneal subbasal nerve density for clinical investigations with laserscanning..., Parissi [/bib_ref] The central cornea and inferior whorl region were scanned. Eight images best representing the central cornea with <20% overlap between images 24 and 3 to 5 images from the inferior whorl region were identified for analysis as previously described [fig_ref] Figure 1: In vivo corneal confocal microscopy images of the corneal sub-basal nerve plexus [/fig_ref]. [bib_ref] The effect of age, gender and body mass index on tear film..., Tummanapalli [/bib_ref] Data were averaged across the eight central images and three to five inferior whorl images. The inferior whorl is the most distal portion of corneal nerves located about 2 mm inferonasal from the central cornea (see [fig_ref] Figure 1: In vivo corneal confocal microscopy images of the corneal sub-basal nerve plexus [/fig_ref] and is known to be a sensitive parameter for detecting peripheral neuropathy. [bib_ref] Greater corneal nerve loss at the inferior whorl is related to the..., Kalteniece [/bib_ref] Corneal nerve parameters were measured with an established automated image analysis software [bib_ref] Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve..., Dabbah [/bib_ref] [bib_ref] An automatic tool for quantification of nerve fibers in corneal confocal microscopy..., Chen [/bib_ref] (ACCMetrics, The University of Manchester Intellectual Property UMIP, Manchester, UK): CNFL (mm/mm 2 ), the total length of all nerve fibers (main trunks and branches) at the central cornea, and inferior whorl length (IWL; mm/mm 2 ), the total length of all nerve fibers and branches in the inferior whorl region given the diagnostic value of CNFL as shown in diabetic peripheral neuropathy. [bib_ref] Corneal confocal microscopy for identification of diabetic sensorimotor polyneuropathy: A pooled multinational..., Perkins [/bib_ref] The IWL is also thought to represent the most distal regions of the corneal nerve and shown to be a sensitive measure in length-dependent peripheral neuropathy in diabetes. [bib_ref] Greater corneal nerve loss at the inferior whorl is related to the..., Kalteniece [/bib_ref] Additional pertinent corneal nerve parameters, including corneal nerve fiber density (CNFD; no/mm 2 ), corneal nerve branch density (CNBD; no/mm 2 ), corneal nerve fiber area (CNFA; mm 2 /mm 2 ), and average nerve fiber length (ANFL = CNF L + IW L 2 , mm/mm 2 ) have also been included given their potential diagnostic utility reflected in diabetic peripheral neuropathy. [bib_ref] Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: Comparing..., Chen [/bib_ref] [bib_ref] Corneal nerve fiber size adds utility to the diagnosis and assessment of..., Brines [/bib_ref] [bib_ref] Diagnostic utility of corneal confocal microscopy and intra-epidermal nerve fibre density in..., Alam [/bib_ref] [bib_ref] Diabetic neuropathy is characterized by progressive corneal nerve fiber loss in the..., Ferdousi [/bib_ref] [bib_ref] Diagnosis of neuropathy and risk factors for corneal nerve loss in type..., Ferdousi [/bib_ref] # Statistical analysis Statistical analysis was conducted using SPSS Statistics 26 (IBM Corp., Armonk, NY). Significance was defined as P ≤ 0.05, unless stated otherwise. Normality was considered when P > 0.05 in the Shapiro-Wilk test. Neurophysiological, functional, and corneal measures were compared between the paclitaxel and oxaliplatin groups with the respective healthy control groups (healthy controls group 1 with paclitaxel, and healthy controls group 2 with oxaliplatin). Chi squared test of independence was used to compare gender proportions between the groups. Normally distributed data were compared using independent samples ttest, whereas nonparametric data were compared using the Mann-Whitney U test. The two largest patient groups for each treatment type (breast cancer for paclitaxel and colorectal cancer for oxaliplatin) were also compared in terms of corneal nerve parameters. A subanalysis of patients with neuropathy (TNSr grade ≥1) versus no neuropathy (TNSr grade 0) as assessed with TNSr was performed within each chemotherapeutic treatment group. Chi squared test of independence was used to compare gender proportions between the two groups. Normally distributed data were compared using independent samples t-test, whereas nonparametric data were compared using the Mann-Whitney U test. Comparisons with the respective healthy control groups matched in the primary analysis using the Kruskal Wallis test with Bonferroni adjustment were also conducted, with an adjusted P value of 0.05 3 used to assess for significance. Partial correlation analysis was used to assess the association between corneal nerve parameters with five main measures of sensory neuropathy severity and nerve function, including grooved pegboard test, NCI-CTCAE, EORTC QLQ-CIPN20, TNSr scores, and sural nerve amplitude in chemotherapy-treated patients (both paclitaxel and oxaliplatin) after adjusting for age. The associations among stage of cancer prior to treatment, number of treatment cycles, mean cumulative dose, period post-treatment, and relative dose intensity by the two chemotherapy types with corneal nerve parameters were also assessed with correlation analysis while adjusting for age. Additionally, correlation between cancer and chemotherapy data with the measures of sensory neuropathy severity and nerve function was conducted. An adjusted P value of 0.05 5 was used to assess for significance in each correlation analysis to control for type 1 errors. # Results The clinical, demographic, and neurological data are presented in [fig_ref] Table 1: Clinical Characteristics, Neurophysiological, and Functional Measures of Study ParticipantsRelative dose intensity [/fig_ref]. Representative figures of the corneal nerve images for the paclitaxel, oxaliplatin, and healthy control groups are shown in [fig_ref] Figure 1: In vivo corneal confocal microscopy images of the corneal sub-basal nerve plexus [/fig_ref]. Both paclitaxel-and oxaliplatin-treated patients had severe reduction in length of the inferior whorl corneal nerves compared with their respective healthy control groups [fig_ref] Table 2: In Vivo Corneal Confocal Microscopy Parameters of Study Participants [/fig_ref]. Only the paclitaxel-treated patients had significantly reduced CNFL in the central cornea compared with healthy controls [fig_ref] Table 2: In Vivo Corneal Confocal Microscopy Parameters of Study Participants [/fig_ref]. ANFL and CNFD were also reduced in paclitaxel-treated patients, whereas reductions in ANFL and CNFA were noted in oxaliplatin-treated patients when compared with healthy controls [fig_ref] Table 2: In Vivo Corneal Confocal Microscopy Parameters of Study Participants [/fig_ref]. Comparisons of the corneal nerve parameters of the two largest cancer groups showed no significant differences between patients with breast cancer who received paclitaxel treatment and patients with colorectal cancer who received oxaliplatin treatment (CNFL: 13.5 ± 3.7 mm/mm 2 vs. 14.2 ± 2.4 mm/mm 2 , P = 0.41; IWL: 13.7 ± 4.0 mm/mm 2 vs. 12.5 ± 3.3 mm/mm 2 , P = 0.21; ANFL: 13.6 ± 3.6 mm/mm 2 vs. 13.3 ± 2.7 mm/mm 2 , P = 0.72; CNFD: 22.7 ± 7.6 no/mm 2 vs. 24.25 ± 6.0 no/mm 2 , P = 0.39; CNBD: 31.3 ± 15.8 no/mm 2 vs. 27.6 ± 11.6 no/mm 2 , P = 0.47; CNFA: 0.0050 ± 0.0014 mm 2 /mm 2 vs. 0.0053 ± 0.0012 mm 2 /mm 2 , P = 0.49). A subanalysis was performed on the treatment groups to explore differences in corneal nerve parameters of those with neuropathy (TNSr grade ≥ 1), those without neuropathy (TNSr grade 0), and healthy controls. Only the paclitaxel group was considered in this analysis as the oxaliplatin group had a limited number of patients without neuropathy for comparison (n = 1). Paclitaxel-treated patients with neuropathy were older (P = 0.002) and had worse fine hand dexterity (P = 0.005) compared with paclitaxeltreated patients without neuropathy [fig_ref] Table 1: Clinical Characteristics, Neurophysiological, and Functional Measures of Study ParticipantsRelative dose intensity [/fig_ref]. CNFL, IWL, ANFL, and CNFD were significantly reduced in those with paclitaxel-induced neuropathy (n = 27) compared with healthy controls (P = 0.001, P = 0.004, P = 0.001, and P = 0.002, respectively; [fig_ref] Figure 2: Scatterplots of [/fig_ref]. There was also significant CNFL, IWL, ANFL, and CNFD loss in the paclitaxel-treated group with neuropathy compared with those without neuropathy (n = 13; P = 0.004, P = 0.009, P = 0.005, and P = 0.004, respectively). All data is reported as mean ± SD. Abbreviations: CNFL, corneal nerve fiber length; IWL, inferior whorl length; ANFL, average nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branch density; CNFA, corneal nerve fiber area. CNBD and CNFA were not considered significantly different between those with neuropathy compared with those without neuropathy (P = 0.034 and P = 0.53, respectively) or healthy controls (P = 0.027 and P = 0.25, respectively). There were no significant differences in CNFL, IWL, ANFL, CNFD, CNBD, and CNFA between the patients without neuropathy and healthy controls (P = 0.75, P = 0.90, P = 0.75, P = 0.99, P = 0.99, and P = 0.56, respectively; Supplementary [fig_ref] Table 2: In Vivo Corneal Confocal Microscopy Parameters of Study Participants [/fig_ref]. The relationship between corneal nerve parameters and peripheral nerve measures were also analyzed. IWL reduction correlated with worse functional outcomes in terms of fine dexterity of the upper extremities in chemotherapy-treated patients [fig_ref] Figure 3: Figure 3 [/fig_ref]. When analyzed by chemotherapy type, corneal nerve parameters did not correlate with stage of cancer prior to treatment, number of treatment cycles, mean cumulative dose, relative dose intensity, or period post-treatment for either the paclitaxel-or oxaliplatintreated group [fig_ref] Figure 3: Figure 3 [/fig_ref]. In investigating the association between chemotherapy dosage and neurophysiological measures, the number of treatment cycles for oxaliplatin correlated significantly with Scatterplot showing association between corneal nerve inferior whorl lengths (IWL) and increased grooved pegboard test completion times after accounting for age in partial correlation analysis in chemotherapy-treated patients. . No significant associations were noted between chemotherapy dosage and cancer staging with neurophysiological measures in paclitaxel-treated patients . ## Tnsr scores (supplementary # Discussion The present study explored the differences in corneal nerve parameters in patients with cancer who have completed treatment with two commonly used neurotoxic chemotherapies. A significant reduction in the corneal nerve fiber length particularly at the inferior whorl regions reflected a loss of nerves in patients who completed treatment with paclitaxel and oxaliplatin treatments. Interestingly, corneal nerve loss particularly in the inferior whorl regions, the most distal portion of the corneal nerves, was associated with functional loss in terms of fine dexterity in the upper extremities, suggesting an association between corneal measures and peripheral functional outcomes. Similar central and inferior whorl corneal nerve changes have been found in diabetic peripheral neuropathy. [bib_ref] Use of the inferior whorl to detecting age-related changes in human corneal..., Zhao [/bib_ref] [bib_ref] A comparative study on the diagnostic utility of corneal confocal microscopy and..., Tummanapalli [/bib_ref] Although previous research has shown evidence of corneal nerve dysfunction in patients receiving neurotoxic chemotherapy, [bib_ref] Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal..., Ferdousi [/bib_ref] [bib_ref] Chemotherapy-induced peripheral neuropathy: A prospective study using methods of small fibre function..., Venkitaraman [/bib_ref] the current study involved a more comprehensive assessment of CIPN, including clinical, symptomatology, and functional tests. The large sample size enabled us to detect a significant difference in the corneal nerve measures with greater power. The current novel findings suggest that in vivo corneal confocal microscopy, a noninvasive and rapid test, combined with a validated automated software 27,28 is able to analyze and detect corneal nerve length reduction in chemotherapy-treated patients, particularly those with peripheral neuropathy after treatment. Similar findings have already been demonstrated in patients with diabetes and early corneal changes have been shown to be predictive of neuropathy in prediabetic and diabetic cohorts. [bib_ref] Early detection of nerve fiber loss by corneal confocal microscopy and skin..., Ziegler [/bib_ref] [bib_ref] Corneal confocal microscopy identifies small-fiber neuropathy in subjects with impaired glucose tolerance..., Azmi [/bib_ref] [bib_ref] Corneal confocal microscopy for identification of diabetic sensorimotor polyneuropathy: A pooled multinational..., Perkins [/bib_ref] Although evidence of an association between corneal nerve reduction and local clinical corneal measures, such as corneal sensitivity or ocular surface discomfort remains equivocal, [bib_ref] Degeneration of corneal sensation and innervation in patients with facial paralysis: A..., Zhang [/bib_ref] this study shows evidence of corneal nerve loss in a neurotoxic systemic condition. The current study cannot exclude an even greater effect of corneal nerve fiber loss during or immediately after cessation of chemotherapy treatment, however, even at a delayed time point, a significant loss in corneal nerve fiber lengths was evident. Whereas the CNFL values of healthy controls in the current study seem to be lower than a few studies, [bib_ref] Early nerve fibre regeneration in individuals with type 1 diabetes after simultaneous..., Azmi [/bib_ref] [bib_ref] Corneal nerve fiber size adds utility to the diagnosis and assessment of..., Brines [/bib_ref] the values are comparable to others using similar methodologies. [bib_ref] Relationship between corneal confocal microscopy and markers of peripheral nerve structure and..., Yan [/bib_ref] [bib_ref] Reduced association between dendritic cells and corneal sub-basal nerve fibers in patients..., Klitsch [/bib_ref] [bib_ref] In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in..., Giannaccare [/bib_ref] [bib_ref] Early corneal nerve fibre damage and increased Langerhans cell density in children..., Ferdousi [/bib_ref] This may also be due to the more advanced ages of healthy controls that were involved in the current study for age-matching with the patients with cancer. [bib_ref] The effect of age, gender and body mass index on tear film..., Tummanapalli [/bib_ref] [bib_ref] Normative values for corneal nerve morphology assessed using corneal confocal microscopy: A..., Tavakoli [/bib_ref] A limitation of this study is not stratifying patients according to dry eye severity, which may impact on nerve morphology. [bib_ref] In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in..., Giannaccare [/bib_ref] Future studies will investigate the association between ocular surface discomfort symptomatology with corneal and peripheral nerve involvement. Another limitation is that only one eye was examined, however, previous studies have shown symmetry in healthy corneal nerve fiber parameters and pattern of loss in the context of peripheral neuropathy. [bib_ref] Interocular comparison by in vivo confocal microscopy of the 2-dimensional architecture of..., Misra [/bib_ref] [bib_ref] Standardized baseline human corneal subbasal nerve density for clinical investigations with laserscanning..., Parissi [/bib_ref] [bib_ref] Corneal nerve loss detected with corneal confocal microscopy is symmetrical and related..., Petropoulos [/bib_ref] Additionally, testing one eye reduced the test burden on patients who have a recent history of cancer and chemotherapy treatment, and enabled shortening of the protocol to assist with flow of assessments. A longitudinal study by Ferdousi and colleagues showed that patients with gastrointestinal cancer had reduced CNFL prior to oxaliplatin or cisplatin treatment, whereas CNFL increased from baseline while undergoing treatment. [bib_ref] Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal..., Ferdousi [/bib_ref] The baseline reduction of CNFL may be attributed to existing subclinical peripheral neuropathy, which may be associated with gastrointestinal cancers. [bib_ref] Quantitative sensory testing at baseline and during cycle 1 oxaliplatin infusion detects..., Reddy [/bib_ref] The current study is limited by its cross-sectional design to investigate this association, as it assessed the effects of peripheral neuropathy persisting after treatment on limb function and corneal nerve morphology. However, a remote effect of cancer is unlikely as paraneoplastic effects typically predate cancer diagnosis by months to years. [bib_ref] Update on neurological paraneoplastic syndromes, Höftberger [/bib_ref] Moreover, paraneoplastic syndromes generally improve with treatment of the underlying tumor. The current study demonstrated corneal nerve changes following treatment cessation, which would be more consistent with a treatment effect rather than a remote effect of cancer. The increase in CNFL observed by Ferdousi and colleagues may be an acute aberrant regenerative response of the corneal nerves to the initial chemotherapy insult. Corneal tortuosity or microneuromas would be insightful measures to be included in future studies to investigate putative aberrant regeneration of corneal nerves. Significant corneal nerve reduction at both the central cornea and inferior whorl in paclitaxeltreated patients as compared to mainly inferior whorl length reduction in oxaliplatin-treated patients versus healthy controls may indicate that paclitaxel potentially affects small nerve fibers of the cornea more extensively, despite paclitaxel having a lower proportion of patients with peripheral neuropathy manifesting clinically compared to oxaliplatin-treated patients. This may suggest differences in pathophysiological mechanisms and clinical expression of neurotoxicity between the two chemotherapies. Prior research has shown that oxaliplatin affects large nerve fiber function as indicated by reduced sensory nerve amplitudes and elevated vibration thresholds, whereas small fiber morphology represented by intra-epidermal nerve fiber density involved in pain and temperature signaling seem to be less affected. [bib_ref] Early changes in tests of peripheral nerve function during oxaliplatin treatment and..., Kroigard [/bib_ref] [bib_ref] A quantitative sensory analysis of peripheral neuropathy in colorectal cancer and its..., De Carvalho Barbosa [/bib_ref] In contrary, corneal nerve fiber involvement has been shown to be reduced in mice treated with oxaliplatin. [bib_ref] Corneal nerve plexus changes induced by oxaliplatin chemotherapy and ergothioneine antioxidant supplementation, Tyler [/bib_ref] However, the evidence for small fiber involvement with paclitaxel treatment is more convincing, as shown by reduction in both intraepidermal and corneal nerve fiber densities. [bib_ref] Corneal innervation as a window to peripheral neuropathies, Ferrari [/bib_ref] [bib_ref] Peptidergic intraepidermal nerve fibers in the skin contribute to the neuropathic pain..., Ko [/bib_ref] Motor nerves are usually only minimally involved in CIPN as found by Bennedsgaard and colleagues, [bib_ref] Oxaliplatin-and docetaxel-induced polyneuropathy: Clinical and neurophysiological characteristics, Bennedsgaard [/bib_ref] which is also reflected in the current study. Large sensory nerve fiber function in nerve conduction studies have been shown to be reduced with CIPN. 14 However, a cutoff value for sensory nerve conduction amplitudes for diagnosing CIPN is currently absent, with clinicians and researchers instead proposing a multimodal approach incorporating symptomatology as well as large and small nerve fiber assessments. [bib_ref] Oxaliplatin-and docetaxel-induced polyneuropathy: Clinical and neurophysiological characteristics, Bennedsgaard [/bib_ref] Future investigation of the ability of sensory nerve conduction studies and in vivo corneal confocal microscopy in differentiating patients with and without peripheral neuropathy during development and progression of the condition in longitudinal studies is warranted. Of clinical significance, the paclitaxel group with established neuropathy also showed significantly reduced corneal nerve parameters compared with healthy controls, whereas those without neuropathy had similar levels of corneal nerve fiber lengths to healthy controls. This implies that corneal nerve dysfunction is more evident in patients with CIPN, and raises the possibility of using this technique to monitor nerve function in patients receiving paclitaxel. Although the age of paclitaxel-treated patients with neuropathy is higher than those without neuropathy, it is unlikely that the difference in corneal nerve parameters is due to an age effect as the discrepancy observed exceeds physiological decrease due to normal aging seen in previous studies (−0.06 mm/mm 2 per year in CNFL, [bib_ref] Normative values for corneal nerve morphology assessed using corneal confocal microscopy: A..., Tavakoli [/bib_ref] −0.171 no/mm 2 per year in CNFD [bib_ref] The effect of age, gender and body mass index on tear film..., Tummanapalli [/bib_ref]. Whereas the current study did not find an association between cumulative dose of chemotherapy with corneal nerve loss, the relationship and impact of dosing on corneal nerve fibers may be more appreciated when patients are undergoing treatment. Hence, longitudinal studies that involve prospective monitoring of patients with cancer receiving neurotoxic chemotherapy treatment from baseline are needed to provide a more insightful understanding of the potential effect of treatment on corneal nerve parameters in parallel with neurophysiological measures associated with the development of neuropathy. This will provide a more definitive answer regarding the association between neurotoxic chemotherapy and corneal nerve changes. Future studies should also explore the corneal effects of other neurotoxic chemotherapy drugs, including vinca alkaloids, epothilones, immunomodulatory therapies, and proteasome inhibitors. The current study presents evidence of corneal nerve loss in patients who have completed paclitaxel and oxaliplatin. Patients treated with either paclitaxel or oxaliplatin with more severe reduction in corneal nerve parameters was associated with reduced fine dexterity of the hands. Larger studies involving a longitudinal design will be of value in the future to investigate the potential of in vivo corneal confocal microscopy in identifying corneal nerve changes during treatment. [fig] Figure 1: In vivo corneal confocal microscopy images of the corneal sub-basal nerve plexus. The approximate anatomical locations of the central region and the inferior whorl on the illustration of an eye are shown, with representative corneal nerve images of age-matched participants treated with paclitaxel (age = 58 years) or with oxaliplatin (age = 56 years), and a healthy control (age = 54 years). Sparser nerves are evident in the paclitaxel and oxaliplatin groups compared to the healthy controls. [/fig] [fig] Figure 2: Scatterplots of (a) corneal nerve fiber length (CNFL), (b) inferior whorl length (IWL), (c) average nerve fiber length (ANFL), (d) corneal nerve fiber density (CNFD), (e) corneal nerve branch density (CNBD), and (f) corneal nerve fiber area (CNFA) in patients treated with paclitaxel who have established peripheral neuropathy and those without neuropathy compared with healthy controls. Healthy controls group 1 represent the group of age-, sex-, and body mass index-matched healthy controls for the paclitaxel group. Scatterplots show the mean and standard deviation bars with significant differences highlighted. [/fig] [fig] CNFL − 0 19: (P = 0.12) −0.10 (P = 0.44) −0.002 (P = 0.99) −0.07 (P = 0.56) 0.15 (P = 0.24) IWL −0.24 (P = 0.04) −0.17 (P = 0.16) −0.22 (P = 0.07) −0.33 (P = 0.007) 0.23(P = 0.08) ANFL −0.25 (P = 0.04) −0.15 (P = 0.21) −0.14 (P = 0.27) −0.24 (P = 0.051) 0.22 (P = 0.08) CNFD −0.15 (P = 0.22) −0.01 (P = 0.92) 0.009 (P = 0.94) −0.13 (P = 0.31) 0.19 (P = 0.14) CNBD −0.19 (P = 0.11) −0.05 (P = 0.67) −0.05 (P = 0.67) −0.12 (P = 0.36) 0.14 (P = 0.27) CNFA 0.01 (P = 0.93) −0.04 (P = 0.74) 0.17 (P = 0.16) 0.06 (P = 0.62) −0.10 (P = 0.43) Data are reported as r (P value), with statistically significant correlations highlighted in bold. Abbreviations: CNFL, corneal nerve fiber length; IWL, inferior whorl length; ANFL, average nerve fiber length; CNFD, corneal nerve fiber density; CNBD, corneal nerve branch density; CNFA, corneal nerve fiber area; NCI-CTCAE, National Cancer Institute Common Toxicity Criteria for Adverse Events; EORTC QLQ-CIPN20, the European Organization for Research and Treatment of Cancer Quality of Life -Chemotherapy-induced Peripheral Neuropathy Questionnaire; TNSr, reduced version of Total Neuropathy Scale. [/fig] [fig] Figure 3: Figure 3. Scatterplot showing association between corneal nerve inferior whorl lengths (IWL) and increased grooved pegboard test completion times after accounting for age in partial correlation analysis in chemotherapy-treated patients. [/fig] [table] Table 1: Clinical Characteristics, Neurophysiological, and Functional Measures of Study ParticipantsRelative dose intensity (actual dose received/actual dose prescribed) is expressed as a percentage. Data is reported as mean ± SD, or median [interquartile range, quartile 1 to quartile 3]. Abbreviations: BMI, body mass index; NCI-CTCAE, National Cancer Institute Common Toxicity Criteria for Adverse Events; EORTC QLQ-CIPN20, the European Organization for Research and Treatment of Cancer Quality of Life -Chemotherapy-induced Peripheral Neuropathy questionnaire; TNSr, reduced version of Total Neuropathy Scale. [/table] [table] Table 2: In Vivo Corneal Confocal Microscopy Parameters of Study Participants [/table] [table] Table 3: Correlation Matrix Summarizing Associations Between Corneal Measures, and Neurophysiological and Functional Measures After Adjusting for Age in Chemotherapy-Treated Patients [/table]

Ultrasound: Validity of a Pocket-Sized System in the Assessment of Swallowing Adequate hyoid and laryngeal displacement facilitate safe and efficient swallowing. Although videofluoroscopy is commonly used for assessment of this biomechanical event, ultrasound provides benefits as a radiation-free modality for this purpose. This study investigated validity of a pocket-sized ultrasound system (Clarius™) in the assessment of hyoid and laryngeal excursion. Hyoid excursion and thyrohyoid approximation were concurrently assessed in 20 healthy adults using ultrasound and videofluoroscopy during saliva, liquid, and puree swallowing. Correlation analyses were performed to evaluate validity. There was a strong and moderate positive association between ultrasound and videofluoroscopic measurements of hyoid excursion during dry and liquid swallowing, respectively. No evidence for a significant association was found for ultrasound and videofluoroscopic measurements of hyoid excursion for puree swallowing and of thyrohyoid approximation for any bolus type. Further work towards improved validity is necessary prior to clinical transfer of the pocket-sized Clarius™ system in clinical swallowing assessment. # Introduction Adequate hyoid and laryngeal displacement are critical for safe and efficient swallowing. Hyoid excursion during swallowing facilitates opening of the upper oesophageal sphincter and, therefore, bolus passage from the pharynx into the oesophagus [bib_ref] Hyoid motion during swallowing: Factors affecting forward and upward displacement, Ishida [/bib_ref]. The intra-swallow approximation of the thyroid cartilage and hyoid bone plays a role in airway protection, through supraglottic shortening and epiglottic deflection [bib_ref] Intrinsic fibre architecture and attachments of the human epiglottis and their contributions..., Vandaele [/bib_ref]. Objectively assessing hyoid and laryngeal displacement is of interest in swallowing evaluation. Videofluoroscopic imaging is a commonly used modality in clinical practice to examine intra-swallow movement of the hyolaryngeal complex. However, its use for prolonged examinations and repeated testing is limited, due to radiation exposure. Further, access to a videofluoroscopy suite may be challenging for a number of populations [bib_ref] Role of videofluoroscopy in evaluation of neurologic dysphagia, Rugiu [/bib_ref]. The use of ultrasound in swallowing assessment yields benefits as a radiation-free imaging modality [bib_ref] Application of ultrasonography in assessing oropharyngeal dysphagia in stroke patients, Hsiao [/bib_ref]. While ultrasound is not a standard assessment tool in daily clinical practice, there are reports in the literature documenting its use in the assessment of hyoid excursion [bib_ref] Application of ultrasonography in assessing oropharyngeal dysphagia in stroke patients, Hsiao [/bib_ref] [bib_ref] Usefulness of submental ultrasonographic evaluation for dysphagia patients, Lee [/bib_ref] and thyrohyoid approximation [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref] [bib_ref] Sonographic analysis of laryngeal elevation during swallowing, Kuhl [/bib_ref]. With technical advancements, pocket-sized ultrasound systems have been developed. As an adjunct to diagnostic swallowing evaluation, these devices could be useful for application in patients who are not able to mobilise out of bed or patients in community settings where transfer to a videofluoroscopy suite is challenging. Further, this new ultrasound technology is smaller and more affordable within the reach of allied health resourcing as compared to larger ultrasound systems including laptop-driven ultrasound devices. Increased clinical availability may be beneficial for some patient populations and can potentially gain importance during times of restricted access to instrumental swallowing assessment in the hospital due to epidemic outbreaks, such as COVID In the literature, there is evidence of validity when measurements of hyoid and laryngeal excursion derived from ultrasound instrumentation and from videofluoroscopy are compared. Two studies documented a strong correlation between ultrasound and videofluoroscopic measurements of hyoid excursion in patients with dysphagia [bib_ref] Application of ultrasonography in assessing oropharyngeal dysphagia in stroke patients, Hsiao [/bib_ref] , while one study reported no significant difference between ultrasound and radiographic measurements of thyrohyoid approximation in subjects with dysphagia [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref]. Data are lacking regarding validity of pocket-sized ultrasound systems; yet validity data are essential prior to implementation of this technology into routine clinical practice. This preliminary study investigated if valid measurements of hyoid excursion and thyrohyoid approximation could be derived from a pocket-sized ultrasound system in healthy subjects, as determined by comparison with videofluoroscopic measures. Validity data of ultrasound in the assessment of healthy swallowing are required as swallowing evaluation aims to differentiate between normal and impaired swallowing function in subjects with suspected dysphagia. Given the undisputed use of videofluoroscopy for visualisation of swallowing biomechanics, a comparison of ultrasound against videofluoroscopy is appropriate for validation purposes. The use of videofluoroscopy allows for concurrent radiographic and ultrasound imaging to ensure that the same swallowing event is captured by the two instruments. # Materials and methods ## Participants This study recruited 20 healthy participants for one session (five participants per age group, namely 20-39, 40-59, 60-79, and 80 + years) with females and males equally represented across the age groups. Participants were recruited via advertisement and an in-house volunteer database. Exclusion criteria, based on participant' report, included history of swallowing impairment or current swallowing difficulty, neurological or muscular disease, head and neck tumour or anatomical abnormalities of the head and neck region, drugs, which might have an impact on swallowing, or pregnancy. The study was approved by the appropriate regional health ethics committee (HEC 2017/20). Verbal and written information about the study was provided and informed consent was obtained from the participants. ## Instrumentation Ultrasound high-resolution live imaging was performed using a curvilinear Clarius™ scanner (C3, Clarius, Burnaby, British Columbia, Canada; frequency range: 2-6 MHz, depth: 3-30 cm, size: 169 mm × 105 mm × 41 mm) that conforms the anatomy of the chin. The scanner connected wirelessly to the Clarius™ application software (Burnaby, British Columbia, Canada) that was installed on an iPad (screen size 20 cm × 15 cm) with ultrasound recordings visualised on the iPad (20 frames/s). For videofluoroscopic imaging, the Fluorostar 7900 (GE OEC Medical Systems GmbH, Wendelstein, Germany) was used (25 frames/s). ## Procedure Participants were seated in a chair placed within the C-arm of the fluoroscope. A calibration disc of known size was taped to the participant's lateral face for calibrated post hoc measurement. Ultrasound and videofluoroscopic imaging was performed concurrently. Data collection included measures of hyoid excursion and thyrohyoid approximation [fig_ref] Figure 1: Radiographic image depicting the structures of interest 1 3 [/fig_ref]. Order of data acquisition was randomised between the two measures. Data acquisition involved swallows of saliva, 5 mL water, and 5 mL apple sauce (Wattie's™). To minimise radiation exposure, participants performed each measure once only per consistency (six swallows per participant in total). The order in which bolus types were presented was kept consistent across participants and sessions as the sequence may influence the findings. Saliva was presented first followed by water and puree to reflect the order routinely followed in clinical practice. Quantities were measured with a syringe; liquid boluses were offered in a 20 mL plastic cup and puree boluses with a spoon. Participants were instructed to hold the bolus in the mouth. Once the scanner was placed, they were asked to swallow as naturally as possible, without accommodating head position to the scanner. The principal researcher collected data from all participants. Ultrasound data collection involved scanning, image selection from the recorded video and measurement. Image selection and measurement were performed online on the iPad immediately after each swallow. A randomly selected 20% of swallows per measure and instrument were measured offline on a second occasion by the principal researcher for assessment of intra-rater reliability. A period of at least 11 days between data collection and offline measurement was implemented to avoid recall. A second rater measured another randomly selected 20% of the data offline for assessment of inter-rater reliability. For both instruments, reliability assessment included image selection from the recorded cine and measurement. Raters were blinded to the measurements performed by the other rater. Both researchers were trained in ultrasound and videofluoroscopic data extraction for purpose of this study. Training was consensus based rather than standardised. Prior to assessment of interrater reliability, raters agreed on measurement techniques with a written reference document with measurement guidelines established. ## Recording Aquasonic transmission gel was used for acoustic coupling before the ultrasound scanner was placed manually on the skin surface of the participant's neck. Consistent scanner position was maintained and minimal pressure against the skin was applied throughout data acquisition [bib_ref] A guide to analysing tongue motion from ultrasound images, Stone [/bib_ref] to minimise the impact of the scanner on swallowing function. Specific pre-set exam types with customised features, such as scanning depth or gain were selected per measure. For optimal image quality, manual accommodation of depth, gain, and display brightness was performed per measure and participant if required. Structures were viewed upside down according to previous studies assessing validity and/or reliability of hyoid excursion [bib_ref] Intra-and inter-rater reliability for analysis of hyoid displacement measured with sonography, Macrae [/bib_ref] and thyrohyoid approximation [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref]. Swallowing events were recorded as individual video-clips of 20 s. For imaging hyoid excursion, settings included a depth between 7 and 10 cm, a frequency of 4 MHz, and single focus. Sagittal sonography was performed with the scanner positioned at right angles to the floor of mouth muscles. The scanner was placed midline, capturing the acoustic shadow of the mandible on one side and on the other side the acoustic shadow of the hyoid [bib_ref] Intra-and inter-rater reliability for analysis of hyoid displacement measured with sonography, Macrae [/bib_ref]. Inferiorly, the surface of the tongue was visible. For recording of thyrohyoid approximation, settings included a depth between 1-7 cm, a frequency of 5 MHz, and dual focus. The scanner was positioned midline or slightly off midline in sagittal plane approximately overlying the thyrohyoid muscle. This allowed visualisation of key features, including the acoustic shadow of the hyoid on one side, and the acoustic shadow of the thyroid cartilage on the other side [bib_ref] Sonographic analysis of laryngeal elevation during swallowing, Kuhl [/bib_ref]. [fig_ref] Figure 2: Scanner placement for assessment of hyoid excursion [/fig_ref] depicts the placement of the scanner during data collection. For videofluoroscopic examination, a low dose continuous cine mode was selected. Recordings comprised mandible (anteriorly), nasal cavity (superiorly), cervical spine (posteriorly), proximal oesophagus, and trachea (inferiorly). ## Data extraction For online measurement of ultrasound data, specific images were selected after each swallow by navigating through the recorded video on the iPad. For data extraction of hyoid excursion, two images were selected, one representing the peak of hyoid displacement and one with the hyoid at rest position post swallow. The peak position image was defined as the one showing the smallest distance between shadow cast by the hyoid and shadow cast by the mental spine of the mandible [fig_ref] Figure 3: Sonogram of the hyoid at rest position [/fig_ref]. The extent which the hyoid travels was expressed as a percentage of the distance at maximal displacement from rest ((rest distance between mental spine of the mandible and hyoid -maximal distance between the two structures)/rest distance between the two structures) × 100 [bib_ref] Intra-and inter-rater reliability for analysis of hyoid displacement measured with sonography, Macrae [/bib_ref]. Data extraction of thyrohyoid approximation involved selection of one image showing hyoid and thyroid cartilage maximally approximated intra-swallow and the other with the two structures at rest, post-swallow [fig_ref] Figure 4: Sonogram of the distance [/fig_ref]. Thyrohyoid approximation was expressed as a percentage of the distance between thyroid cartilage and hyoid at maximal approximation from rest [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref] [bib_ref] Sonographic analysis of laryngeal elevation during swallowing, Kuhl [/bib_ref]. Videofluoroscopic recordings were analysed using ImageJ software (U.S. National Institutes of Health, Bethesda, Maryland, USA). Images were calibrated for measurement. For data extraction of hyoid excursion, the percentage change from the position at rest post swallow and at maximal anterior displacement was calculated [fig_ref] Figure 5: Hyoid excursion assessed using videofluoroscopy [/fig_ref]. For data extraction of thyrohyoid approximation, one image was selected depicting the hyoid and thyroid cartilages at maximal approximation intra-swallow and one showing these structures at rest, post-swallow . Percentage approximation was calculated from rest. # Statistical analysis Means and standard deviations were calculated for both measures and both instruments across participants. Using R software, intra-class correlation estimates (ICC) were calculated based on mixed effects analyses [bib_ref] Fitting linear mixedeffects models using {lme4}, Bates [/bib_ref] , if the assumptions for analyses were met. Intra-rater reliability was calculated using a two-way mixed effects model (ICC, inter-rater reliability using a two-way random effects model (ICC [bib_ref] Hyoid motion during swallowing: Factors affecting forward and upward displacement, Ishida [/bib_ref] for agreement of single measures. A likelihood ratio test was used to test for a potential bolus effect. The full model which included bolus as a fixed effect was compared to the reduced model that did not contain bolus as a fixed [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref] [bib_ref] Sonographic analysis of laryngeal elevation during swallowing, Kuhl [/bib_ref] for evaluation of thyrohyoid approximation. One calliper was placed at the beginning of the anterior border of the shadow of the hyoid (shadow on the left of the windows) or at the opacity representing the hyoid. The other calliper was placed at either the onset of the shadow cast by the thyroid cartilage (shadow on the right of the images) or at the bright opacity at the superior border of the thyroid cartilage. Of each of the two points, the one that was visible in both images was selected effect. If there was a significant bolus effect, analysis using the full model was continued to remove variability due to bolus consistency. If there was no bolus effect, the reduced model was used. Interpretation of reliability findings was based on published criteria: poor reliability (ICC < 0.50), moderate reliability (ICC 0.50-0.75), and good reliability (ICC > 0.75). A Pearson's correlation coefficient (r) was calculated using R softwareto determine strength and direction of a linear relationship between ultrasound and videofluoroscopic measurements [bib_ref] What we need to know when calculating the coefficient of correlation?, Udovičić [/bib_ref]. A p-value of ≤ 0.05 was considered significant. If the assumptions of a Pearson's correlation analysis were violated, a non-parametric Kendall's correlation coefficient (tau) was calculated. Published guidelines were used for interpretation. Limits of agreement were calculated post hoc to quantify the range of agreement between ultrasound and videofluoroscopic measurements as measurement methods differed across imaging modalities. A priori limits of agreement were not defined as the intent of the study was not to evaluate the potential of ultrasound to replace videofluoroscopy. Differences between paired measurements derived from the two instruments were plotted against the mean of the paired measurements (Bland-Altman plot) [bib_ref] Measurement in medicine: the analysis of method comparison studies, Altman [/bib_ref]. If the assumptions for agreement analyses were violated, post hoc analyses were not further continued. Hyoid excursion assessed using videofluoroscopy. Dashed measurement lines for calculation of the distance from hyoid to mandible at rest (a) and at maximal hyoid displacement (b). The white drawings were used to define the measurement point at the mandibular prominence. The inferior-anterior part of the hyoid and the mandibular prominence were used as measurement points to approximate the measurement methods of the radiographic images to those for ultrasound and based on reported methodology in the literature [bib_ref] Coordinate mapping of hyolaryngeal mechanics in swallowing, Thompson [/bib_ref] Fig. [bib_ref] Application of ultrasonography in assessing oropharyngeal dysphagia in stroke patients, Hsiao [/bib_ref] Thyrohyoid approximation assessed using videofluoroscopy. Dashed measurement lines depicting the distance between anteriorinferior aspect of the hyoid and the anterior edge of the inferior end of the thyroid cartilage at rest (a) and at maximal approximation (b). As opposed to ultrasound, the inferior rather than the superior border of the thyroid cartilage was chosen as the upper border was often not sufficiently distinct for measurement. Additionally, even if it was visible, the upper border of the thyroid cartilage superimposed the hyoid at maximal excursion in some cases; hence, calculation of percentage approximation would yield more than 100% # Results Ten females and 10 males completed the study protocol. Descriptive statistics of ultrasound and videofluoroscopic data are reported in [fig_ref] Table 1: Videofluoroscopic and ultrasound measures [/fig_ref]. For reliability of videofluoroscopic data extraction, measures of thyrohyoid approximation were excluded from analysis if the thyroid cartilage was insufficiently visible for measurement due to weak contrast (n = 5 for intra-rater reliability; n = 3 for inter-rater reliability) or if the calibration disc was not captured within the frame (n = 1 for intra-rater reliability). As illustrated in , the findings indicate good intra-rater reliability for videofluoroscopic data extraction; inter-rater reliability ranged from poor to moderate. Intra-rater reliability of ultrasound data extraction ranged from poor to moderate, interrater reliability was moderate. Videofluoroscopic data were excluded from validity analyses if the visibility of the calibration disc or of target structures were insufficient for measurement purposes. Out of 60 acquired measurements for hyoid excursion, 58 were analysed. For thyrohyoid approximation, 44 of the 60 measurements were included into analyses. As depicted in, there was evidence of an association between ultrasound and videofluoroscopic measurements of hyoid excursion during dry and liquid swallowing. The positive correlation was strong for dry swallowing and moderate for liquid swallowing. No evidence of a significant association during puree swallowing was found. There was no evidence of a significant association between ultrasound and videofluoroscopic measurements of thyrohyoid approximation during swallowing of any bolus types. For hyoid excursion during dry, liquid, and puree swallowing, the upper limits of agreement for ultrasound measurements were calculated at 10.86 percentage change for dry swallows, 14.50 percentage change for liquid swallows, and 16.48 percentage change for puree swallows. The lower limits were calculated at − 6.27 percentage change for dry swallows, − 6.71 percentage change for liquid swallows, and − 15.15 percentage change for puree swallows. This indicates that ultrasound measures of hyoid excursion during dry swallowing, for example, may range from 10.86 percentage change above to 6.27 percentage change below videofluoroscopic measures. Upper limits of agreement for ultrasound measurements of thyrohyoid approximation were calculated at 44.67 percentage change for dry swallows, 35.67 percentage change for liquid swallows, and 42.92 percentage change for puree swallows. The lower limits were calculated at − 25.94 percentage change for dry swallows, − 26.18 percentage change for liquid swallows, and − 27.72 percentage change for puree swallows. The Bland Altman plots are depicted in Figs. 7 and 8. The plots indicate significant bias between ultrasound and videofluoroscopy for hyoid excursion during dry and liquid swallowing, as the theoretical mean difference of zero on the Y-axis does not lie within the 95% confidence interval of the mean difference [fig_ref] Figure 7: Bland Altman plot for hyoid excursion during dry [/fig_ref]. # Discussion Ultrasound measurements of hyoid excursion and thyrohyoid approximation assessed using the Clarius™ system in healthy subjects poorly reflect measurements derived from videofluoroscopy, with the exception of hyoid excursion during saliva swallowing. There are several factors that may explain these findings. Validity of any measure requires consistent measurement acquisition. Thus, validity findings may be explained by insufficient reliability of ultrasound measurement acquisition. Poor to moderate reliability may suggest that the image quality of the Clarius™ system is insufficient for evaluation of hyoid and laryngeal excursion. Yet, more reliability data of the Clarius™ system are required for an in-depth interpretation of the findings, given that reliability was evaluated for only a small sub-set of the data. It cannot be excluded that due to difficulties in visualisation of laryngeal structures in some participants, structures other than the thyroid cartilage such as the epiglottis or artefacts were erroneously used for measurement. While previous studies performed ultrasound and videofluoroscopic imaging separately [bib_ref] Application of ultrasonography in assessing oropharyngeal dysphagia in stroke patients, Hsiao [/bib_ref] [bib_ref] Ultrasonographic evaluation of hyoid-larynx approximation in dysphagic stroke patients, Huang [/bib_ref] , concurrent data acquisition was performed in this study to eliminate test-retest variability. However, concurrent collection of ultrasound and videofluoroscopic data may partially explain insufficient validity and reliability, as this method posed a technical challenge that may have negatively affected image quality. Stable scanner placement, known to be critical for quality of ultrasound recordings [bib_ref] A guide to analysing tongue motion from ultrasound images, Stone [/bib_ref] , may have been compromised as the researcher kept an arm-length distance from the participant for radiation safety [bib_ref] Technical aspects of a videofluoroscopic swallowing study, Peladeau-Pigeon [/bib_ref]. For future studies, advantages of the use of a stabilisation unit for scanner , and puree swallowing (c) assessed using ultrasound and videofluoroscopy (VFSS). The unit of the X-and Y-axis is percentage change. The thick dashed red line represents the mean difference between ultrasound and videofluoroscopic measurements; the thin dashed red lines represent the 95% confidence interval of the mean difference. The thick dashed blue lines represent the upper and lower limits of agreement; the thin dashed blue lines represent the 95% confidence intervals placement during videofluoroscopic imaging would need to be balanced against the issues related to the use of these devices, such as the risk of loss of contact between the scanner and the skin [bib_ref] Variability in ultrasound measurement of hyoid hone displacement and submental muscle size..., Perry [/bib_ref]. Study findings suggest that the two instruments not only poorly relate but also produce considerably different measurements of hyoid excursion and thyrohyoid approximation, as indicated by the Bland-Altman plots. For measurement of thyrohyoid approximation, the superior border of the thyroid cartilage was used in ultrasound, while the inferior border was used for videofluoroscopic images. This difference in measurement methods across instrumentation may explain, in part, that ultrasound and videofluoroscopic values of thyrohyoid approximation were considerably different. Another factor that may have compromised validity and reliability is the ultrasound technology. A thorough comparison of technical aspects between pocket-sized and larger ultrasound devices is not possible, as technical details are not available for other published studies. Having used an ultrasound system that is in the early stage of development, substantial technical issues were encountered with its frequent use during data collection. Unstable connection between the Clarius™ scanners and the iPad resulted in frequent interruption of data collection. Other technical issues, such as a malfunctioning scrolling function for video review likely impacted the study findings. There are limitations of this preliminary research, including the sample size and number of measurements. Results may have been negatively impacted by technical difficulties. Future studies will benefit from improved radiographic contrast for visualisation of structures such as the thyroid cartilage. With a clearer visualisation of the lower border of the thyroid cartilage, equal measurement methods across instruments would be possible for evaluation of thyrohyoid approximation. This may increase agreement across instruments. However, prior to a study assessing validity in # Conclusions Given the importance of validity and reliability of instrumentation used for diagnostic purposes, the clinical use of the Clarius™ system for swallowing assessment is not indicated at this time. Future research is required to clarify whether our findings reflect technical limitations of the Clar-ius™ system or of pocket-sized technology more generally. This will further elucidate if new ultrasound technology may be used in routine clinical assessment of swallowing. Further research is required to clarify whether reliability of ultrasound data explains insufficient validity. [fig] Figure 1: Radiographic image depicting the structures of interest 1 3 [/fig] [fig] Figure 2: Scanner placement for assessment of hyoid excursion (at the left of the image) and thyrohyoid approximation (at the right of the image) [/fig] [fig] Figure 3: Sonogram of the hyoid at rest position (a), and at maximal displacement (b) for evaluation of hyoid excursion. For measurement, the line of best fit (Line A) was drawn along the anterior border of the shadow cast by the hyoid (the shadow at the right of the images). For Line B, one calliper was placed at the posterior border of the onset of the shadow created by the mental spine of the mandible (shadow on the left of the images). The second calliper was placed at the intersection point with Line A at the onset of the shadow cast by the hyoid [/fig] [fig] Figure 4: Sonogram of the distance (Line D) between hyoid and upper border of thyroid cartilage at rest (a) and at maximal approximation (b) [/fig] [fig] Figure 5: Hyoid excursion assessed using videofluoroscopy. Dashed measurement lines for calculation of the distance from hyoid to mandible at rest (a) and at maximal hyoid displacement (b). The white drawings were used to define the measurement point at the mandibular prominence. The inferior-anterior part of the hyoid and the mandibular prominence were used as measurement points to approximate the measurement methods of the radiographic images to those for ultrasound and based on reported methodology in the literature [23] [/fig] [fig] Figure 7: Bland Altman plot for hyoid excursion during dry (a), liquid (b) [/fig] [fig] Figure 8: Bland Altman plot for thyrohyoid approximation during dry (a), liquid (b), and puree swallowing (c) assessed using ultrasound and vide- [/fig] [table] Table 1: Videofluoroscopic and ultrasound measures: mean (standard deviation) VFSS videofluoroscopic swallowing study. Intra-and inter-rater reliability for videofluoroscopic and ultrasound measures ICC intra-class correlation coefficient, CI confidence interval, VFSS videofluoroscopic swallowing study, [] assumptions for analysis are not met. [/table]

Bacterial F-type ATP synthases follow a well-choreographed assembly pathway [bib_ref] A second missense mutation in the mitochondrial ATPase 6 gene in Leigh's..., De Vries [/bib_ref] [bib_ref] ATP synthase and the actions of inhibitors utilized to study its roles..., Hong [/bib_ref] [bib_ref] Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease, Cha [/bib_ref] [bib_ref] Electric field driven torque in ATP synthase, Miller [/bib_ref] [bib_ref] Assembly of the Escherichia coli F O F 1 ATP synthase involves..., Deckers-Hebestreit [/bib_ref] [bib_ref] Mitochondrial diseases and ATPase defects of nuclear origin, Houštěk [/bib_ref] [bib_ref] Assembly of the Escherichia coli F O F 1 ATP synthase involves..., Deckers-Hebestreit [/bib_ref] [bib_ref] Structural basis for a unique ATP synthase core complex from Nanoarcheaum equitans, Mohanty [/bib_ref] [bib_ref] The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals..., Cross [/bib_ref] [bib_ref] The preferred stoichiometry of c subunits in the rotary motor sector of..., Jiang [/bib_ref] [bib_ref] Constant c 10 ring stoichiometry in the Escherichia coli ATP synthase analyzed..., Ballhausen [/bib_ref] [bib_ref] Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H..., Mitome [/bib_ref] [bib_ref] The c 15 ring of the Spirulina platensis F-ATP synthase: F 1..., Pogoryelov [/bib_ref] [bib_ref] Structure of the mycobacterial ATP synthase F O rotor ring in complex..., Preiss [/bib_ref] [bib_ref] Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria, Watt [/bib_ref] [bib_ref] Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei, Schulz [/bib_ref] [bib_ref] Neither helix in the coiled coil region of the axle of F..., Hossain [/bib_ref] [bib_ref] The ATP synthase-a splendid molecular machine, Boyer [/bib_ref] [bib_ref] Partial purification of active delta and epsilon subunits of the membrane ATPase..., Smith [/bib_ref] [bib_ref] Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine..., Smith [/bib_ref] [bib_ref] lnhibitory properties of endogenous subunit ε in the Escherichia coli F 1..., Laget [/bib_ref] [bib_ref] Functional production of the Na + F 1 F O ATP synthase..., Brandt [/bib_ref] [bib_ref] An intermediate step in the evolution of ATPases -a hybrid F O..., Fritz [/bib_ref] [bib_ref] Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and..., Yi [/bib_ref] [bib_ref] Assembly of F O sector of Escherichia coli H + ATP Synthase, Hermolin [/bib_ref] [bib_ref] Assembly of the Escherichia coli F O F 1 ATP synthase involves..., Deckers-Hebestreit [/bib_ref] [bib_ref] Modular assembly of yeast mitochondrial ATP synthase, Rak [/bib_ref] [bib_ref] Reconstitution of a functional coupling factor from the isolated subunits of Escherichia..., Dunn [/bib_ref] [bib_ref] Reconstitution of ATPase activity from the isolated α, β and γ subunits..., Futai [/bib_ref] # Results Role of ATP for the in vitro assembly process of bacterial F-type ATP synthases. As the assembly mechanism of the bacterial F-type ATP synthase, in particular, for the soluble F 1 module, is mostly still unclear, we aim to shed light on this process. We determine individual subunit interactions, relevant conditions and also investigate whether the assembly of all different subunits follows a specific order. In a first step, we purified recombinant subunits (α, β, δ, and γε) of the soluble part F 1 individually. Attempts to express the γsubunit in E. coli separately yielded insoluble aggregates (inclusion bodies). Therefore, we cloned the genes atpG and atpC bicistronically in the expression vector and purified the central stalk γε to generate higher complex stability and protein solubility . To investigate individual assembly steps we incubated different subunit combinations under different assembly conditions and determined with LILBID-MS and High-performance liquid chromatography (HPLC) experiments, which complexes did or did not form. The resulting (sub)complexes represent key steps in the assembly process. To ensure the relevance of the observed in vitro subcomplexes for the in cellula processes in the cell and for comparison of structure, complex stability, and bioactivity, we then set out to produce homologous subcomplexes in cellula. Incubation of recombinant α and β in vitro in ammonium acetate buffer without any additives shows no specific α/βoligomerization into higher subcomplexes , suggesting that our experiment is missing something essential. The cytoplasmic concentrations of ATP in active cells are known to be approximately in the range of 2-5 mM [bib_ref] Role of charged residues in the catalytic sites of Escherichia coli ATP..., Ahmad [/bib_ref] , which prompted us to test the relevance of ATP for this assembly. As MS resolution can be affected by additives, we stayed in the lower range of the native concentration and incubated the same proteins with 2 mM ATP and MgCl 2 . (Effect of ATP/Mg 2+ on LILBID-MS spectra is shown in . demonstrates that the addition of ATP/Mg 2+ leads to specific αβ heterodimer formation only (no unspecific homodimers) with a charge state distribution (−1 to −3). Interestingly, no higher α/ β subspecies can be identified, even though ATPases are known to form a hexameric head, containing three αand β-subunits, respectively. As in cellula comparison, we purified and isolated an αβ complex from E. coli, which we then analyzed under the same in vitro conditions. The in cellula αβ complex showed the same heterodimer and no higher oligomeric states either (Supplementary [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref]. It should be noted that the in vitro formed αβ heterodimers need the presence of ATP/Mg 2+ not only for formation but in order to remain stable, as can be seen from measurements, for which the buffer is slowly diluted during the LILBID-MS run . Reduction of the ATP/ Mg 2+ concentration during LILBID-MS goes along with an increase in the signal resolution but reduced complex stability. Interestingly the in cellula αβ complex seem less prone to dissociation at reduced ATP/Mg 2+ concentration (Supplementary [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] , which is the first hint that assembly of α and β in vitro and in cellula might lead to slightly different heterodimers. For comparison with the LILBID-MS results we investigated the in vitro assembly of subunit α and β by an alternative analytical method: size exclusion chromatography (SEC) by HPLC, using the SEC column with a protein separation range of 10-700 kDa . The chromatograms show a shift into a shorter elution peak time confirming αβ heterodimer formation only upon the addition of 2 mM ATP and MgCl 2 . LILBID and SEC experiments both showed that the additives ATP/ Mg 2+ are crucial for the in vitro assembly of subunits α and β of Na [formula] + -F 1 F O -ATP synthases of A. woodii. [/formula] Assembly studies of single isolated subunits with next neighbors. After determination of the appropriate assembly conditions, we are now in a position to analyze the entire assembly process including subunits α, β, δ, and γε. As it was not yet clear, if the assembly follows a clear step by step order we tested every possible combination of subunits for binding in the same manner as described above and investigated which prior binding steps are prerequisite for other binding steps to follow (all binding experiments are listed in [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref]. [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] gives an overview of all possible subcomplexes which we observed after incubation of different combinations of subunits. This provides information about next neighbor interactions and the order of assembly steps within the F 1 complex. As mentioned above we can observe αβ heterodimer assembly under the right assembly conditions-but no higher-order oligomers, if just these two subunits are present . Similarly, we observe that upon incubating either subunit α or β with the γε complex, a single subunit α or β can bind to the γε complex, forming αγε or βγε complexes, respectively. These complexes are seen under soft laser conditions (laser energy 8 mJ). Already medium laser desorption conditions, allow the ε-subunit to dissociate from the γε complex [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref] , giving rise to αγ or βγ complexes. This shows that α or β bind to the γ-subunit, whereas the ε-subunit is only weakly bound to γ and not directly involved in the interaction with α or β. As subunit α and β alone can only form heterodimers and only a single α or β can bind to γ if incubated alone, the next step was to purify and incubate recombinant α, β, and γε in presence of 2 mM ATP/MgCl 2 . Mass spectrometric analysis showed the formation of the α 3 β 3 γε-subcomplex which we could identify at a mass of 378 ± 13 kDa (theoretical protein mass 378.272 kDa) and a charge state distribution from −1 to −5 . In contrast, no higher subcomplex formation was observed if the same experiment was performed without ATP/Mg 2+ . This implies that these additives are vital for the entire in vitro reconstitution process providing complex stability. Including the δ subunit in the incubation then led to its association with the preformed α 3 β 3 γε complex resulting in the fully assembled F 1 -complex α 3 β 3 δγε with a determined protein mass of 399 ± 8 kDa (theoretical mass: 399.809 kDa) and a charge state distribution of −1 to −3 . The spectrum shows the fully assembled F 1 -complex, as well as subcomplexes α 2 β 2 γε, αβγε, and αβγ, which could stem from incomplete assembly or dissociation. Surprisingly our spectra show no subcomplexes with an odd number of α or β subunits, indicating that the αβ heterodimer is the stable basic binding block from which the hexamer is built up. No δ is seen binding to any of the single subunits, which is in line with studies showing the interaction of δ with α-subunits only if complexed with other F 1subunits [bib_ref] Assembly of the stator in Escherichia coli ATP synthase. Complexation of α..., Senior [/bib_ref]. No δ binding is seen for any of the mentioned subcomplexes either [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] and [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref] , suggesting the fully assembled αβ-hexamer to be a prerequisite for binding of δ. Combined the observed on-pathway subcomplexes en-route to the F 1 complexes allow proposing the ATPase assembly to occur only via the binding of preformed αβ heterodimers onto the γ-subunit, to form the hexameric head, which then allows for binding of δ. As γ could not be purified separately we could not perform binding experiments with and without ε to determine, if ε is a prerequisite for binding of the αβ dimers onto γ, or if the hexameric head might form around γ independently of ε. For comparison, we then attempted to purify and isolate all proposed on-pathway subcomplexes in cellula. All expected complexes [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] could be purified. In addition, we could purify complexes lacking ε, but still include the hexameric head. This allowed us to conclude that the hexameric head forms around γ independently of ε, which therefore has multiple entry points into the assembly pathway. Mass spectrometric analysis [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] reveals, as for the in vitro complexes, only complexes with the same amount of α and β subunits, supporting the αβ heterodimer as a basic building block. Incubation of δ with [formula] + -------------- a b c d 3- δ δ 1- Fig. 3 [/formula] ATP/Mg 2+ dependence of the in vitro assembly process of the full F 1 complex of Na + -F 1 F O -ATP synthases of A. woodii. The α-(light blue), βsubunit (dark blue), γε-complex (dark and light green) were incubated at 4°C for 1 h either without (a) or with (c) 2 mM ATP and 2 mM MgCl 2 . Then the δ-subunit (yellow) was added (b, d). a, b No specific assemblies into higher F 1 -subcomplexes could be detected without ATP/Mg 2+ . The appearance of the γ subunit alone is due to dissociation. (See as well . c, d In presence of ATP/Mg 2+ the fully assembled F 1 can be identified with additional F 1 subcomplexes (e.g., α 2 β 2 γε, αβγε, αβγ) implying pairwise specific αβ heterodimer association. The δ-subunit can only bind when a complex containing the hexameric head domain α 3 β 3 is already preformed. Source data are provided as a Source Data file. the in cellula complex as well confirms our in vitro results, which showed δ to only bind onto the complete αβ hexamer. Interestingly, the in cellula α 3 β 3 γε complex [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] shows a significantly higher complex stability even without the addition of ATP/Mg 2+ than the in vitro complex (Supplementary [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] , f). This difference could be very interesting, as it might show that there are deviations in the in cellula and in vitro assembly, possibly owing to structural differences of the proteins. An alternative explanation would be steric hindrances due to the tags, which are part of our in vitro purified proteins. The in vitro studies were performed on N-terminal StrepI-α and His 6 -β, C-terminal His 6 -δ, and γε (His 6 -tag located on subunit ε), whereas the in cellula complex was expressed with only one N-terminal His 6 -tag on the β-subunit. For comparison, we purified a complex, which includes the same tags on every subunit as our isolated purified subunits, which we name α 3 β 3 γε* for distinction. The mass spectrum in [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] shows that the tags are not responsible for destabilization of the complex. Remarkably the in cellula complexes are not only stable without ATP/Mg 2+ addition that is crucial for the in vitro complexes, but even the last assembly step association of δ to in cellula purified α 3 β 3 γε and α 3 β 3 γε* pre-complexes forming the full F 1 -complex [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] is ATP/Mg 2+ independent. The role of ATP hydrolysis vs. nucleotide binding for the in vitro assembly process of bacterial F 1 -ATPase. The important role of ATP hydrolysis for the function of ATP synthases is unquestioned and we have as well observed the significance of ATP for complex assembly. Nevertheless, it is not yet clear, whether ATP hydrolysis or only nucleotide binding is required for the F 1 assembly process. Therefore, we repeat the described assembly experiments with α, β, and γε, but replace ATP by non-hydrolysable ATP analogs, such as ADP, adenylyl-imidodiphosphate (AMP-PNP) and adenosine 5′-[γ-thio]triphosphate with a concentration of ATP/ATP analogs and Mg 2+ at a concentration of 2 mM. The obtained MS spectra with non-hydrolysable ATP analogs shows the successful formation of larger F 1subcomplexes. Interestingly all ATP analogs allow the formation of α 3 β 3 γε apart from AMP-PNP, which generates only α 2 β 2 γε subcomplexes and not the expected α 3 β 3 γε complex; which could be due to differences in the chemical geometry of AMP-PNP. We speculate that the free electron pair of nitrogen might sterically inhibit the assembly of the last αβ heterodimer to the complete α 3 β 3 hexamer. Generally, this confirms that ATP hydrolysis is not required for assembly, but nucleotide binding is the essential factor triggering the in vitro complex formation. Importance of charged residues in the catalytic sites of bacterial F-type ATP synthases. After determining the crucial effect of ATP binding for ATPase assembly, we want to shed more light on this process. Recent studies have shown the importance of charged residues for the ability of Pi -binding at the catalytic sites of the αβ interface of the hexameric head. The replacement of charged amino acids with neutral residues induced reduced cell growth and loss of ATPase activity, suggesting the electrostatic interactions between amino acids to be crucial for initial phosphate binding in the catalytic sites [bib_ref] Role of charged residues in the catalytic sites of Escherichia coli ATP..., Ahmad [/bib_ref] [bib_ref] Involvement of ATP synthase residues αArg-376, βArg-182, and βLys-155 in P i..., Ahmad [/bib_ref] [bib_ref] Modulation of charge in the phosphate binding site of Escherichia coli ATP..., Ahmad [/bib_ref] [bib_ref] Mutagenesis of residue βArg-246 in the phosphatebinding subdomain of catalytic sites of..., Ahmad [/bib_ref]. We want to deduce if these mutations affect ATPase activity via their influence on the ability of the complex to bind/ hydrolyze ATP or if the effect might be even more drastic and influence the complexes assembly. Therefore, we introduced homologous point mutations in conserved regions in α[R363Q, R363K] and β[K159Q, R186Q, R251Q] in the A. woodii operon [fig_ref] Figure 5: Amino-acid sequence alignment of αand β-subunit residues for E [/fig_ref]. To investigate the effect of these charged residues in the catalytic site we first analyze the binding efficiency of ATP to α, β, and different mutants with nano electrospray-ionization (nESI) [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] , which allows observing the number of bound ATP. The deconvoluted MS-spectrum in [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] Role of ATP hydrolysis vs. nucleotide-binding in the assembly process to F 1 of bacterial F-type ATP synthases. LILBID spectra of in vitro α, β, γε assembly: The α-(light blue), β-subunit (dark blue), γε-complex (dark and light green) were incubated with 2 mM MgCl 2 and 2 mM of a nucleotide at 4°C for 1 h. Complex formation is observed after incubation with ATP (a), AMP-PNP (b), ADP (c), and ATP-γ-S (d). Full α 3 β 3 γε assembly can be identified next to small amounts of other F 1 subcomplexes (e.g., α 2 β 2 δγε α 2 β 2 γε, αβγε, αβγ), for ATP as well as the non-hydrolysable ATP analogs, apart from AMP-PMP, where the highest observed complex is α 2 β 2 γε. Source data are provided as a Source Data file. The dominating species is clearly the one with one bound ATP, indicating specific binding. In contrast, peaks trailing for the mutant β[K159Q] [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] suggest non-specific ATP binding only. [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] shows the differences in ATP binding for the investigated α and β subunits and their respective mutants. Seeing that charged residues in the catalytic site can influence ATP-binding, we wanted to assess, if reduced ATP-binding affinity had an effect on the in the vitro assembly process. Therefore, we performed the same HPLC and MS experiments as for the wildtype α-β incubations. In vitro assembly of the α and β mutant constructs was then probed by LILBID-MS. Interestingly, we observe that under the same experimental conditions, which show stable heterodimers of wildtype α and β , we do not detect stable αβ heterodimers for α[WT] incubated with β[K159Q] [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] or any of the β mutants [fig_ref] Figure 5: Amino-acid sequence alignment of αand β-subunit residues for E [/fig_ref]. This suggests that all β mutations affect stability to the extent that dimers can still be observed with HPLC (to a different degree) but are much reduced in the LILBID spectra. Similarly, we see clearly decreased dimerization for β[WT] with α[R363Q] [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. A point mutation at the same position which retains the charge of the original amino acid (α[R363K]) shows no effect, as it does not hinder the formation of the αβ heterodimer . As our experiments so far indicate that the formation of the αβ heterodimer is decisive for further complex formation, the mutations, which impede the heterodimer, should affect the formation of higher-order F 1 -complexes as well. Our mass spectra confirm that incubation of α[WT], β[K159Q] and the central stalk γε does not lead to the α 3 β 3 γε complex [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] , which we observed with the wildtype β [fig_ref] Figure 5: Amino-acid sequence alignment of αand β-subunit residues for E [/fig_ref]. In contrast assembly of the mutant α[R363Q], β[WT] and the central stalk γε generates a larger complex-but interestingly not the expected hexameric head but subcomplex α 2 β 2 γε [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. This indicates that replacing the arginine with the uncharged glutamine in subunit α has an effect on the assembly process, which is less pronounced than the effect of the above-mentioned substitutions in subunit β, but still hinders the formation of the correct αβ hexamer. Order of assembly steps into F 1 . The combined results obtained with LILBID and nESI-MS and HPLC allow us to establish all subcomplexes which form in vitro en route to the full complex as well as the necessary assembly conditions. As these assemblies take place in a minimal environment, compared to a cell, we need to guarantee that the observed subcomplexes are not just the result of aggregation, but can occur in the same manner in the cell. Therefore, we expressed the subunit combinations, which we found in our assembly experiments directly in E. coli cells for comparison. We were able to obtain all anticipated subcomplexes with the same stoichiometries [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. Based on these findings, we can follow the individual assembly steps and establish their order for the formation of bacterial soluble F 1 domain of F-type ATP synthases . Overall our results show that, αand β-subunits assemble specifically to αβ heterodimers only in presence of nucleotides and Mg 2+ and while surprisingly not forming higher subcomplexes (e.g., αβ 2, α 2 β, α 2 β 2 etc.) least of all the expected hexameric head (α 3 β 3 ). The α and β subunits can both bind individually to the central stalk γ, but only little binding is observed and in a maximal copy number of one [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref] , unless α and β dimerize first. Three pairs of αβ heterodimers bind either to the central stalk γ or a preformed γε complex to generate stable α 3 β 3 γ or α 3 β 3 γε complexes, respectively. The relevance of these in vitro results is shown in our heterologously expressed subcomplexes α 3 β 3 γ and α 3 β 3 γε which we can isolate as stable and bioactive complexes in cellula [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. The δ-subunit binds to the in cellula or in vitro pre-complexes containing the hexameric head and [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] , which generates the full F 1 complex. As isolated subunit γ could not be purified in E. coli for stability reasons, we purified γε , so our assembly studies did not include binding of ε and γ analogously to all the other binding experiments. The complete aggregation of the γ-subunit into inclusion bodies in the bacterial matrix during the overexpression could suggest that the ε-subunit is essential for the stabilization of the central stalk , which would place binding of ε to γ as a first step parallel to the formation of the αβ heterodimer, before further assembly steps occur. Nevertheless, expression of in cellula subcomplexes showed that α 3 β 3 γ can be expressed as a stable subcomplex, which indicates that binding of ε is not an essential prerequisite for any further assembly to the F 1 complex. ε could therefore enter the assembly network at different times, as we indicate in . Interestingly, we observe some differences between our in cellula and in vitro complexes: the in cellula preformed complexes are stable in a buffer without nucleotide-and Mg 2+ and the binding of δ to any complexes including the hexameric head can then be performed in vitro without nucleotide-and Mg 2+ addition. The same assembly experiment with the in vitro formed α 3 β 3 γε complex requires nucleotide-and Mg 2+ addition, to hinder disaggregation of the complexes. This could be explained, if the F 1 -subcomplexes formed in cellula and in vitro shared the same assembly pathway and stoichiometry, but a structural change occurring in the cell to stabilize the formed complex, potentially by more tightly retaining bound nucleotides, were missing in vitro, which is a question we will come back to later. depicts a full summary of the different assembly steps of the soluble part F 1 as determined by our experimental data. The entire assembly process follows a choreographed pathway, which allows for efficient construction of the F 1 complex. ATPase activity of in vitro assembled A. woodii αβ and α 3 β 3 γε complex. In general, many macromolecular complexes need molecular chaperones, which assist for example in the conformational folding or assembly processes. In mitochondria, it is known that the F 1 -assembly of the hexamer of alternating αand β-subunits requires two specific chaperones, Atp11p and Atp12p, that bind transiently to β and α 33,39 . Our previous results demonstrate that the bacterial F 1 -assembly can occur independently of chaperones. This was a surprising finding, as a generally accepted hypothesis expects the bacterial F 1 -assembly to as well depend on chaperones. So far one such Atp12p homolog has been identified in proteobacteria [bib_ref] Assembly factors of F 1 F O -ATP synthase across genomes, Pícková [/bib_ref]. The question arises, if chaperones are not needed at all, or if their presence might be required, if not for assembly, then to guarantee correct function and activity. To answer this question, we investigated our in vitro assembled complexes for bioactivity. We performed ATP hydrolysis experiments with all of the separately expressed constructs (α, β, γε, and ε) and the in vitro assembled αβ and α 3 β 3 γε complexes using an enzyme-coupled activity assay [bib_ref] The stimulating role of subunit F in ATPase activity inside the A..., Singh [/bib_ref] [bib_ref] Purification of ATP synthase from Acetobacterium woodii and identification as a Na..., Reidlinger [/bib_ref]. For in vitro complex formation, the subunits were incubated with 4 mM MgCl 2 and ATP with subunit stoichiometries of 1:1 for the αβ heterodimer, and 3:3:1 for α 3 β 3 γε. All subunits and subcomplexes showed moderate ATPase activity [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref]. We could determine ATPase activities for the βand α-subunit of 1.5 ± 0.4 and 2.0 ± 1.4 mmol [ATP]/(mol[protein] · s), respectively. These are quite similar and do not reflect the threefold higher ATPase activity of α previously seen in E. coli 32 . As isolated subunit ε was already known to bind ATP [bib_ref] Isolated ε subunit of Bacillus subtilis F 1 -ATPase binds ATP, Kato-Yamada [/bib_ref] and has to undergo large conformational changes to regulate the ATPase [bib_ref] The role of subunit epsilon in the catalysis and regulation of F..., Feniouk [/bib_ref] we hypothesized that ε as well undergoes hydrolysis. Interestingly, the isolated ε-subunit showed a hydrolytic activity of 34 ± 14 mmol [ATP]/(mol[protein] · s), which is one magnitude higher than that of the single catalytic β-subunit. The enzymatic hydrolytic activity of γε was measured at 30 ± 13 mmol [ATP]/(mol[protein] · s) placing the overall activity of γε very similar to ε, suggesting no contribution from γ. The ATPase activity calculated for the in vitro assembled αβ heterodimer is 6.9 ± 2.2 mmol [ATP]/(mol[protein] · s), assuming incubation leads to full complex formation. As this might not be the case, all molar activities of in vitro complexes have to be seen as a conservative estimate-if full complexation could be guaranteed the observed values would likely be higher. Nevertheless, the observed hydrolytic activity is about twice the sum of the individual α and β activities, supporting the relevance of the αβ heterodimer as a building block and working unit of the ATPase. Surprisingly this value stays clearly below the hydrolytic activity of ε. The reconstitution based on the incubation of α, β, and γε resulted in a hydrolytic enzymatic activity of 60 ± 4 mmol [ATP]/ (mol[protein] · s). Interestingly, the activity of this in vitro assembly is not much higher than the sum of the ATPase activities of the utilized subunits. The increased activity (factor 1.5) of the now complexed subunits stems mainly from the increase of activity seen for the formation of the three required αβ heterodimers. Similarly, the in cellula purified αβ and α 3 β 3 γε* were investigated. The ATPase activity for both is noticeably beyond anything we observed for in vitro complexes (1.60 ± 0.14 and 9.06 ± 0.15 mol [ATP]/(mol[protein] · s), respectively) [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref]. These values cannot be compared directly with those Order of assembly steps into bacterial F 1 . a As a first step α-subunits (light blue) and β-subunits (dark blue) form αβ heterodimers, which requires the presence of nucleotides and Mg 2+ . b As next step three of these heterodimer building blocks assemble pairwise to the central stalk γ (dark green). Only after the formation of the full hexameric head, the δ-subunit (yellow) is able to bind. The ε-subunit (light green) can join into the assembly process at all intermediate step en-route to the F 1 complex. [formula] α β γ ɛ δ ATP Mg 2+ ATP Mg 2+ ATP Mg 2+ a b [/formula] obtained for in vitro complexes, for which the individual subunits do not fully form complexes. Nevertheless, the activity of the in cellula αβ heterodimer alone is already much higher than that of the in vitro assembled α 3 β 3 γε complex. Similarly, the in cellula α 3 β 3 γε * shows an activity way beyond the sum of its components. This indicates that the in vitro conditions allow for the correct assembly of the functional subunits, which then retain their individual ATPase activity, while the cellular environment allows for modifications, which increase the ATPase activity of the αβ heterodimer and the whole ATPase, way beyond that of the sum of its parts. Stability studies of in cellula vs in vitro assembled heterodimers with ion mobility MS. The observed differences in bioactivities as well as stability without ATP/Mg 2+ for our in cellula and in vitro complexes suggest that additional effects besides assembly are required to form an active complex. We assume conformational changes to play a role, possibly triggered by chaperones. The relevance of the αβ heterodimer formed as a first step in the ATPase assembly and the differences observed in our in cellula and in vitro studies, makes the heterodimer a very interesting candidate to investigate if the changes in activity can be correlated to structural changes. Noticeable structural rearrangements should be accompanied by a change in collision cross-section (CCS), which can be monitored by ion mobility (IM) MS, while changes to the intrinsic stability can be revealed by differences in collision-induced unfolding (CIU) experiments. To validate our assumption, we investigate the heterodimer for differences between in cellula and in vitro formed complex. We select in both cases the 22-times charged αβ heterodimer in our nESI mass spectra as precursor ion which is analyzed via IM MS in dependence of the applied collision voltage. IM allows the separation of ions based on their mobility through an inert gas under the influence of an electric field. Increasing the collision voltage can lead to CIU and then dissociation of the complex. The energy required to cause CIU can be correlated to the complex stability. In the resulting IM fingerprint of the in cellula assembled αβ heterodimer, the IM signal appears for low collision voltages at 7595 Å 2 and 7630 Å 2 for the in cellula and the in vitro complex, respectively [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref] These values correspond to the compact feature of the folded complexes and already suggest slight structural differences. An increase of collision voltage leads to protein unfolding, which is accompanied by an increase in CCS. The original IM signal decreases partially in favor of a signal at 7802 Å 2 (in cellula) and 7851 Å 2 (in vitro), representing the corresponding unfolded structure of the αβ heterodimer. 50% of the in vitro assembled αβ heterodimer unfolds at collision voltages of 152.6 V and appears at a slightly higher CCS compared to the in cellula assembled αβ, which preserves the compact state for >50% even at the highest collision voltages. This indicates that the in vitro structure is less stabilized against unfolding than the in cellula assembled αβ. This can be seen more clearly in the difference plot (in vitro minus in cellula, [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref] , which helps to compare specific features of each fingerprint directly. In addition, it reveals a significantly broader IM signal for the compact, as well as the unfolded state for the in vitro assembled αβ heterodimer indicating a less homogenous structure for the in vitro than for the in cellula αβ heterodimer. We conclude that the more defined structure and increased stability of the in cellula heterodimer are structural properties, which can be correlated to the increased ATPase activity. # Discussion The aim of this study is to better understand the assembly process of an ATPase-a large heterogeneous macromolecular complex, with eight different subunits. We determined conditions required for the whole in vitro assembly process into the soluble part F 1 of A. woodii ATPase from single purified F 1 -subunits. We followed the assembly process in vitro step by step from subunits via the subcomplexes, which form (or do not form) after incubation of different subunit combinations. To verify that the observed complexes on pathway to the F 1 complexes are meaningful and can assemble in the same stoichiometries in the cell, we expressed a variety of in cellula F 1 subcomplexes. We could purify all of the on-pathway complexes in cellula, which allowed comparison of in vitro vs. in cellula formed complexes. In vitro data were collected in biological quadruplicates (n = 4) except ε with three biological replicates (n = 3) and in cellula data were collected in biological triplicates for αβ (n = 3) and technical duplicates for α 3 β 3 γε* (n = 2). Source data underlying [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref] and b are provided as a Source Data file. Although the presence of nucleotides and Mg 2+ is known to be required for F 1 assembly of E. coli 31 , for the thermophilic bacterium PS3 the formation of an α 3 β 3 hexamer has been shown to occur independently of ATP/Mg , showing that this is not a universal requirement. Here we find for the mesophilic A. woodii that the presence of ATP/Mg 2+ is essential for complex formation , up to the α 3 β 3 γε. We reveal that it is ATP binding that triggers the in vitro assembly and not ATP hydrolysis . We could demonstrate that the incubation of α and β leads to the formation of the heterodimer without the formation of any higher αβ oligomers. Only incubation in the presence of the central stalk γε allowed further assembly into larger complexes including the hexameric head-on the pathway to the F 1 [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref]. The lack of complexes with odd numbers of α and β subunits , [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] shows that the formation takes place with the αβ heterodimers as basic building blocks. As we were able to express and purify intact α 3 β 3 γ complexes without the presence of ε we can conclude that it is the central stalk subunit γ, which is crucial for generating a stable α 3 β 3 hexagon out of αβ heterodimers, while ε can join at different stages of the assembly. Including δ in our assembly studies could show that δ does not bind to isolated α or β [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref]. Only after the formation of the hexameric head, the subunit δ can bind, resulting in the full F 1 complex [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] , which can occur ATP/ Mg 2+ independent [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref]. Overall, our in vitro assembly experiments in combination with the complexes which can be assembled in cellula give a full picture of a wellchoreographed assembly process, as shown in . Point mutations in the catalytic interface of α (at position R363), and β[K155, R182, R246] which replace positively charged amino acids with neutral residues, demonstrated that such modifications can but do not have to have a direct effect on ATP binding efficiency. HPLC runs still showed the in vitro assembly of αβ heterodimers for all combinations of one wildtype and one mutated subunit [fig_ref] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation... [/fig_ref] , albeit with affected complex stability, as incomplete dimer assembly is observed for those mutants with reduced ability to bind ATP. This effect on stability is even more pronounced in the LILBID-MS results, which show reduced or no αβ dimer for all mutants which substitute a charged with a neutral amino acid [fig_ref] Figure 5: Amino-acid sequence alignment of αand β-subunit residues for E [/fig_ref]. Substitution of one charged amino acid for another α[R363K] had no influence. This demonstrates that those point mutations, which remove a charge in the αβ interface, cause a decrease of αβ heterodimer stability. In vitro assembly experiments with α and any β mutant, which included γε did not yield any higher complexes. An interesting finding was that for the α mutant α[R363Q], stable αβ heterodimers and even higher F 1 subcomplexes, including one or two heterodimers [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref] can still form, albeit no complex with a complete hexameric head can be detected [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. A similar disturbance was found for incubations of wildtype subunits with AMP-PNP, which might sterically influence the assembly. This shows that even if αβ dimer formation is possible, the assembly of the hexameric head, which has to function via large conformational changes in a very tightly controlled manner, is a precise process, which can be hindered by small disruptions. Our in vitro assembly studies prove that no direct involvement of chaperones is necessary for the correct reconstitution of the F 1 complex from the individual subunits . Based on the known relevance of chaperones for complex formation in cells this is a surprising finding. Therefore, the question arises: If chaperones are not essential for the in vitro assembly of the F 1 - b In vitro assembled αβ heterodimer. Exemplary spectra are shown in [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref]. At low collision voltages the IM signal of the αβ heterodimer appears at 7595 Å 2 for the in cellula and at 7630 Å 2 for the in vitro assembled αβ heterodimer, respectively. At higher collision voltages the signal of the in vitro assembled αβ heterodimer unfolds, resulting in a signal at 7851 Å 2 . At 152.6 V 50% has reached the unfolded state. In contrast, increasing the collision voltage in the case of the in cellula formed αβ heterodimer leads to a signal appearing at 7802 Å 2 . 50% unfolding is never reached-the maximal unfolding of 47% is observed at 200 V. c The difference plot of both fingerprints illustrates the change regarding unfolding behavior between in vitro and in cellula assembled heterodimers. d-f CCS distribution for different collision voltages for in vitro (d) and in cellula (e) assembled heterodimers, and the difference plot (f). The overall feature of the in vitro αβ is clearly broader than for the in cellula αβ. ATPase, could they be relevant to ensure the bioactivity and function of the enzyme? To shed light on this question, we correlate our assembly results with an enzyme-coupled activity assay for single isolated F 1 -subunits, in cellula and in vitro formed complexes [fig_ref] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled... [/fig_ref]. The first building block is the αβ heterodimer. The in vitro assembled αβ shows an activity, which is about twice as high as the mere sum of the α and β activity. This effect is not big, so it could be based on the ATP hydrolysis taking place in the αβ interface, which would offer a better binding pocket for the nucleotide. A much larger effect is seen in the comparison with the in cellula αβ dimer. Mass spectrometric analysis and HPLC measurements confirm the efficient, albeit not complete heterodimer assembly, so exact comparison of in cellula and in vitro activity is difficult. Assuming that about half of the possible dimers form in vitro, a conservative estimate would put the activity of the in cellula purified protein complex at least a factor of 100 above that of the vitro assembled complex. This suggests a principle difference in the heterodimers, leading to much higher bioactivity of complexes formed in a cell. This could be explained by a structural difference of the in vitro and the in cellula formed heterodimer. To further investigate this assumption, we perform stability studies with in cellula [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref] , d) and in vitro [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref] , formed αβ heterodimers by means of mass spectrometry CIU experiments, which we monitor by IM MS. At moderate collision voltages, the IM plots show signals at similar CCS about 7630 Å 2 for the in vitro and 7595 Å 2 for the in cellula-formed complex. Noticeable is the comparably broad signal distribution for the in vitro complex as opposed to the in cellula dimers. Comparison of both IM plots is most intuitive in the difference spectrum of both IM plots [fig_ref] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in... [/fig_ref]. This implies that the in cellula assembled αβ heterodimer is present with a more homogenous structure, while the in vitro formed complexes seem less defined. The comparison of both complexes during the CIU experiment demonstrates that in vitro assembled αβ unfolds at a lower voltage than the in cellula formed heterodimer, validating that the in cellula formed complex is intrinsically more stable than the in vitro construct. These are interesting observations in the light of the subunit β's ability to undergo four different conformations with different nucleotide-binding affinities, tight (T), loose (L), closed (C), and open (O) resulting in ATP hydrolysis/synthesis [bib_ref] Understanding ATP synthesis: structure and mechanism of the F 1 -ATPase, Leyva [/bib_ref]. The stable and homogeneous conformation found for the in cellula complex might suggest a potentially chaperone-induced bias towards a single conformation, which supports assembly. In our, in vitro assembly assays no such bias towards a specific conformation is induced, explaining the differences in stability and hydrolysis activity. This structural difference propagates to the whole in vitro F 1 which can be seen in the dependence on ATP/Mg 2+ of the in vitro F 1 complexes to prevent dissociation (Supplementary [fig_ref] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula [/fig_ref] , which is less of an issue for their in cellula counterparts [fig_ref] Figure 7: Role of charged residues in the catalytic site for the assembly process... [/fig_ref]. This will as well be the reason behind the much higher ATPase activity of the in cellula complexes. A precise investigation of the structural conformation of the subunits/ subcomplexes compared to in cellula complexes will be the focus of future studies. What is known about the role of the ε subunit so far, is its relevance in the coupling of the soluble part F 1 with the membrane-embedded F O and its regulatory role in the inhibition of ATP hydrolysis activity [bib_ref] The epsilon subunit of Escherichia coli coupling factor 1 is required for..., Sternweis [/bib_ref] [bib_ref] Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine..., Smith [/bib_ref] [bib_ref] lnhibitory properties of endogenous subunit ε in the Escherichia coli F 1..., Laget [/bib_ref] [bib_ref] A systematic assessment of mycobacterial F 1 -ATPase subunit ε's role in..., Wong [/bib_ref]. Interestingly, our ATP activity assays revealed that isolated purified ε itself shows an ATP hydrolysis activity, which is surprisingly even higher than for subunit β. The ε-subunit in bacterial F 1 F O is known to perform its regulatory function, by taking a hairpin or extended conformation [bib_ref] Assembly of the Escherichia coli F O F 1 ATP synthase involves..., Deckers-Hebestreit [/bib_ref] [bib_ref] Large conformational changes of the ε subunit in the bacterial F 1..., Tsunoda [/bib_ref] , and isolated subunit ε of Bacillus subtilis was found to be able to bind ATP [bib_ref] Isolated ε subunit of Bacillus subtilis F 1 -ATPase binds ATP, Kato-Yamada [/bib_ref]. Our results suggest that this extensive conformational change is driven by ATP hydrolysis. All in all, we could show that the soluble F 1 part can be reconstituted in vitro from the purified F 1 -subunits in presence of nucleotide and Mg 2+ . Our studies have provided an exciting overview of the step-by-step assembly via well-defined subcomplexes towards the full complex, with a preformed αβ heterodimer as an essential building block. Our results show the special roles, especially of subunits β and ε for the functional complex. Differences in the ATPase activity of the generated F 1 complex generated in vitro and in cellula seem to be based on structural differences suggesting that bacterial chaperones and/or other cellular effects (i.e., molecular crowding) may not be needed for correct stoichiometric assembly of the ATPase but assist to fine-tune the assembly into a fully functional F 1 . # Methods Cloning expression vectors of the A. woodii F 1 ATP synthase. Using pKB3-His (a pET21a[+] vector derivative) as a master plasmid, which was kindly provided by Karsten Brandt (Müller laboratory 27 at Goethe University), we performed PCR for cloning of deletions or point mutations and ligation independent cloning with the In-Fusion HD Cloning Kit (Takara Bio). For cloning of single genes encoding for subunits and subcomplexes of F 1 (atpHAGDC for subunit δ, α, γ, β and ε) with an N-or C-terminal tag (StrepI-or His 6 -tag) were cloned into a commercially available pET21a(+)-vector (Novagen). All generated plasmids are listed in Supplementary . A list of primers, used for cloning the respective plasmids is given in Supplementary . Stellar competent cells (Takara Bio) were used for plasmid amplification. All constructs including mutations were verified by sequencing (Microsynth Seqlab). Purification of heterologous F 1 ATP synthase subunits and subcomplexes of A. woodii containing a His 6 -tag or a Strep-tag. All constructs were transformed in E. coli BL21gold(DE3) competent cells (AgilentTechnologies) following the provided manual. Lysogeny Broth (LB) Agar (Lennox) transformation plates supplemented with 100 µg/mL ampicillin were used for the transformation of all constructs. Cell colonies were grown at 37°C overnight and stored at 4°C for up to 2 weeks. A single clone was picked to inoculate a pre-culture of LB containing the respective antibiotic and was grown at 37°C and 150 rpm overnight. Pre-culture was inoculated in the main culture of Terrific Broth medium supplemented with respective antibiotics. Cells were cultivated at 37°C and 150 rpm until an OD 600nm of 0.5-0.7 was reached. After inducing protein overexpression in the main culture with 0.25 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) the protein expression was performed at 20°C and 150 rpm overnight. The cells were harvested by centrifugation at 4000 × g for 20 min at 4°C. The isolated cell pellets were resuspended in 20 mL wash buffer I (50 mM KP i , 200 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol, pH 7.4) supplemented with 1 mM EDTA and DNAse I. Using a French Pressure Cell Press cells were mechanically disrupted at a pressure of 700 bar and cell debris were removed by centrifugation (50,000 × g for 60 min at 4°C). After the addition of 2 mM MgCl 2 cell lysate was incubated with 5 mL Ni-NTA Agarose (binding capacity 50 mg/mL) (MACHEREY-NAGEL) for 30 min. The column was washed with five column volumes wash buffer I to remove unbound proteins and bound protein was eluted with 2.5 column volumes wash buffer I containing 300 mM imidazole. The elution fractions were analyzed with (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, concentrated with an Amicon Ultra centrifugal filter units, and purified by SEC with a Superdex 75 10/300 (GE-Healthcare). Protein samples were shock frozen in liquid nitrogen and stored at −80°C. The same workflow procedure was performed for all protein constructs which contained a StrepI-tag with the exception of using a 5 mL Strep-Tactincolumn (binding capacity 15 mg/mL resin) (Iba) with the respective wash buffer II (50 mM KP i , 200 mM NaCl, 10% (v/v) glycerol, pH 7.4) and elution with the wash buffer II containing 2.5 mM d-Desthiobiotin. In vitro assembly and mass spectrometric analysis. For the in vitro studies, all in E. coli heterologously purified subunits α, β, δ, and γε were incubated in a ratio of 3:3:1:1 for assembly into the full F 1 -complex, or subsets for subcomplexes in 50 mM KP i , 200 mM NaCl, 2 mM ATP, 2 mM MgCl 2 , 10% (v/v) glycerol, pH 7.4 at 4°C for 1 h. For LILBID-MS measurements the buffer was exchanged to 100 mM ammonium acetate, 2 mM ATP, 2 mM MgCl 2 , pH 7.4 at 4°C before measurements using desalting columns with a cutoff of 7 kDa (Zeba Micro Spin Desalting Columns, Thermo Scientific). Approximately 5 µL samples were used per measurement. LILBID-MS droplets were generated by a piezo-driven droplet generator (MD-K-130, Microdrop Technologies GmbH, Germany). This generator produces droplets of 50 µm diameter with a frequency of 10 Hz at 100 mbar. The droplets were transferred to a vacuum and irradiated by an IR laser at 2.94 µm, a vibrational absorption wavelength of water, which leads to the explosive expansion of the sample droplet. The energy output has a maximum energy of 23 mJ per pulse and a pulse length of 6 ns. The overall gradient between the first lens (repeller) and the third lens (grounded lens) is always 6.6 kV. The applied voltage on the second lense NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28828-1 ARTICLE NATURE COMMUNICATIONS | (2022) 13:1218 | https://doi.org/10.1038/s41467-022-28828-1 | www.nature.com/naturecommunications (extractor) is set between 4.0 kV and 6.0 kV. Either the first two lenses were pulsed from 0 kV to their final voltages, or both lenses were set at the same voltage of 4.0 kV and the repeller was pulsed up to 6.6 kV in total. The released ions then are analyzed using a home-built time-of-flight spectrometer [bib_ref] LILBID and nESI: different native mass spectrometry techniques as tools in structural..., Peetz [/bib_ref]. These settings correspond to the standard settings at the LILBID ion source. The presented spectra show the averaged signals of several hundred up to thousand droplets. Data processing was done using the software Massign [bib_ref] Massign: an assignment strategy for maximizing information from the mass spectra of..., Morgner [/bib_ref]. Electrospray ionization mass spectrometry (ESI-MS) was performed on a Synapt G2S (Waters Corpn., Wilmslow, Manchester, UK) equipped with a high mass quadrupole upgrade. Pd/Pt sputtered nESI tips were pulled in-house from borosilicate glass capillaries on a Flaming/Brown Micropipette Puller (P-1000; Sutter Instrument Co.). For the analysis of purified F 1 -subunits, capillary and cone voltages were set to 1.85 kV and 150 V, respectively. The source block temperature was set to 30°C. Neither trap nor transfer collision cell was used. Directly prior to MS analysis, 10 μL of the protein solution (30 µM) was buffer exchanged into 100 mM ammonium acetate, 100 µM ATP, 100 µM MgCl 2 , and pH 7.4 at 4°C using Zeba Micro Spin Desalting Columns (Thermo Scientific). For the IM MS experiments, αβ heterodimers were analyzed in positive ion mode using a capillary voltage of 2.1 kV. The rest of the settings for MS analysis were adjusted as follows: cone voltage 100 V at an offset of 150 V, 20°C source temperature. The instrument was calibrated by a conventional CsI solution. IM experiments were done using a traveling wave setup operating at a wave height of 40 V, a traveling wave velocity of 700 m/s, a nitrogen gas flow of 90 mL/min, and a drift cell pressure of 3.5 mbar. MS-MS was performed to isolate the αβ heterodimers, which were selected at m/z = 4950 using an LM resolution of 12 and an HM resolution of 15. CIU experiments were performed by increasing the trap collision energy (CE) in steps of 5 V from 85 to 200 V. Data analysis of ESI-MS and IMS experiments was done using the software MassLynx V4.1, TWIMExtract 51 , CIUSuite 52 , and UniDec 53 . All mass spectra were normalized to a maximal intensity which is displayed in arbitrary units (a.u.). ATP hydrolysis assay. The enzyme-coupled activity assay performed in this work was adapted from references [bib_ref] The stimulating role of subunit F in ATPase activity inside the A..., Singh [/bib_ref] [bib_ref] Purification of ATP synthase from Acetobacterium woodii and identification as a Na..., Reidlinger [/bib_ref]. Generally, assays were run in 96-well f-bottom microtiter plates (Greiner Bio-One). The ATP hydrolysis assay was performed to analyze the ATP hydrolysis activity of the single F 1 -subunits, the in vitro assembled complexes of F 1 , and in cellula purified F 1 -subcomplexes. During this assay, ATP was constantly regenerated by an enzyme-coupled reaction, while the oxidation of NADH was spectroscopically followed via the decrease of absorbance at 340 nm. Measurements were performed in for subunits and in vitro assembled subcomplexes, for which the following amounts of the respective proteins were used: 5.5 µM in case of α and β, 1.8 µM γε, 5 µM ε. In the enzymatic assays of in vitro complexes 5.5 µM protein was used for α as well as β and 1.8 µM γε or 5 µM of the ε-subunit, respectively, to allow for subunit stoichiometries of 1:1 for the αβ heterodimer, and 3:3:1 for α 3 β 3 γε. In cellula produced αβ and α 3 β 3 γε* were employed with 0.5 µM and 0.25 µM, respectively. Protein solutions were diluted to 100 µL total volume (100 mM TRIS, 100 mM maleic acid, pH 7.5, 5 mM MgCl 2 , 3 mM phosphoenolpyruvate, 4 mM ATP, 0.5 mM NADH, 10 units L-lactate dehydrogenase, 10 units pyruvate kinase), filtered and degassed for 700 s in 3 s intervals at 25°C. The decrease in absorbance at 340 nm per second was determined by fitting a linear fit of the slope. All measurements were performed in biological triplicates except α 3 β 3 γε* with two technical duplicates. Kinetic data were analyzed with Origin 2018b and Excel 2016. HPLC analysis. For the assembly studies and chromatographic separations 60 µg protein was applied on an SEC column (bioZen 1.8 µm SEC-3) coupled with UV detection in the elution buffer A (50 mM KP i , 200 mM NaCl, pH 7.4) or elution buffer B (50 mM KP i , 200 mM NaCl, 2 mM ATP, 2 mM MgCl 2 , pH 7.4) at 4°C, respectively. [fig] Figure 2: Summary of all subcomplexes obtained in vitro and in cellula. a List of all subcomplexes observed with LILBID-MS after in vitro assembly of isolated subunits in the presence of 2 mM ATP and 2 mM MgCl 2 . b SDS-PAGE (NuPAGE 4-12% Bis-TRIS) of single purified subunits α, β, γε, and δ. Affinity purification of α with Strep-Tactin-and β, γε, and δ with Ni-chelating chromatography. N = 3 independent experiments. c List of in E. coli heterologously purified F 1 and subcomplexes. All complexes were analyzed with LILBID-MS and SDS-PAGE (SupplementaryFig. 7). Source data underlyingaare provided as a Source Data file. [/fig] [fig] Figure 5: Amino-acid sequence alignment of αand β-subunit residues for E. coli and A. woodii. a α-subunit residues. b β-subunit residues. Positions of positively charged amino acids that were mutated are marked in blue. [/fig] [fig] Figure 6: Dependence of ATP-binding of α, β and their mutants and the formation of αβ heterodimers. a, b ATP-binding: deconvoluted nESI-MS spectra of ATP-binding of β[WT] (a) and β[K159Q] (b). Corresponding spectra for all α and β mutants are shown in Supplementary Fig. 4. c Summary of ATP-binding of β[K159Q], β[R186Q], β[R251Q], β[K159Q, R186Q, R251Q], α[WT], α[R363Q], and α[R363K] relative to β[WT]. Data are presented as mean values with error bars showing the SD, with individual data superimposed. d-h HPLC measurements: in vitro αβ heterodimer assembly of α[WT] (light blue) with β[WT] (dark blue) and β mutants. i HPLC measurement: In vitro αβ heterodimer assembly of β[WT] (dark blue) with α[R363Q] (light blue). Data were collected in biological replicates (n = 3). Source data are provided as a Source Data file. [/fig] [fig] Figure 7: Role of charged residues in the catalytic site for the assembly process analyzed by LILBID-MS. a In vitro assembly of α[WT] (light blue) with β[K159Q] mutant (dark blue) shows no significant formation of the αβ heterodimer, which we observe for the α[WT] and β[WT] (Fig. 1b). b The addition of the central stalk γε (dark and light green) does not lead to the formation of the desired α 3 β 3 γε complex. c In vitro assembled α[R363K] (light blue) and β[WT] (dark blue) revealed reduced αβ heterodimer assembly. d The biggest complex that is observed after addition of γε (dark and light green) is an α 2 β 2 γε subcomplex with charge states (−1 to −4). Source data are provided as a Source Data file. | (2022) 13:1218 | https://doi.org/10.1038/s41467-022-28828-1 | www.nature.com/naturecommunications [/fig] [fig] Figure 9: ATP hydrolysis of isolated subunits and in vitro and in cellula assembled complexes. a Isolated α, β, ε, γε and in vitro assembled αβ and α 3 β 3 γε complexes. b In cellula αβ and α 3 β 3 γε * complexes. Data are presented as mean values with error bars showing the SD, with individual data superimposed. c Spectroscopic comparison of the decrease in absorbance at 340 nm per time [s] in the enzyme-coupled ATPase activity assay of the in vitro assembled αβ from isolated α and β (5.5 µM each) relative to the in cellula produced αβ (0.5 µM protein complex) in 100 µL (100 mM TRIS, 100 mM maleic acid, 5 mM MgCl 2 , 3 mM phosphoenolpyruvate (PEP), 4 mM ATP, 0.5 mM NADH, 10 units L-lactate dehydrogenase (L-LDH), 10 units pyruvate kinase (PK), pH 7.5). [/fig] [fig] Figure 10 CIU: fingerprints of the in cellula and in vitro assembled αβ heterodimer in the presence of ATP and Mg 2+ . a In cellula assembled αβ heterodimer. [/fig]

Management of Retained Genital Piercings: A Case Report and Review The prevalence of genital piercing among women is increasing. As the popularity increases, the number of complications from infection, injury, and retained jewelry is likely to rise. Techniques to remove embedded jewelry are not well described in the literature. The purpose of this report was to describe a case of a patient with a retained clitoral glans piercing, discuss a simple technique for outpatient removal, and review current evidence regarding associated risks of clitoral piercings. A 24-year-old female presented to the emergency department with an embedded clitoral glans piercing. Local anesthetic was injected into the periclitoral skin and a small superficial vertical incision was made to remove the ball of the retained barbell safely. In conclusion, among patients with retained genital piercing, outpatient removal of embedded jewelry is feasible. While the practice of female genital piercing is not regulated, piercing of the glans of the clitoris is associated with increased injury to the nerves and blood supply of the clitoris structures leading to future fibrosis and diminished function compared to piercing of the clitoral hood. # Introduction Genital piercing is increasing in frequency among many demographic groups. Genital piercings are unique as they may have a functional role in addition to aesthetical value, with some women reporting heightened sexual arousal and increased orgasm potential after clitoral piercings [bib_ref] First glimpse of the functional benefits of clitoral hood piercings, Millner [/bib_ref]. In women, clitoral piercing may play a therapeutic role in overcoming past genital trauma or dysfunctional sexual relationships [bib_ref] Body piercing: medical consequences and psychological motivations, Stirn [/bib_ref]. As the popularity for genital piercing grows, the number of related complications is likely to increase. In a survey of men and women with genital piercings, 53% of participants reported complications as a result their procedure [bib_ref] Selfreported characteristics of women and men with intimate body piercings, Caliendo [/bib_ref]. Bleeding, infection, allergic reactions to jewelry, keloid formation, and scarring have been reported among the complications of female genital piercings [bib_ref] First glimpse of the functional benefits of clitoral hood piercings, Millner [/bib_ref] [bib_ref] Complications of genital piercings, Dalke [/bib_ref] [bib_ref] Complications of body piercing, Meltzer [/bib_ref]. Embedded jewelry after clitoral piercing is rare and the management is not well described in the literature. In this case report, we describe a case of a woman who presented to the emergency department with an embedded clitoral piercing, which was removed with a simple outpatient procedure. ## Case A 24-year-old female presented to the emergency room with pain at the site of her clitoral piercing for 1 day. She reported that earlier that day, she noted that the piercing did not move freely on with touching the area. The patient was sexually active one day prior. The patient's own attempts to remove the piercing were unsuccessful due to pain. She had also returned to the tattoo parlor where the piercing was performed 8 months earlier who recommended she seek medical care. On examination, the labia were normal without lesions. There was a straight metal barbell protruding at a perpendicular angle from the glans of the clitoris. On closer inspection, one of the removable balls was missing from the barbell externally and on palpation, the other ball attached to the straight barbell was appreciated underneath the glans of the clitoris. There was no drainage, bleeding, or evidence of infection. There was no evidence of scarring or keloid formation at the site of the piercing. The patient was unable to tolerate initial attempts to remove due to pain. She gave consent for attempted removal of the piercing in the emergency 2 Case Reports in Obstetrics and Gynecology department. She was premedicated with 0.2 milligrams of intramuscular hydromorphone and the area was thoroughly cleansed with betadine. Then, 3 cc of 1% lidocaine without epinephrine was injected into the periclitoral skin and tested to ensure analgesia. A #11 scalpel was used to make a 2millimeter superficial incision within the nonkeratinizing squamous epithelium overlying the clitoral glans over the embedded ball of the piercing [fig_ref] Figure 1: Illustration of removal of embedded clitoral glans piercing removed successfully with an... [/fig_ref]. The embedded end of the piercing was then removed easily with gentle traction with forceps on the free end. The area was hemostatic after the procedure and did not require any sutures. She was discharged home from the emergency department with follow-up in gynecology clinic. # Discussion Genital piercing is not a new phenomenon. There are historical records of male genital piercing from as early as the 1st century B.C., and this practice has been alluded to in writings from the Mayan, Victorian, and World War II eras [bib_ref] Body piercing: where and how, Waugh [/bib_ref]. While genital piercing is not a new practice, information about the safety and complications did not reach the healthcare literature until the mid-1990s [bib_ref] Selfreported characteristics of women and men with intimate body piercings, Caliendo [/bib_ref]. Recent reports estimate that female clitoral piercing is increasing among many demographic groups [bib_ref] First glimpse of the functional benefits of clitoral hood piercings, Millner [/bib_ref] [bib_ref] Selfreported characteristics of women and men with intimate body piercings, Caliendo [/bib_ref] [bib_ref] Complications of genital piercings, Dalke [/bib_ref] [bib_ref] Complications of body piercing, Meltzer [/bib_ref] [bib_ref] Body piercing: where and how, Waugh [/bib_ref]. Despite its longevity throughout time, there is still much to be learned about genital piercings, especially in women. The practice of female genital piercing is not standardized. Common anatomical locations for female genital piercings include the labia majora, labia minora, and clitoris. The clitoris may be pierced through the prepuce, either horizontally or vertically or through the glans of the clitoris. Alternative female genital piercing locations that have been described include a vertical clitoral piercing exiting superiorly on the mons pubis, placement of a ring entering through the urethra and exiting between the urethral and vaginal openings ("Albertina Piercing") [bib_ref] The urologist's guide to genital piercing, Anderson [/bib_ref] [bib_ref] Genital piercings: diagnostic and therapeutic implications for urologists, Nelius [/bib_ref] , and posterior fourchette piercing. There are several complications of genital piercings that have been well documented in the literature, including bleeding, infection, trauma, and development of scar tissue. In a review by Anderson et al. they reported that genital piercing complications occur due to 4 reasons: poor technique during insertion, lack of adequate hygiene and care for the piercing, body changes after long-term jewelry wear, and partner damage [bib_ref] The urologist's guide to genital piercing, Anderson [/bib_ref]. For clitoral piercings specifically, piercings through the clitoral hood or prepuce are associated with decreased rates of infection, injury to the clitoris, and fibrosis. Genital piercing of the clitoral glans has been associated with higher rates of loss of sensation and diminished sexual function due to increased risk of damage to the nerves and vascular structures that supply of clitoris (dorsal artery and vein and deep artery and vein) [bib_ref] Genital piercings: diagnostic and therapeutic implications for urologists, Nelius [/bib_ref]. Traumatic injuries occurring at the time of insertion or afterwards, such as during sexual intercourse, are not uncommon [bib_ref] The urologist's guide to genital piercing, Anderson [/bib_ref] [bib_ref] Genital piercings: diagnostic and therapeutic implications for urologists, Nelius [/bib_ref]. Surgical repair is generally preferred to ensure that the delicate anatomic structure is retained [bib_ref] Complications of genital piercings, Dalke [/bib_ref]. However, a recent case report described the usage of 2-octyl cyanoacrylate (Dermabond) after a traumatic clitoral avulsion during a sexual encounter with full return of orgasmic function [bib_ref] Clitoral avulsion successfully repaired with 2-Octylcyanoacrylate, Berger [/bib_ref]. Ensuring good hygiene at the site of the piercing is essential. Chronic irritation and inflammation can lead to keloid and granuloma formation overtime. This may be prevented with use of stainless steel jewelry exclusively and meticulous self-care practices [bib_ref] Complications of genital piercings, Dalke [/bib_ref]. In patients with scar tissue or keloids, surgical removal or intralesional steroids have been helpful [bib_ref] First glimpse of the functional benefits of clitoral hood piercings, Millner [/bib_ref] [bib_ref] Complications of genital piercings, Dalke [/bib_ref]. While retained or embedded jewelry is reported as a known complication of genital piercings, there is little published information about patients who are at risk and management strategies. To our knowledge, this is the first report detailing the removal of an embedded clitoral piercing. Embedded jewelry from piercings has been reported in other areas of the body, most commonly for lingual piercings. Piercings of the tongue and clitoris are alike as barbell jewelry is commonly used in both which consists of a central stud with two peripheral balls [bib_ref] Complications of genital piercings, Dalke [/bib_ref] [bib_ref] Genital piercings: diagnostic and therapeutic implications for urologists, Nelius [/bib_ref] [bib_ref] A complication of lingual piercing: a case report, López-Jornet [/bib_ref]. The length of the central bar should exceed the thickness of the tongue or clitoris to permit free movement and prevent the jewelry from becoming trapped underneath the skin [bib_ref] A complication of lingual piercing: a case report, López-Jornet [/bib_ref]. If one of the peripheral studs on the barbell becomes loose and the jewelry length is inadequate, or the tissue is too wide, as in the case of swelling, this may cause the jewelry to become embedded. Several case reports have reported presence of an embedded barbell within the body of the tongue occurring several months after the piercing first occurred [bib_ref] A complication of lingual piercing: a case report, López-Jornet [/bib_ref] [bib_ref] Retained tongue stud: an unusual complication of tongue piercing, Mandal [/bib_ref]. In these cases, surgical excision of the embedded jewelry was performed under both general and local anesthesia. Similar to our case, small incisions were made on the dorsal surface of the tongue and the jewelry was removed intact with no long-term sequelae [bib_ref] A complication of lingual piercing: a case report, López-Jornet [/bib_ref] [bib_ref] Retained tongue stud: an unusual complication of tongue piercing, Mandal [/bib_ref]. A limitation of this report is that the patient was not followed longitudinally to assess for longterm sequelae of the clitoral piercing and removal procedure, including scarring, diminished sexual function, inability to achieve orgasm, dyspareunia, or vaginal pain. Patients should be counseled on the possibility of these outcomes prior to undergoing a removal procedure. In patients with clitoral piercings, the barbell length should be long enough to ensure free movement and patients should ensure that the two peripheral balls that secure the jewelry are screwed tight to avoid the jewelry becoming embedded. Healthcare providers should be aware of the increasing prevalence of genital piercing and the complications inherent to this practice. Complications after genital piercings are common, reported in over 50% of patients who had genital piercings. While the practice of female genital piercing is not standardized or supported by a wealth of evidence based literature, prior studies do suggest that piercing of the glans of the clitoris is associated with higher rates of injury to the neurovascular structures leading to future fibrosis, decreased function, and injury [bib_ref] The urologist's guide to genital piercing, Anderson [/bib_ref] [bib_ref] Genital piercings: diagnostic and therapeutic implications for urologists, Nelius [/bib_ref]. Therefore, clitoral piercings through the prepuce should be considered superior given the lower rate of injury and risk of diminished sexual function and patients should be aware of these risks prior to the piercing procedure. Retained jewelry is a known complication of genital piercings, but there is a paucity of published information on how to manage this problem. The best removal method in these delicate and sensitive areas must be tailored to the patient, the embedded object, and the physician's skill set. In this case, removal was performed safely in an outpatient setting avoiding the morbidity of general anesthesia and the expense of operative management. As the popularity for genital piercings increases, clinicians should be prepared to handle the potential complications. [fig] Figure 1: Illustration of removal of embedded clitoral glans piercing removed successfully with an outpatient procedure. [/fig]

Primary Versus Salvage Reverse Total Shoulder Arthroplasty for Displaced Proximal Humerus Fractures in the Elderly: A Systematic Review and Meta-analysis Background: There is currently no established consensus on best treatment for complex proximal humerus fractures (PHFs) in the elderly. Reverse total shoulder arthroplasty (RTSA) is a viable option in this population but many times is used as a salvage procedure. Methods: A systematic review of studies comparing RTSA as a primary treatment for PHF versus as a salvage procedure following failed open reduction internal fixation (ORIF), humeral intramedullary nailing, hemiarthroplasty (HA) or nonoperative treatment was conducted using PRISMA guidelines. Pooled outcomes and sub-group analyses assessing range of motion, patient reported outcomes and complications were examined using RevMan. Results: Five articles were included in final analysis with 104 patients in the primary RTSA group and 147 in the salvage RTSA group compromising 251 total patients. Primary RTSA had a statistically significant advantage in range of motion (forward flexion and external rotation), patient reported outcomes, and complications compared to salvage RTSA. Conclusions: Based on the best available evidence, primary RTSA may result in slightly better patient reported outcomes, range of motion and a lower rate of complication when compared to salvage RTSA. Further high-quality prospective studies are needed to confirm the findings of the current review. # Introduction Proximal humerus fractures (PHFs) account for approximately six percent of all adult fractures and disproportionately affect older patients and women. [bib_ref] Epidemiology of adult fractures: a review, Court-Brown [/bib_ref] As the population ages, the incidence of PHFs is projected to triple by 2030. [bib_ref] Update in the epidemiology of proximal humeral fractures, Palvanen [/bib_ref] While most PHFs are nondisplaced or one part fractures that can be treated non-operatively, [bib_ref] Functional outcome after minimally displaced fractures of the proximal part of the..., Koval [/bib_ref] [bib_ref] Risk factors for fractures of the proximal humerus: results from the EPIDOS..., Lee [/bib_ref] the optimal treatment strategy for more complex fractures (e.g., three and four-part) remains unclear. [bib_ref] Interventions for treating proximal humeral fractures in adults, Handoll [/bib_ref] Traditionally, open reduction and internal fixation (ORIF) or hemiarthroplasty (HA) have been the surgical treatment of choice, but they have many potential complications such as nonunion and poor tuberosity healing. [bib_ref] Tuberosity malposition and migration: reasons for poor outcomes after hemiarthroplasty for displaced..., Boileau [/bib_ref] While many times HA treatment provides excellent outcomes, when complications arise they are frequently complex. This exacerbated in the elderly population where bone quality is diminished, and comorbidities are common. Consequently, achieving adequate fixation in this population is technically challenging as low bone mineral density is one of the biggest predictors of fixation failure. [bib_ref] Predicting failure after surgical fixation of proximal humerus fractures, Krappinger [/bib_ref] Since the controversial Proximal Fracture of the Humerus: Evaluation by Randomization (ProFHER) trial in 2015 suggested non-operative treatment of these injuries is equivalent to surgery, [bib_ref] Surgical vs nonsurgical treatment of adults with displaced fractures of the proximal..., Rangan [/bib_ref] identifying both surgical candidates and optimal fixation technique continues to be contentious with no consensus on best treatment strategy. Reverse total shoulder arthroplasty (RTSA) has provided surgeons with a reliable option for managing complex PHFs. Traditionally, RTSA was used primarily as a salvage procedure after failed HA, ORIF, or nonoperative fracture sequelae when no other options remained. [bib_ref] Reverse total shoulder arthroplasty for failed open reduction and internal fixation of..., Grubhofer [/bib_ref] [bib_ref] Reverse total shoulder arthroplasty for posttraumatic sequelae, Hattrup [/bib_ref] [bib_ref] Five-year outcome after conversion of a hemiarthroplasty when used for the treatment..., Holschen [/bib_ref] [bib_ref] Reverse shoulder arthroplasty as a salvage procedure after failed internal fixation of..., Hussey [/bib_ref] [bib_ref] The use of the reverse shoulder prosthesis for the treatment of failed..., Levy [/bib_ref] [bib_ref] Reverse shoulder prosthesis as revision surgery after fractures of the proximal humerus,..., Lollino [/bib_ref] [bib_ref] Can reverse shoulder arthroplasty in post-traumatic revision surgery restore the ability to..., Maier [/bib_ref] [bib_ref] Reverse total shoulder arthroplasty for acute fractures and failed management after proximal..., Martin [/bib_ref] [bib_ref] The treatment of proximal humerus nonunions in older patients with reverse shoulder..., Martinez [/bib_ref] [bib_ref] Early clinical and radiological outcomes of reverse shoulder arthroplasty with an eccentric..., Merolla [/bib_ref] [bib_ref] Reverse shoulder arthroplasty for malunions of the proximal part of the humerus..., Raiss [/bib_ref] [bib_ref] Reverse shoulder arthroplasty for the treatment of nonunions of the surgical neck..., Raiss [/bib_ref] [bib_ref] Reverse total shoulder arthroplasty for type I fracture sequelae after internal fixation..., Schliemann [/bib_ref] [bib_ref] Increased-offset reverse shoulder arthroplasty for the treatment of failed posttraumatic humeral head..., Uri [/bib_ref] [bib_ref] Proximal humeral malunion treated with reverse shoulder arthroplasty, Willis [/bib_ref] [bib_ref] Reverse total shoulder replacement for nonunion of a fracture of the proximal..., Zafra [/bib_ref] However, these patients frequently experience only mild improvements in their activities of daily living (ADLs) [bib_ref] Can reverse shoulder arthroplasty in post-traumatic revision surgery restore the ability to..., Maier [/bib_ref] [bib_ref] Comparison of functional outcomes of reverse shoulder arthroplasty versus hemiarthroplasty in the..., Young [/bib_ref] and face a high risk of complications. [bib_ref] Reverse total shoulder replacement for nonunion of a fracture of the proximal..., Zafra [/bib_ref] [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Treatment of fracture sequelae of the proximal humerus: comparison of hemiarthroplasty and..., Alentorn-Geli [/bib_ref] Several systematic reviews have suggested that RTSA may be superior to HA for acute PHFs [bib_ref] Hemiarthroplasty versus reverse shoulder arthroplasty for treatment of proximal humeral fractures: a..., Shukla [/bib_ref] [bib_ref] Meta-analysis suggests that reverse shoulder arthroplasty in proximal humerus fractures is a..., Wang [/bib_ref] [bib_ref] Is reverse total shoulder arthroplasty more effective than hemiarthroplasty for treating displaced..., Gallinet [/bib_ref] and in recent years RTSA has become an increasingly popular initial option for these injuries. [bib_ref] Short-term projected use of reverse total shoulder arthroplasty in proximal humerus fracture..., Law [/bib_ref] [bib_ref] Reverse total shoulder arthroplasty for the treatment of proximal humeral fractures: patterns..., Acevedo [/bib_ref] [bib_ref] National trends in proximal humerus fracture treatment patterns, Sabesan [/bib_ref] [bib_ref] Survey study suggests that reverse total shoulder arthroplasty is becoming the treatment..., Savin [/bib_ref] [bib_ref] Arthroplasty treatment of proximal humerus fractures: 14-year trends in the United States, Schairer [/bib_ref] As such, the purpose of this systematic review was to compare outcomes of RTSA used as a primary versus salvage procedure for complex PHFs among the elderly population. # Methods We conducted a systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] ## Literature search A literature search of EMBASE, MEDLINE, PubMed and the Cochrane trials registry was conducted using the following keywords in combination: "shoulder fractur*", "humer* fractur*, proximal", and "reverse adj2 shoulder" for MEDLINE and "reverse shoulder arthroplasty", "proximal humerus", and "fracture" for EMBASE and Cochrane for studies published between 2015-2020. Systematic reviews from our search were retrieved, and their references were reviewed for any additional studies that could be included. ## Eligibility criteria All studies comparing RTSA as a primary treatment for PHF with RTSA as a salvage procedure for failed treatment of PHF were eligible for inclusion. Failed treatment included non-operative, hemiarthroplasty, intramedullary nailing, or ORIF. Only prospective and retrospective comparative studies evaluating functional outcomes (e.g., simple shoulder test, constant score, American Shoulder and Elbow Surgeons [ASES] score) were included. Study inclusion criteria included minimum of 12-month follow-up and a minimum of 10 patients in each treatment group. A study was excluded if data from the same patients were included in another eligible study with longer follow-up. Conference abstracts were also excluded. ## Study selection Two reviewers screened the titles and abstracts generated by the literature search for eligibility. If there was any uncertainty or ambiguity regarding eligibility, the study was included for full-text review. The reviewers independently assessed each full report to determine whether inclusion criteria were met. Disagreements were resolved by discussion with the senior author, when necessary. ## Data extraction Two reviewers extracted relevant data from each included study and recorded them into Microsoft Excel worksheets. Data collected in the worksheets included first author, journal, year of publication, level of evidence, number of patients, follow-up duration, average age of patient, definition of proximal humerus fracture and outcomes observed. A comments section was included for any other relevant data pertinent to each study. Common outcome data were entered into a metaanalysis software package 37 for pooled analysis. ## Assessment of risk of bias in eligible studies The Newcastle-Ottawa Scale (NOS)was used to evaluate the quality of eligible prospective and retrospective cohort studies. The NOS assesses each study on 3 domains: selection, comparability, and outcome. Two reviewers independently assessed the methodological quality of eligible studies. Any disagreements were resolved with consensus discussion. # Statistical methods Descriptive statistics were calculated with categorical data presented as frequency with percentages and continuous data as mean AE standard deviation (SD). Weighted means with their corresponding SDs were calculated for all parameters. For studies that did not report the mean and SD directly, these values were imputed from the p-value, confidence interval (CI) and range using well-established statistical techniques. [bib_ref] Estimating the mean and variance from the median, range, and the size..., Hozo [/bib_ref] Mean differences were calculated for continuous outcomes. Ninety-five percent CIs were reported for all point estimates. Pooled estimates were calculated using Review Manager (RevMan) 5.3 (Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014).A funnel plot was used to assess for publication bias. An a priori subgroup analyses was planned to examine the differences in range of motion (ROM) and patient reported outcome measures (data permitting) following primary RTSA versus RTSA as a salvage after each of: ORIF, HA and non-operative treatment (resulting in non-union or malunion). # Results ## Literature search and study characteristics The results of our comprehensive search, the selection process, and the number of studies excluded with the corresponding rationale are depicted in . Five studies were identified that satisfied all inclusion and exclusion criteria. [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] [bib_ref] Early vs. late reverse shoulder arthroplasty for proximal humerus fractures: does it..., Seidl [/bib_ref] [bib_ref] Outcomes and revision rates of primary vs. secondary reverse total shoulder arthroplasty..., Katthagen [/bib_ref] Three studies were retrospective cohorts [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Early vs. late reverse shoulder arthroplasty for proximal humerus fractures: does it..., Seidl [/bib_ref] [bib_ref] Outcomes and revision rates of primary vs. secondary reverse total shoulder arthroplasty..., Katthagen [/bib_ref] while the other two were matched casecontrol. [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] All studies were published between 2016-2020. A total of 147 patients underwent RTSA as a salvage procedure (78% female), while 104 patients underwent RTSA for acute PHF (79% female) for a total of 251 patients. Mean follow-up time was 31.8 months (SD AE 2.5) and mean age was 73.0 years (SD AE 2.1). Two of the studies included failed hemiarthroplasty, ORIF, and non-operative treatment as part of the salvage group, 41,42 one included failed hemiarthroplasty, ORIF and intramedullary nailing, [bib_ref] Outcomes and revision rates of primary vs. secondary reverse total shoulder arthroplasty..., Katthagen [/bib_ref] and two studies only included patients who failed ORIF. [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] Common outcomes between the studies included forward flexion, 26,40-43 external rotation, [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] [bib_ref] Early vs. late reverse shoulder arthroplasty for proximal humerus fractures: does it..., Seidl [/bib_ref] [bib_ref] Outcomes and revision rates of primary vs. secondary reverse total shoulder arthroplasty..., Katthagen [/bib_ref] ASES, [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] [bib_ref] Early vs. late reverse shoulder arthroplasty for proximal humerus fractures: does it..., Seidl [/bib_ref] UCLA score, [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] and Constant Score. [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty as primary versus revision procedure for..., Dezfuli [/bib_ref] [bib_ref] Outcomes and revision rates of primary vs. secondary reverse total shoulder arthroplasty..., Katthagen [/bib_ref] The characteristics of each study are summarized in [fig_ref] Table 1: Baseline Characteristics for All Included Studies [/fig_ref]. ## Study quality The studies involved were comparable with respect to important demographic variables (i.e. age, follow-up, definition of fracture). Overall study quality was moderate to high (see Supplemental Digital Content 1, which provides complete results of the NOS). Funnel plots were used to assess for publication bias and were found to be symmetrical (see Supplemental Digital Content 2). ## Range of motion The mean forward flexion (FF) and external rotation (ER) were both significantly higher in the primary RTSA group compared to the salvage RTSA group with a mean difference (MD) of 11.7 degrees for FF (95% CI, 6.0-17.4; P < 0.0001) [fig_ref] Figure 2: Pooled mean difference of range of motion in patients undergoing primary RTSA... [/fig_ref] ) and 1.5 for ER (95% CI 0.2-2.8; P ¼ 0.02) [fig_ref] Figure 2: Pooled mean difference of range of motion in patients undergoing primary RTSA... [/fig_ref] ## Functional outcomes Primary RTSA for PHFs was found to have significantly higher ASES (MD, 7.6; CI, 1.7-13.5; P ¼ 0.01) [fig_ref] Figure 3: Pooled mean difference of qualitative function measures in patients undergoing primary RTSA... [/fig_ref] , UCLA (MD, 3.6; 95% CI, 1.6-5.5; P ¼ 0.0004) [fig_ref] Figure 3: Pooled mean difference of qualitative function measures in patients undergoing primary RTSA... [/fig_ref] ) and Constant (MD, 6.8; 95% CI, 2.2-11.3; P ¼ 0.003) [fig_ref] Figure 3: Pooled mean difference of qualitative function measures in patients undergoing primary RTSA... [/fig_ref] ) scores when compared to salvage RTSA for failed treatment of PHFs. There was no statistically significant difference between the groups for the SST [fig_ref] Figure 3: Pooled mean difference of qualitative function measures in patients undergoing primary RTSA... [/fig_ref]. ## Complications Breakdown of specific complications is included in . The overall complication rate in the primary RTSA and salvage RTSA groups was 4.8% (5/104) and 18.7% (28/150), respectively. This included five intraoperative complications in the salvage group and one in the primary group. The odds of having a complication were 78% lower amongst those undergoing RTSA primarily versus as a salvage procedure (P ¼ 0.002) [fig_ref] Figure 4: Pooled odds ratio for [/fig_ref]. The most common complications in the salvage group included dislocation (6), periprosthetic fracture (5) ## , infection (3), implant loosening (3), cortical perforation (2) and component malposition (2). ## Reoperation Reoperation was defined as any subsequent surgical procedure that occurred as a direct result of the initial operation including incision and drainage for infection or hematoma removal. If the text did not explicitly state a procedure occurred, it was not counted. The overall reoperation rate in the primary RTSA group was 2.9% (3/105) and 10.7% (16/150) in the salvage group (P ¼ 0.04) [fig_ref] Figure 4: Pooled odds ratio for [/fig_ref]. # Discussion The results of the current systematic review demonstrate that primary RTSA may result in greater forward flexion, fewer complications and superior functional outcomes when compared to RTSA as a salvage procedure. The clinical significance of these results is especially timely as the ProFHER trial, [bib_ref] Surgical vs nonsurgical treatment of adults with displaced fractures of the proximal..., Rangan [/bib_ref] which reported no significant difference in outcome between operative and non-operative management of displaced proximal humerus fractures involving the surgical neck, continues to receive widespread media attention, despite well-documented concerns over study methodology. [bib_ref] To operate or not to operate, that is the question: the proximal..., Ghert [/bib_ref] [bib_ref] A review of current surgical practice in the operative treatment of proximal..., Dean [/bib_ref] Consequently, this may result in a greater proportion of patients being inappropriately treated non-operatively initially, leading to a potential increase in the rate of fracture sequalae such as malunion and non-union. [bib_ref] Impact of the PROFHER trial findings on surgeons' clinical practice: an online..., Jefferson [/bib_ref] An exploratory subgroup analyses of primary RTSA compared to salvage RTSA after either failed HA or ORIF for PHFs demonstrated that patients may have better ROM and functional outcomes with primary RTSA in both subgroups. Two recent studies have noted that patients undergoing salvage RTSA for failed non-operative management may have better outcomes when compared to those undergoing RTSA for failed HA of PHFs. [bib_ref] Reverse shoulder arthroplasty in acute fractures provides better results than in revision..., Cicak [/bib_ref] [bib_ref] Reverse shoulder arthroplasty for fracture sequelae: How the initial fracture treatment influences..., Santana [/bib_ref] These findings would suggest that patients at high risk of failing HA may fare better with non-operative management initially with the option to undergo RTSA in the future, if indicated. While this review found statistical significance favoring primary RTSA compared to RTSA as a salvage procedure, the clinical significance of these results is less clear. The mean forward flexion among the primary RTSA group was 125 compared to 110 in the salvage RTSA group. Based on previous reports, it has been noted that 120 of forward flexion is required to perform the majority of ADLs that involve the shoulder. [bib_ref] Defining functional shoulder range of motion for activities of daily living, Namdari [/bib_ref] Therefore, the 15 mean difference between the primary and salvage RTSA group demonstrated in the current review may represent an important clinical difference. When . Summary of Complications. considering external rotation (ER), studies evaluating functional ER measure ROM with the shoulder abducted 90 and have found approximately 60 AE 10 is necessary to perform most ADLs. [bib_ref] Defining functional shoulder range of motion for activities of daily living, Namdari [/bib_ref] The studies included in this review measured ER with arm at the side with neither primary nor salvage RTSA coming within 25 of this threshold (30.3 and 19.9, respectively). However, Simovitch and colleagues found that among patients undergoing RTSA, the minimal clinically important difference (MCID) for active external rotation was À5.3 AE 3.1 degrees, suggesting that restoration of external rotation may not be as important as pain relief, joint stability, and a low complication rate when evaluating the success of RTSA in this patient population. [bib_ref] Quantifying success after total shoulder arthroplasty: the minimal clinically important difference, Simovitch [/bib_ref] Patients in the primary RTSA group had statistically significantly higher Constant, ASES, and UCLA scores. However, it is difficult to determine whether these differences are clinically important. The MCID for the Constant score among patients undergoing RTSA for rotator cuff arthropathy or glenohumeral arthritis has been reported to be as high as 8.0 51 and as low as 5.7. [bib_ref] Quantifying success after total shoulder arthroplasty: the minimal clinically important difference, Simovitch [/bib_ref] As such, the observed mean difference of 6.1 among the primary RTSA cohort in the current review may be clinically significant. [bib_ref] Quantifying success after total shoulder arthroplasty: the minimal clinically important difference, Simovitch [/bib_ref] The mean difference in ASES of 7.6 observed between the primary RTSA and salvage RTSA groups in the current study closely approximates the previously reported MCIDs of 8.4 and 10.3 but falls short of achieving clinical significance. [bib_ref] Quantifying success after total shoulder arthroplasty: the minimal clinically important difference, Simovitch [/bib_ref] [bib_ref] What change in American shoulder and elbow surgeons score represents a clinically..., Werner [/bib_ref] Finally, the observed mean difference in UCLA score between the primary and salvage groups did not fall within the reported MCID of 7.0. [bib_ref] Quantifying success after total shoulder arthroplasty: the minimal clinically important difference, Simovitch [/bib_ref] It is important to note that among the few published studies that evaluate the MCID for the aforementioned outcome measures in patients with RTSA, the majority do so for the rotator cuff tear arthropathy population. Extrapolating these findings to patients receiving RTSA for PHFs may overestimate the difference required for clinical significance in the trauma population and even small differences may be important to these patients given the difficulty in achieving good outcomes in these complex fractures. It has been reported that the initial costs associated with undergoing HA and RTSA are approximately $30,000 and $55,000 respectively. [bib_ref] Cost analysis of hemiarthroplasty versus reverse shoulder arthroplasty for fractures, Solomon [/bib_ref] [bib_ref] Arthroplasty for the surgical management of complex proximal humerus fractures in the..., Nwachukwu [/bib_ref] However, these costs can vary significantly depending on the cost of the implant (between $1,000-10,000). [bib_ref] A costeffectiveness analysis of reverse total shoulder arthroplasty versus hemiarthroplasty for the..., Osterhoff [/bib_ref] Several recent studies have found RTSA to be more cost-effective over the long term with an incremental costeffectiveness ratio of <$14,000 per quality adjust life year (QALY) gained compared to HA. [bib_ref] Arthroplasty for the surgical management of complex proximal humerus fractures in the..., Nwachukwu [/bib_ref] [bib_ref] A costeffectiveness analysis of reverse total shoulder arthroplasty versus hemiarthroplasty for the..., Osterhoff [/bib_ref] In fact, Osterhoff et al. noted that the cost-effectiveness of RTSA is similar to other highly successful orthopaedic procedures such as total hip and knee arthroplasty. [bib_ref] A costeffectiveness analysis of reverse total shoulder arthroplasty versus hemiarthroplasty for the..., Osterhoff [/bib_ref] In the current review patients undergoing primary RTSA were found to have a significantly lower rate of complication when compared to salvage RTSA. Saltzman and colleagues reported similar findings with primary RTSA and revision RTSA having complication rates of 25% and 69%, respectively. [bib_ref] Complication rates comparing primary with revision reverse total shoulder arthroplasty, Saltzman [/bib_ref] Given the costeffectiveness, lower rate of complication and superior functional outcomes of primary compared to salvage RTSA, the use of RTSA as a first line treatment option among patients who are at increased risk of treatment failure, such as older patients, those with osteoporosis, varus displacement and medial comminution may be justified. [bib_ref] Predicting failure after surgical fixation of proximal humerus fractures, Krappinger [/bib_ref] [bib_ref] Proximal humeral fracture treatment in adults, Maier [/bib_ref] This study has several limitations. First, the results of the current review are based on the results of five small comparative studies. Second, all the studies included in this review were retrospective in nature. Therefore, the conclusions are subject to the inherent biases associated with these types of studies, such as selection bias. For example, patients undergoing acute RTSA may have had more complex fracture patterns, therefore leading to a possible underestimation of our findings. This is compounded by the fact that only two of the five studies specified the type of fracture pattern for their inclusion criteria. [bib_ref] Reverse shoulder arthroplasty for proximal humeral fractures: outcomes comparing primary reverse arthroplasty..., Shannon [/bib_ref] [bib_ref] Outcomes of reverse total shoulder arthroplasty for proximal humeral fractures: primary arthroplasty..., Sebastia-Forcada [/bib_ref] Furthermore, none of the studies commented on surgeon experience with RTSA in the trauma setting as this could have affected the outcome. It has previously been noted that RTSA done by less experienced surgeons can result in a higher rate of complications. [bib_ref] Do the indications, results, and complications of reverse shoulder arthroplasty change with..., Walch [/bib_ref] Additionally, the time from index to salvage procedure was not reported in the majority of included studies; a shorter interval would suggest that primary RTSA may be worthwhile among those patients who are at higher risk of early failure. Finally, potential confounding factors such as comorbidities and bone quality were not evaluated in the current review due to the lack of reporting among included studies. # Conclusion Based on the current available evidence, elderly patients with displaced PHF may have significantly greater range of motion, a lower risk of complications, and slightly better patient reported outcomes with primary RTSA compared to those undergoing RTSA as a salvage procedure. The clinical significance of these results is less clear and requires additional prospective studies to confirm our findings. ## Authors' note ## Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. # Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. ## Orcid id Patrick A Nelson https://orcid.org/0000-0002-5484-8336 # Supplemental material Supplemental material for this article is available online. [fig] Figure 2: Pooled mean difference of range of motion in patients undergoing primary RTSA compared with salvage RTSA. A, forward flexion; B, external rotation. RTSA, reverse total shoulder arthroplasty; SD, standard deviation; CI, confidence interval. [/fig] [fig] Figure 3: Pooled mean difference of qualitative function measures in patients undergoing primary RTSA compared with salvage RTSA. A, American Shoulder and Elbow Surgeons Shoulder Score (ASES); B, UCLA Shoulder Score; C, Constant Score; D, Simple Shoulder Test (SST). RTSA, reverse total shoulder arthroplasty; SD, standard deviation; CI, confidence interval. [/fig] [fig] Figure 4: Pooled odds ratio for (A) complications and (B) reoperations among patients undergoing primary RTSA compared with salvage RTSA. RTSA, reverse total shoulder arthroplasty; CI, confidence interval. [/fig] [table] Table 1: Baseline Characteristics for All Included Studies. Abbreviations: RTSA, reverse total shoulder arthroplasty; SD, standard deviation; JSES, Journal of Shoulder and Elbow Surgery; HA, hemiarthroplasty; ORIF, open reduction internal fixation; ASES, American Shoulder and Elbow Surgeon score; SF-12, 12-item short form health survey; SPADI, shoulder pain and disability index; SST, simple shoulder test; UCLA, University of California at Los Angeles score; ROM, range of motion; DASH, disabilities of the arm, shoulder hand score; SSV, subjective shoulder value; JOT, Journal of Orthopaedic Trauma; ABJS, Archives of Bone and Joint Surgery; SANE, single assessment numerical evaluation; HO, heterotopic ossification. [/table]

Challenges and Promises for Planning Future Clinical Research Into Bacteriophage Therapy Against Pseudomonas aeruginosa in Cystic Fibrosis. An Argumentative Review Which PA hosts should be investigated in vitro and in vivo?Experiments investigating phage efficacy in vitro and in vivo in CF should test mucoid and nonmucoid multidrug-resistant (MDR) PA host strains from patients with CF (MDR PA defined according to Centers for Disease Control and Prevention, CDC, Atlanta, USA).Which experimental models should be used in vitro to mimic CF biological conditions?Models mimicking CF biological conditions include testing airway surface liquid models, alginate consequences, dynamic biofilm, and epithelial cell monolayers from patients with CF. Composite tests should evaluate phage efficacy, and safety from prolonged phage exposure, PA phage resistance, and fitness costs on PA hosts.How should phages be characterized for treating CF?Phage characterization and selection should follow and report rigorous steps (including efficiency of plating, host range, bioinformatics genomic sequence, burst size, latency period, activity, efficiency, and stability).What do non-pulmonary models add to in vivo studies?Non-pulmonary models in vivo (Galleria mellonella larvae) add essential information for assessing, easily and at a low cost, prolonged phage exposure, phage-PA host-related toxicity (lipopolysaccharide release from lysis), and PA host resistance at least 120 hours after phage administration to PA infected larvae. Theses studies need also to use standardized negative control groups to assess phage safety. Although early aggressive and prolonged treatment with specific antibiotics can extend survival in patients with cystic fibrosis (CF) colonized by opportunistic Pseudomonas aeruginosa (PA), antibiotics fail to eradicate the infecting multidrug-resistant (MDR) PA strains in CF. Century-long research has suggested treating patients with bacteriophages (phages, prokaryotic viruses) naturally hosted by bacteria. Although the only phage types used in therapy, lytic phages, lyse PA aggregated in biofilm matrix by depolymerase degrading enzymes, how they can effectively, safely, and persistently do so in patients with CF is unclear. Even though advanced techniques for formulating phage cocktails, training phages and collecting phage libraries have improved efficacy in vitro, whether personalized or ready-to-use therapeutic approaches or phages and antibiotics combined are effective and safe in vivo, and can reduce PA biofilms, remains debatable. Hence, to advance clinical research on phage therapy in clinical trials, also involving mucoid and non-mucoid multidrug-resistant PA in CF, and overcome problems in Western international regulations, we need reliable and repeatable information from experiments in vitro and in vivo on phage characterization, cocktail selection, personalized approaches, and phages combined with antibiotics. These findings, challenges, and promises prompted us to undertake this argumentative review to seek up-to-date information from papers describing lytic phage activity tested in vitro on PA laboratory strains, and PA strains from chronic infections including CF. We also reviewed in vivo studies on phage activity on pulmonary and non-pulmonary animal host models infected by laboratory or CF PA strains. Our argumentative review provides essential information showing that future phage clinical research in CF should use wellcharacterized and selected phages isolated against CF PA, tested in vitro under dynamic conditions in cocktails or combined with antibiotics, and in vivo on non-pulmonary and pulmonary host models infected with mucoid and non-mucoid CF MDR PA. Our findings should encourage pharmaceutical industries to conduct clinical trials in vitro and in vivo testing patented genomic engineered phages from phage libraries combined with antibiotics to treat or even prevent multidrug-resistant PA in CF, thus helping international regulatory agencies to plan future clinical research on phage therapy in CF. # Introduction Pseudomonas aeruginosa (PA), an opportunistic environmental pathogen, typically colonizes 30% of children, and up to 80% of 25-year-old and older adults with cystic fibrosis (CF) [bib_ref] Pathophysiology and management of pulmonary infections in cystic fibrosis, Genbank ; Gibson [/bib_ref] [bib_ref] Early eradication of Pseudomonas aeruginosa in patients with cystic fibrosis, Stuart [/bib_ref] , thus inexorably causing chronic lung infection, pulmonary function decline, and death [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] [bib_ref] Pseudomonas aeruginosa chronic colonization in cystic fibrosis patients, Murray [/bib_ref] [bib_ref] Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. a short review, Moreau-Marquis [/bib_ref]. In CF lung, PA typically produces biofilm, a deleterious, complex tower-or mushroom-like matrix enclosing sessile PA aggregates. Surface motile planktonic PA cells leave the biofilm, colonize new lung sites, and initiate new sessile PA micro-colonies triggering repeated lung infections requiring antibiotics (Supplementary ; [bib_ref] Bacterial biofilms: a common cause of persistent infections, Costerton [/bib_ref] [bib_ref] The application of biofilm science to the study and control of chronic..., Costerton [/bib_ref]. After prolonged and repeated broad-spectrum antibiotic courses, enhanced resistance to antibiotics develops and leads to nonmucoid and mucoid multidrug-resistant (MDR) PA strains [bib_ref] Biofilms: survival mechanisms of clinically relevant microorganisms, Donlan [/bib_ref] [bib_ref] Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in..., Li [/bib_ref] [bib_ref] Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis, Gómez [/bib_ref] [bib_ref] The formation of biofilms by Pseudomonas aeruginosa: a review of the natural..., Rasamiravaka [/bib_ref]. MDR PA as defined by Centers for Disease Control and Prevention in Atlanta [bib_ref] Antimicrobial-resistant pathogens associated with healthcareassociated infections summary of data reported to the..., Sievert [/bib_ref] is non-susceptible (resistant or intermediate) to at least one agent in three or more antimicrobial classes: βlactam, aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems. Despite international recommendations on CF infections, including MDR organism prevention and control [bib_ref] Infection prevention and control guideline for cystic fibrosis: 2013 update, Saiman [/bib_ref] , and recent findings on PA evolutionary adaptation, diversification, and resistance factors in CF lungs (Supplementary ; [bib_ref] Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections, Winstanley [/bib_ref] , research interest is waning on new antibiotics to include in pipeline programs [bib_ref] Future antibiotics scenarios: is the tide starting to turn?, Theuretzbacher [/bib_ref] [bib_ref] New treatment options against gram-negative organisms, Bassetti [/bib_ref]. Hence, to combat CF MDR PA infections, we urgently require more efficient therapeutic approaches. As a new weapon to treat CF lung infection caused by MDR PA and other multi-resistant superbugs, extensive research has reappraised bacteriophage (phage) therapy [bib_ref] Old dogma, new tricks 21st century phage therapy, Thiel [/bib_ref] [bib_ref] Novel approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis, Hurley [/bib_ref]. Phages, bacterial parasitic viruses abounding in the environment, are the oldest known viruses dating back 3.5 billion years [bib_ref] The oldest fossils and what they mean, Schopf [/bib_ref] [bib_ref] Tailed bacteriophages. The order Caudovirales, Ackermann [/bib_ref]. Although every bacterium probably hosts its own phages, they are deemed unable to produce infection in human organisms [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref]. Phages were discovered independently about a century ago in stool samples taken from patients shortly before recovering from dysentery [bib_ref] An investigation on the nature of ultra-microscopic viruses, Twort [/bib_ref] [bib_ref] Sur un microbe invisible antagoniste des bacilles dysentériques, D&apos;hérelle [/bib_ref] [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref]. These findings, therefore, suggested that phages might be useful for antibacterial therapy [bib_ref] Phage therapy gets revitalized, Reardon [/bib_ref]. Even though Eastern European health institutions officially permitted, and researchers clinically applied phage therapy in patients with CF [bib_ref] Pseudomonas aeruginosa bacteriophage in treatment of P. aeruginosa infection in cystic fibrosis..., Shabalova [/bib_ref] [bib_ref] Bacteriophages as potential new therapeutics to replace or supplement antibiotics, Kutateladze [/bib_ref] [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref] , in Western Europe international regulatory bodies were unable to solve ethical concerns or issue clinical guidelines on phage therapy in patients with CF [bib_ref] European regulatory conundrum of phage therapy, Verbeken [/bib_ref]. Hence, phage therapy gave way to the newly discovered antibiotics [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref]. Unlike antibiotics, phages attack the host-pathogen surface, and inject their genome into the host cell leaving the commensal bacteria unchanged, thus gaining the nickname "intelligent antibiotics" (Loc-Carrillo and [bib_ref] Pros and cons of phage therapy, Loc-Carrillo [/bib_ref] [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref]. After injecting their genome, phages can have two different lifecycles. In the lytic cycle, the bacterial molecular system produces viral components such as capsid, nucleic acid, and structural proteins. Eventually, these assembled components release new virions thus causing bacterial lysis [bib_ref] The structure and infective process of a contractile Pseudomonas aeruginosa bacteriophage, Bradley [/bib_ref]. In the lysogenic cycle, the phage lingers as a plasmid or integrates into the bacterial genome giving rise to a prophage. These quiescent states (plasmid or prophage) last until specific stimuli force them to enter the lytic cycle [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref]. Because lysogenic (temperate) phages can transfer DNA host fragments possibly containing gene-encoding toxins or antibiotic resistance thus generating new virulent bacteria, the only phages exploited for therapy are lytic phages. Lytic phages lyse PA and through specific depolymerizing enzymes degrade bacterial biofilm thus efficiently killing the host bacteria [bib_ref] Bacteriophages and their immunological applications against infectious threats, Criscuolo [/bib_ref]. What we do not know is which readyto-use (prêt-à-porter) naturally-occurring (ancestral) or trained (evolved) phages or phages isolated on specific patient PA strains (sur-mesure, personalized approach) alone or in cocktails could increase phage virulence (lytic activity) against CF PA tested in vitro and in vivo [bib_ref] The phage therapy paradigm: pret-a-porter or sur-mesure?, Pirnay [/bib_ref] [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref] [bib_ref] Bacteriophage-based therapy in cystic fibrosis-associated Pseudomonas aeruginosa infections: rationale and current status, Hraiech [/bib_ref]. Other open questions include phage gene function, lifecycles, and phage-pathogen interactions. We also need more information on which characterized and genome-sequenced lytic phages, or trained phages or phages stored in therapeutic commercial mixtures, certified as safe from central phage banks or libraries could safely combat MDR PA strains in patients with CF [bib_ref] Phage approved in food not as a therapeutic?, Sarhan [/bib_ref] [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. Although the Food and Drug Administration recommends lytic phages [bib_ref] What is needed for phage therapy to become a reality in western..., Brüssow [/bib_ref] , before testing in vivo clinical efficacy in CF PA, we lack essential information on the property criteria that should be reported to justify choosing one phage rather than another [bib_ref] Information phage therapy research should report, Abedon [/bib_ref]. These criteria address methods to assess in vitro phage functional characterization (burst size, latency period), activity (host range assay used), bio-informatics genomics (accession numbers from gene sequencing databases, International nucleotide sequence database consortium, INSDC) 1 , how to select and test bacterial hosts to avoid prophage-induced contamination in phage stocks, phage stability, efficacy, and bacterial host resistance in vitro and in vivo [bib_ref] The use of phages for the removal of infectious biofilms, Azeredo [/bib_ref] [bib_ref] Recent advances in bacteriophage therapy: how delivery routes, formulation, concentration and timing..., Ryan [/bib_ref] [bib_ref] Natural solution to antibiotic resistance: bacteriophages "The Living Drugs, Jassim [/bib_ref] [bib_ref] Bacteriophage-based therapy in cystic fibrosis-associated Pseudomonas aeruginosa infections: rationale and current status, Hraiech [/bib_ref] [bib_ref] Bacteriophage procurement for therapeutic purposes, Weber-Dabrowska [/bib_ref]. Criteria for assessing in vivo phage safety include also experiments testing removal (purity) of lipopolysaccharides (LPS) derived from PA lysis, and phage administration routes and doses [bib_ref] Information phage therapy research should report, Abedon [/bib_ref]. Although the first randomized controlled trial (RCT) provided evidence on phage therapy in patients with MDR PA-related chronic otitis [bib_ref] A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis..., Wright [/bib_ref] , and CF MDR PA is a typical candidate for phage therapy, no previous reviews have addressed this topic. Nor are trials underway on MDR PA in CF. Pivotal studies on genomic technologies theorized that genomic engineered phages, genetically manipulated to modify their host range and induce specific depolymerase biofilm-degrading enzymes, interact better than their naturally-occurring species to lyse antibiotic-resistant bacterial hosts [bib_ref] Dispersing biofilms with engineered enzymatic bacteriophage, Lu [/bib_ref] [bib_ref] Reversing bacterial resistance to antibiotics by phage-mediated delivery of dominant sensitive genes, Edgar [/bib_ref] [bib_ref] Inhibition of biofilm formation by T7 bacteriophages producing quorum-quenching enzymes, Pei [/bib_ref] [bib_ref] Phage therapy gets revitalized, Reardon [/bib_ref] [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref] [bib_ref] Genetically engineered phages: a review of advances over the last decade. Microbiol, Pires [/bib_ref]. Equally important, convincing clinical evidence suggests that combining phages with antibiotics restores antibiotic sensitivity in various MDR PA strains (evolutionary synergy) [bib_ref] Evolutionary dynamics of separate and combined exposure of Pseudomonas fluorescens SBW25 to..., Escobar-Páramo [/bib_ref] [bib_ref] Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa, Chan [/bib_ref]. We, therefore, need experiments to select phages that use as receptors the bacterial membrane zones responsible for expelling antibiotics (multidrug efflux systems) [bib_ref] Phage-host interplay: examples from tailed phages and gram-negative bacterial pathogens, Chaturongakul [/bib_ref] [bib_ref] Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa, Chan [/bib_ref] [bib_ref] Evolutionary rationale for phages as complements of antibiotics, Torres-Barceló [/bib_ref] [bib_ref] Synergy and order effects of antibiotics and phages in killing Pseudomonas aeruginosa..., Chaudhry [/bib_ref]. Our several-year-long multicenter experience on microbiology in patients with CF [bib_ref] Pseudomonas aeruginosa Infection in cystic fibrosis caused by an epidemic metallo-β-lactamase-producing clone..., Pollini [/bib_ref] [bib_ref] Pyrosequencing unveils cystic fibrosis lung microbiome differences associated with a severe lung..., Bacci [/bib_ref] [bib_ref] A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis, Moretti [/bib_ref] , and our promising preliminary observations on phages from a collection stored at Bambino Gesù Children's Hospital tested against mucoid and nonmucoid clinical CF MDR PA strains, prompted us to update our knowledge on phage therapy in CF PA strains. Gaining crucial information on phage pedigree properties [bib_ref] Information phage therapy research should report, Abedon [/bib_ref] , standardization and integration into models of drug development, and on potential phage therapy in CF [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref] from reliable and repeatable experiments on phage therapy in combating CF PA, could direct phase III RCTs for testing phage efficacy and safety in hospital settings. We designed this argumentative review to seek up-to-date reliable information on in vitro and in vivo evidence for and against lytic phage efficacy in various PA strains useful for planning future research into phage therapy for patients with CF. To do so we reviewed, appraised and synthesized information from publications describing lytic phages tested in vitro in PA from various sources, and phage activity, resistance and safety in vivo on non-pulmonary and pulmonary host models infected by various laboratory and clinical CF PA strains. # Methods In November 2016, to seek papers to include in our review on phage lytic activity in vitro and in vivo in treating CF PA, we used a multiple sensitive search strategy in PubMed and in a meta-engine (TRIPDATABASE, https://www.tripdatabase. com/), without language, country, and time limits. After retrieving 125 publications, two authors (EVF and PR) screened them by title and abstract, excluded 94 publications, mainly because they were irrelevant to the review question, retrieved and assessed 33 full texts for eligibility, and included 24 publications in the review [fig_ref] FIGURE 1 |: A flow chart showing multiple search strategies and reasons for excluding and... [/fig_ref]. Two authors MR and PR mapped and coded information from the papers included using the same author terms (Supplementary Data Sheets 1, 2), and agreed on reporting eight items in in vitro studies, and 11 items in in vivo studies, according to our experience, and published suggestions on what research should report [bib_ref] Information phage therapy research should report, Abedon [/bib_ref]. They then identified lines of arguments in key results, problems, ## What do pulmonary models add to in vivo studies? Pulmonary models in vivo on mice add essential information for assessing at a sufficient given time the best measures to test phage efficacy and safety (cytokines, lactate dehydrogenase enzyme levels, PA clearance in broncho-alveolar lavage testing, inflammatory cell levels in spleen cultures, luxtagged PA strains in lungs by imaging systems). Future studies on specific candidate animals mimicking CF lung conditions (i.e. pigs and ferrets) might test phage nebulized in CF PA infected lungs, phage titers, delivery rates and immunogenicity. How to improve clinical research on phages combined with antibiotics in vitro and in vivo for treating CF? Also when engineered phages are combined with antibiotics in in vitro studies and in vivo host models infected by MDR and extensively drug-resistant CF PA, antibiotics should be given at minimum inhibitory concentrations and sequentially at 12 hours after phages administered at a pre-specified formulated product concentration, phage dose and nebulized administration route. ## In vivo studies In vitro studies In vitro and in vivo studies How to improve phage cocktails, and develop a personalized approach in vitro and in vivo for treating CF? In in vitro studies and in vivo models, phage cocktails need to be selected according to phage host-receptor affinity, and a personalized approach (new phages isolated deliberately or trained evolved phages or engineered adapted phages against multidrug-resistant PA strain hosts). To test PA resistance, experiments should be prolonged over 48 hours. and limitations. All three authors discussed key results, weighted advantages and disadvantages in study findings, and combined the synthesis to gain major information essential for planning and advancing future clinical research in CF (Supplementary Tables 3, 4; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. # Results Of the 24 publications included in the review, 14 referred to experiments in vitro, 4 to studies in vivo, and 5 to studies in vitro and in vivo (20 studies in vitro, and 9 in vivo; Supplementary Tables 3, 4). Of the 20 publications describing experiments in vitro, 14 were conducted in Western European countries, 3 in Eastern European countries, 2 in the Far East, and 1 in the USA. Although our research fixed no time limit, all the studies included in the review were published in the past 16 years. Only one early study published in 2001 reported the use of a lysogenic phage, unsuitable in human trials [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref]. Of the 24 papers reviewed, only 10 specified the criteria used to justify how they selected phages: 6 selected them according to their range of activity [bib_ref] Characterization of JG024, a Pseudomonas aeruginosa PB1-like broad host range phage under..., Garbe [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] , 1 according to previous studies [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] , 1 using custom-developed phages against resistant PA strains [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref] , 1 using custom-developed phages against a laboratory PA [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] , and 1 according to phage ability to delay and inhibit phage-resistant cells . Our review, therefore, failed to identify a standardized method for justifying phage selection. Only three studies in vitro reported phage functional characterization estimated by latent phase and burst size (the mean number of phages released per bacterial cell; [bib_ref] Characterization of JG024, a Pseudomonas aeruginosa PB1-like broad host range phage under..., Garbe [/bib_ref] [bib_ref] Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their..., Danis-Wlodarczyk [/bib_ref]. Only two studies in vitro selected and tested phage activity and host range by efficiency of plating (EOP) [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] , 11 used plaque assays, spot test or streak test [bib_ref] Characterization of JG024, a Pseudomonas aeruginosa PB1-like broad host range phage under..., Garbe [/bib_ref] [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number..., Larché [/bib_ref] [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref] [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref] [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref] [bib_ref] Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their..., Danis-Wlodarczyk [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] , and seven left the test used unspecified (Supplementary ; [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref] [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref] [bib_ref] Small colony variants and single nucleotide variations in Pf1 region of PB1..., Lim [/bib_ref]. Of the nine in vivo studies, only three used the EOP [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] , and four used plaque assays or spot test [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. Two further studies left the test used unspecified (Supplementary ; [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref]. Of the 24 papers reviewed, 8 studies in vitro left phage bank unreported, and we retrieved and reported genomic sequences from online bank accession numbers [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref] [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref] [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref] [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] [bib_ref] Small colony variants and single nucleotide variations in Pf1 region of PB1..., Lim [/bib_ref]. For nine papers in vitro [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref] [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] [bib_ref] Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number..., Larché [/bib_ref] [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref] [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] , and three papers in vivo [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref] phage genomic sequence data were unreported or completely or partially irretrievable. Only one investigator reported using a phage taken from a standard reference microorganism central certified collection [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref]. Only three studies reported phage stability assessment on newly-isolated phages alone and in a phage cocktail ;. Of the nine studies in vivo, six in PA-infected mouse models instilled phages intranasally, whereas the three in larva models delivered phages into the larval hemolymph. Only three of the nine in vivo studies reported the formulated products used for administering the phage suspension to host animal models (Supplementary ; [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref]. Few papers, therefore, gave essential information on phage functional characterization, selection, activity, genomic sequence, stability, and the formulated products used in phage suspensions useful for further studies on phage therapy in CF PA. Several investigators reported in vitro newly-isolated or trained phage lytic efficacy alone or in cocktails in disrupting the biofilm matrix (including dynamic biofilm models), reducing biomass and planktonic cell numbers in laboratory PA, non-CF and CF mucoid, or non-mucoid PA strains, and MDR PA [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number..., Larché [/bib_ref] [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref] [bib_ref] Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their..., Danis-Wlodarczyk [/bib_ref] , and penetrating PA alginate at a high concentration [bib_ref] Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas..., Hanlon [/bib_ref]. Even though phages penetrated alginate, its overproduction in mucoid CF and non-CF PA strains, under simulated CF lung conditions, reduced phage infection efficiency (Supplementary ; [bib_ref] Characterization of JG024, a Pseudomonas aeruginosa PB1-like broad host range phage under..., Garbe [/bib_ref]. These findings underlined the risk that phage efficacy could be influenced by alginate overproduction especially important in patients with CF. During phage treatment against various PA strains, four studies reported immediate phage resistance to ancestral or newly-isolated or trained single phages (including crossresistance to new phages) within 1 and 5 days [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref] [bib_ref] Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their..., Danis-Wlodarczyk [/bib_ref] [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref] , and only one study reported the right time to test resistance or cross-resistance to trained phages when standard phage training ends [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref]. In 1 of these studies, trained phages halved the percentage of resistant bacterial cells, leading to lower bacterial growth (fitness cost) in chronic than in intermittent PA isolates [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref]. Another four in vitro studies investigated PA resistance to phage cocktails [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref] [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref]. Although phage cocktails were tested in vitro for no more than 48 h, phage cocktails showed less PA resistance than phages used alone [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref]. One study, using three phage cocktails inducing similar resistance in several CF PA strains, failed to identify the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas protein system [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref]. In another study, designed as an ex vivo study mimicking a clinical condition, [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] reported that CF PA colonies resistant to a phage cocktail were not resistant to phages per se. Only two studies in vitro addressed phage resistance in MDR and extensively-drug-resistant (XDR) PA (some from CF or unspecified origin; [bib_ref] Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number..., Larché [/bib_ref]. Another in vitro study reported that combining simultaneously a single phage with a bactericidal antibiotic (tobramycin), PA resistance decreased [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref]. No study investigated resistance to phages combined with antibiotics for CF MDR or XDR PA infections ; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Collectively, few studies reported CF PA phage resistance on phages alone or in cocktails, which have high PA susceptibility. These findings gave no help on selecting, and personalizing phages to use in cocktails to combat chronic CF PA infections. Of the nine papers describing phage efficiency in in vivo studies, three used non-pulmonary host larvae models (Galleria mellonella) [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref] , and six pulmonary models (mice) (Supplementary ; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. Only one of the three studies on non-pulmonary models, tested both single phages and a phage cocktail in non-CF and CF PA-infected larvae, and assessed adverse events from phage injection testing a standardized negative control group, PA uninfected larvae . All these studies highlighted the need to prolong PA-infected larvae exposure to phages, given singly or in cocktails, for up to 120 h. Few studies on in vivo non-pulmonary models provided reliable findings on phage efficiency, adverse events and host survival, which PA should be selected to avoid massive toxic compound release, or which standardized negative control groups should be assessed to clarify safety from phage injection (Supplementary . The six papers testing phages in vivo on mouse PA-infected host models mimicked pulmonary conditions (Supplementary ; [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. In three studies the investigators proved phage efficacy in preventive and curative mouse models [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. In the preventive model, they showed that single ancestral and trained phages prevented PA lung infection. In the curative model, by quantifying imaging (PAKlumi) and bronchoalveolar lavage (BAL) PA counts, they showed that PA loads decreased, rescuing 100% of mice [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref]. Of the six studies using mouse host models, 4 administering phage-cocktails (two or more phages) to mouse lung infected by various PA strains, all reported that phage cocktails reduced PA loads [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref] and one, comparing PA-infected phage-treated mice with a group of PA-infected mice alone, also showed reduced inflammatory markers in BALs . One of these four studies, provided an index to predict phage efficacy reliably especially for phages isolated with a personalized approach [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref]. No study tested in vivo phages in cocktails on MDR CF PA, or phage therapy in other animals (pigs and ferrets), possibly better candidates than mice to mimick pulmonary models. Collectively the four studies testing efficiency for 2-5 phage cocktails in various CF and non-CF PA strains gave inconclusive results (Supplementary . Frontiers in Microbiology | www.frontiersin.org ## Phages combined with antibiotics in in vitro studies Only three studies in vitro and no studies in vivo investigated whether a single phage combined with various antibiotics (tobramycin, colistin, and streptomycin) reduced a laboratory PA biomass or planktonic cells [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref]. Although showed that combined therapy was as effective as the tested antibiotics alone, the combined treatment reduced the emergence of phage PAresistant cells. In their study, Torres-Barceló et al. also recommended ensuring maximum antibacterial efficacy, by giving antibiotics 12 h after phages rather than simultaneously and obtained a synergistic effect in decreasing laboratory PA biofilm biomass or density. Testing a single phage on several strains, Danis-Wlodarczyk et al. showed colistin antagonism to the phage (Supplementary ; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. No study tested phage cocktails combined with antibiotics. In defining experimental efficiency for phages and antibiotics combined, no study mentioned essential information such as host receptor affinity, personalized approaches, well-selected phage cocktails, antibiotics selected by minimal inhibitory concentration (MIC) in vitro on MDR PA, or in vivo on PA-infected animal host models. ## Problems related to phage efficacy and safety in in vitro and in vivo sudies Of the 24 studies, only 14 reported experimental problems (10 in vitro and 4 in vivo). Three investigators reported in vitro problems related to PA biofilm: bacterial regrowth [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] , increased PA biomass in the phage-treated group , and PAO1 phage insensitivity (Supplementary ; [bib_ref] Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their..., Danis-Wlodarczyk [/bib_ref]. Although [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref] suggested overcoming these problems by using short-term phage therapy, preferably in cocktails to avoid PA resistance, no other studies mentioned possible solutions. Four groups reported in vivo phage inefficacy including genetically-related phage inability to lyse the same PA strain [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] , G. mellonella larval death several hours after phage infection [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] , and phage inefficacy in the delayed and prophylactic approaches (Supplementary ; [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. In their in vivo study in a larva model, Olszak et al. to infect larvae, before phage infection, gave weakly virulent PA strains at high loads, and consequent PA lysis caused massive toxic compound release followed by larvae death at 96 h. Although no study suggested how to overcome problems in in vivo phage therapy, [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] # Discussion Our argumentative review provides critically appraised, up-todate previously unavailable information for, and against, in vitro and in vivo lytic phage efficacy in various CF PA strains, useful for planning future clinical research into phage therapy for patients with CF [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Although our multiple search strategy avoided language and time limits, the most promising and pertinent information came mainly from studies published by eminent research groups around the world over the past seven years, presumably owing to MDR PA spread, and disinterest in producing new antibacterial drugs [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref] [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. By collecting information from 24 relevant publications describing lytic phage activity and efficacy in vitro, and efficacy and safety in vivo experiments on pulmonary and non-pulmonary host models infected with PA from various sources, our review fills some major knowledge gaps on phages that can effectively combat PA strains infecting lungs in patients with CF. Our findings also provide new insights that will prompt renewed clinical research in CF PA to test wellselected (ancestral or trained) lytic phages to include in cocktails, develop personalized phage therapy, address phage-antibiotic combinations, and envisage even genomic engineered phages combined with antibiotics to treat MDR and XDR PA in patients with CF [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Among research that especially takes the field ahead in phage treatment for CF PA, our review provides useful information on the various methods used for testing host range and lytic phage activity against a variety of target bacteria (spot tests, plaque assays, and EOP). Ample evidence suggests that the essential, reliable step for confirming lytic phage cocktail efficiency as preliminarily tested by spot test or plaque assay, is to test single phages for effectiveness with EOP (Supplementary Tables 3, 4; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref]. Leaving EOP in vitro unassessed, as many papers in our review did, risks selecting phages that despite their broad lytic activity, as assessed in vitro by other methods (spot tests that simply reflect a bactericidal effect, or plaque assays), fail to infect CF PA planktonic cells [bib_ref] Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and..., Pires [/bib_ref]. Surprisingly, among the nine in vivo studies reviewed, three studies conducted in the past two years left methods for assessing lytic phage host range unspecified or unreported [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref] , and three reported only plaque assay or spot tests [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. As [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] underlined, EOP for seven over nine phages correlates in vitro with activity in vivo in PA-infected mice, thus predicting lytic phage efficacy. These findings were later confirmed by two investigators who concluded that EOP is the standard method also for confirming lytic phage activity against other Gram-negative bacteria, namely Escherichia coli and Salmonella reference collections [bib_ref] Isolation of phages for phage therapy: a comparison of spot tests and..., Mirzaei [/bib_ref]. A key problem in ensuring the efficacy of newly-isolated lytic phages in cocktails is selecting and mixing specific phages having high lytic activity that can also disrupt and reduce the CF PA biofilm matrix, by producing or inducing biofilm matrixdegrading enzymes [bib_ref] Bacteriophage-encoded depolymerases: their diversity and biotechnological applications, Pires [/bib_ref]. In biofilm aggregates, enzymes produced by the CF PA community breakdown the polysaccharides that hold the biofilm together, thus actively releasing surface bacteria (planktonic cells) that colonize fresh substrates (Supplementary . In our review, two studies reported that two different 2-phage cocktails disrupt and reduce, though by only 2-log [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] the CF PA biofilm, also showing age-independent biofilm efficacy . Phage cocktails, probably by mixing specific biofilm-exopolysaccharide-degrading enzymes, increase lytic phage efficacy by reducing bacterial population densities [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref] , and preventing bacterial resistance (Supplementary ; [bib_ref] A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static..., Alves [/bib_ref]. Overall these findings imply that phage cocktail therapy might be useful in preventing PA lung colonization, and in reducing pulmonary exacerbations in patients with CF [bib_ref] Pseudomonas aeruginosa biofilm formation in the CF lung and its implications for..., Anderson [/bib_ref]. Accordingly, new evidence confirming that phage cocktail efficacy depends on selecting the correct phage-degrading-enzyme mixtures comes also from those who claim that in clinical practice phage therapy will probably have limited usefulness in eliminating bacterial infections caused by biofilm aggregates, i.e., microbial communities inhabiting chronic infections. Conversely, phage therapy could be of great use in reducing cells that detach themselves from the biofilm and revert to the virulent planktonic phenotype [bib_ref] Bacteriophages and their immunological applications against infectious threats, Criscuolo [/bib_ref]. Another additional benefit from phage therapy comes from a study included in our review showing reduced inflammatory markers in BALs from PA-infected phage-treated mice compared with controls , as others confirm in a mouse model infected by pulmonary Burkolderia cenocepacia [bib_ref] Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection, Carmody [/bib_ref]. Another major in vitro and in vivo experimental requirement related to formulating efficient newly-isolated phage cocktails to combat PA lung infection in patients with CF is to select highly virulent lytic phages having good, varying target hostreceptor affinity [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Choosing phages that differ in host receptor affinity avoids competition in absorption to the bacterial surface, and improves synergic phage cocktail efficacy [bib_ref] Learning from bacteriophages-advantages and limitations of phage and phage-encoded protein applications, Drulis-Kawa [/bib_ref]. Hence, surprisingly, among the 24 papers on CF PA reviewed, only one underlines the need to select phages according to their host receptor affinity, to avoid antagonism, and ensure a phage synergistic effect (Supplementary ; [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref]. This information is particularly lacking in the otherwise highly informative in vivo study conducted by [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] in which the phage mixture probably competed for bacterial receptors (Supplementary . Our review also provides new insights into CF PA resistance, including defense mechanisms that allow phages to adapt to changing host systems, thus incurring significant fitness costs. One study in our review (although it failed to test resistance on a CF PA strain) emphasizes that a phage-cocktail resistant PAO1 emerges in experiments investigating simultaneous and sequential phage-cocktail exposure. In this study, fitness costs probably arose when phage-cocktail resistant PA overproduced alginate or extracellular polymeric substances (EPS) [bib_ref] Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas..., Hall [/bib_ref]. How PA phage resistance develops in CF or how PA naturally alters its genotype or phenotype to combat phage virulence is debatable [bib_ref] In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential..., Mendes [/bib_ref] [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. Bacteria have developed an astonishing array of defense strategies to combat phages at each step during the infection. These include adsorption-blocking systems, systems for preventing phage DNA entry into the host cell, bacterial restrictionmodification systems, phage abortive infection, and genetically modified systems such as the renowned CRISPR-Cas protein system [bib_ref] Bacteriophage resistance mechanisms, Labrie [/bib_ref]. The bacterial CRISPR-Cas system aims to inactivate invading phage genetic material. Although a study conducted in recent years suggests that many clinical PA isolates develop the CRISPR-Cas system [bib_ref] The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally..., Cady [/bib_ref] , in our review [bib_ref] The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages, Essoh [/bib_ref] observed in the 13 phageresistant PA strains studied from different clusters isolated from patients with CF no CRISPR-Cas system (Supplementary . Althoughunderline that specific phage genes inactivate the CRISPR-Cas system, our review on CF PA lacks information on phage-borne CRISPR inactivation systems. Besides using phages in cocktails to treat CF PA, another strategy for overcoming PA resistance in patients with CF includes phage training. Only one in vivo study [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] reported that trained (man-guided evolved) phages were more efficient than untrained (ancestral) phages in killing CF MDR mucoid PA lung-infected mice, and in reducing bacterial resistance in vivo. They also observed that in the curative phage treatment model, trained phages and ancestral phages act cooperatively with the mouse immune response to eliminate MDR PA acute lung infections. Others subsequently highlighted contrasting results, and possible drawbacks in using trained phages in vitro , 4; [bib_ref] Back to the future: evolving bacteriophages to increase their effectiveness against the..., Betts [/bib_ref] [bib_ref] Pre-adapting parasitic phages to a pathogen leads to increased pathogen clearance and..., Frederiksen [/bib_ref]. Whereas Betts et al. observed that during training bacteria exposed to phage can, in turn, evolve crossresistance toward ancestral and newly-isolated phages, Friman et al. showed that evolved phages were more efficient than untrained phages in killing CF PA strains, and in reducing bacterial resistance. Underlining an equally important problem, [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] demonstrated that phage training is not always feasible, stating in their in vivo study that they were unable to evolve a highly-virulent phage. Insofar as phage optimization by only two single nucleotide mutations extends host range and is sufficient to improve phage virulence toward CF MDR PA clinical strains [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] , trained phages possess broad lytic activity and virulence, and might be useful in advancing future research on phage genetic changes [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Whether lytic phages can be used to prevent as well as treat PA infections in patients with CF remains unclear until we have long-term results, and use pulmonary models on the best animal candidates. Of the 24 studies reviewed, two showed that preventive treatment requires not heat-killed but active phages [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] , thus providing evidence that phage efficiency depends on phage lifecycles. Of the nine in vivo studies we reviewed, four investigated phage treatments for preventing PA infections [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. Despite the impressive efficacy reported, some investigators underline that the preventive approach is of minor clinical interest because it cannot predict the specific PA strain eventually infecting the patient [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref]. Another open question is whether phage therapy can eradicate MDR PA from the airways in patients with CF. Findings in recent years provide evidence that phage therapy can prevent the high-density MDR PA increase and antibiotic treatment failure [bib_ref] Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa, Chan [/bib_ref] [bib_ref] Evolutionary rationale for phages as complements of antibiotics, Torres-Barceló [/bib_ref] [bib_ref] Synergy and order effects of antibiotics and phages in killing Pseudomonas aeruginosa..., Chaudhry [/bib_ref]. A significant and sustained reduction in the number and activity of infecting PA could undoubtedly improve clinical conditions in patients with CF [bib_ref] Bacteriophages of Pseudomonas aeruginosa: long-term prospects for use in phage therapy, Krylov [/bib_ref]. More information advancing clinical microbiological research in CF, and optimizing phage-cocktail efficacy in treating PA concerns the need to personalize phage therapy by using a standardized approach. Two papers in our review underline that the most efficacious phages are those isolated on purpose (personalized phage cocktails) against the patients' infecting, and eventually antibiotic-resistant PA strains (sur-mesure, patienttailored therapy; [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref]. Yet despite its advantages, the personalized approach has several drawbacks because it is time consuming (phages need to be isolated, purified and completely characterized). Difficulties also arise in assessing phage effectiveness on PA strains isolated in certain medical facilities, and fromḡenetically poorlydifferentiated local clones, thus introducing subjectivity in assessing phage lytic activity [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. The lytic phages described in our review, and in current practice, often lack detailed criteria for justifying phages chosen for experiments, their genomic sequences, and database accession numbers. These problems reduce the chances for selecting an optimal set of lytic phages to mix in a cocktail, impede experimental repeatability, and make it hard to generalize the results (Supplementary Tables 3, 4; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] How to name and classify your phage: an informal guide, Adriaenssens [/bib_ref]. Our review leaves unclear whether and to what extent phage efficacy depends on administration routes. No studies have compared the various phage administration routes in treating MDR PA or CF PA in vitro or in vivo models. Although the best way to reach PA-infected host lungs may be by delivering phages as a liquid or a dry powder [bib_ref] Anti-tuberculosis bacteriophage D29 delivery with a vibrating mesh nebulizer, jet nebulizer, and..., Carrigy [/bib_ref] [bib_ref] Anti-pseudomonal activity of phage PEV20 in a dry powder formulation. A proof-of-principle..., Chang [/bib_ref] , the murine CF PA-lung-infected models we reviewed only used intranasal instillation [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Predicting in vivo efficacy of therapeutic bacteriophages used to treat pulmonary infections, Henry [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref]. New studies need to address the potential benefits from giving phage by inhalation instead of intranasally. What we mainly need to know is which device to choose for phage inhalation and the right animal models for reproducing human CF airways anatomically and physiologically [bib_ref] Animal models of cystic fibrosis pathology: phenotypic parallels and divergences, Lavelle [/bib_ref]. Only one study in vitro has compared two nebulizers and one inhaler to define the best active liquid phage delivery in treating Mycobacterium tuberculosis. The investigators concluded that delivering phages with a vibrating mesh nebulizer is significantly more effective than using a jet nebulizer (p < 0.01), whereas a soft mist inhaler may be useful for self-administration of a phage aerosol [bib_ref] Anti-tuberculosis bacteriophage D29 delivery with a vibrating mesh nebulizer, jet nebulizer, and..., Carrigy [/bib_ref]. Apart from the type of device, another important question to address is the time for nebulizing phages. Known mathematical models can help to assess the best time for nebulizing so as to increase PA clearance from the airways [bib_ref] Evaluation of nose-only aerosol inhalation chamber and comparison of experimental results with..., Nadithe [/bib_ref]. Although again in a murine model, new information in a noncomparative study on an MDR PA-neutropenic lung-infected mouse model suggests that a dry powder phage formulation might have advantages over a liquid formulation, and could be applied also in CF MDR PA-infected animals . New promising information advancing in vivo experiments overcoming murine model limitations (no mucus hypersecretion in mice, and the cystic fibrosis transport regulator, CFTR, gene is expressed only in the proximal trachea) envisages specific animal candidates (pigs and ferrets; [bib_ref] Animal models of cystic fibrosis pathology: phenotypic parallels and divergences, Lavelle [/bib_ref]. Like humans, these animals exhibit the same high CFTR expression in serous cells of submucosal glands in the cartilagineous airways, thus mimicking CF lungs. By using these experimental procedures in in vivo models, to deliver the best sufficient active phage titers for targeting MDR CF PA in the lungs, we could also address phage pharmacokinetics [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. Similarly, by using various phage administration routes to address efficacy on combating MDR CF PA lung infections, and negative or adverse phage effects, we could also investigate another unaddressed problem, phage pharmacodynamics [bib_ref] Information phage therapy research should report, Abedon [/bib_ref]. Pharmacokinetics and pharmacodynamics can help transfer research results into clinical care. Even more important, we need to test in vivo phage adverse effects related to immunogenicity in pig and ferret models in studies with a follow-up long enough to allow chronic pulmonary inflammation determined by bacterial persistence to develop [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Animal models of cystic fibrosis pathology: phenotypic parallels and divergences, Lavelle [/bib_ref] [bib_ref] Phage therapy: an alternative to antibiotics in the age of multi-drug resistance, Lin [/bib_ref] [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. Pig and ferret models can also address research on nebulized phage treatment to test what might happen in immune interference from innate immune responses, neutrophil elastase, secretory IgA and macrophages engulfing phage particles after repeated phage administration in CF lungs [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. Another problem that has in the past discouraged research on phage therapy in general clinical use, is how to define phage doses to test phage safety. Our review, thanks to the published studies in vivo, gives up-to-date information for designing future CF PA clinical studies possibly avoiding risky phage-induced reactions and phage-PA interactions. Many phages collected in libraries and several phages in the studies included in our review came from human waste (sewage; Supplementary Data Sheets 1, 2). As human beings, we normally encounter phages throughout our lifetime, owing to the complex interactions between bacteria and phage in our colon, upper respiratory system, and on our skin [bib_ref] The prospect for bacteriophage therapy in Western medicine, Merril [/bib_ref]. In assessing safety in vivo, to investigate possible adverse events we therefore need to include standard negative control groups (PA-uninfected or phage-uninfected animals). For example, experiments testing sham solution injected or nebulized in PA-and-phage-uninfected animals can detect injection-related adverse events, whereas those testing PAuninfected and phage-treated animals investigate potential phage virulence. Surprisingly, of the nine in vivo studies examined in this review six used unstandardized negative control groups, and reported no safety problems, and only one study assessed safety with standardized negative control groups (Supplementary ; [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref]. This major study also investigated safety in vivo in positive (PA-infected) larvae at high CF PA loads. An unexpected result was that the weakly-virulent PA infected larvae required a higher lethal dose than the highlyvirulent PA (10 5 vs. 10), and more phage inoculum to keep the multiplicity of infection (MOI) value of 100. They reported the highest mortality at 48 h in the weakly virulent CF PA owing to toxic compounds released by bacterial lysis. In their study [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] also used inactivated phages to treat larvae infected with PA strains at a lethal dose, and showed no differences in larvae mortality compared with control infected caterpillars, thus confirming that larval survival was entirely due to phage lytic activity rather than to host immune stimulation (Supplementary . In a previous study, [bib_ref] Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections, Debarbieux [/bib_ref] gave bioluminescent-non-mucoid non-CF PAK-infected mice a high phage concentration, monitored their behavior for 10 days, and again reported no adverse effects (Supplementary . Similar results came from a study conducted by Wang and colleagues in bacteremic mice infected with an imipenemresistant PA strain, who concluded that inoculating phages at high doses produced no adverse effects in mice [bib_ref] Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant..., Wang [/bib_ref]. In the same experiment, Wang et al. underline that to avoid severe adverse effects potentially caused by microbial debris, including the endotoxin LPS released during phage propagation, phages prepared for therapeutic applications must be highly purified [bib_ref] Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus..., Biswas [/bib_ref] [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref]. A possible solution to avoid LPS-related mortality, rather than using phages having a high burst size (the average number of phages produced by infected cells) thus producing many virions guaranteeing that sufficient phage numbers reach the infection site [bib_ref] Isolation of phages for phage therapy: a comparison of spot tests and..., Mirzaei [/bib_ref] , in patients with CF we suggest selecting a poorly virulent phage cocktail, with a low burst size nebulized directly to the infection site. Giving patients nebulized drugs to treat lung infections is a standardized procedure in routine clinical practice. Ample evidence shows that phages can be nebulized or delivered as a dry powder [bib_ref] In vitro lung delivery of bacteriophages KS4-M and KZ using dry powder..., Golshahi [/bib_ref] [bib_ref] Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections, Matinkhoo [/bib_ref] [bib_ref] Bacteriophage delivery by nebulization and efficacy against phenotypically diverse Pseudomonas aeruginosa from..., Sahota [/bib_ref]. We now await information from tests using a powdered preparation including two phages (phiKZ, active on PA, and KS4-M, lysing Burkholderia cepacia) given by inhalation to patients with mixed infections [bib_ref] In vitro lung delivery of bacteriophages KS4-M and KZ using dry powder..., Golshahi [/bib_ref]. If inhaled phages reach the infection site directly, possible characterized and selected phage candidates might include those having a low burst size, thus causing the infection to progress at a slower rate, hence probably diluting LPS release over time. Another concern, insofar as efficacious phage therapy involves giving active, vital virus [bib_ref] Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: first steps towards..., Morello [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref] , is the need to assess whether treatment remains effective and safe over time. For example, it should avoid complications related to phage-resistant hosts, bacteria whose pathogenicity has been altered by transduction phenomena, and phagehost genetic rearrangements [bib_ref] Bacteriophages of Pseudomonas aeruginosa: long-term prospects for use in phage therapy, Krylov [/bib_ref]. Although these deleterious events are extremely rare, they might develop especially during the prolonged phage therapy needed for a chronic disease [bib_ref] Generalized transduction by lytic bacteriophages, Waddell [/bib_ref]. Hence the need to use phage mixtures delivered from certified reserve storage (phage banks or libraries) including characterized phages having a wide host range, and differing in PA receptor affinity [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref]. These phage mixtures could become an extremely valuable clinical resource for ensuring safety, should help laboratory clinicians rapidly to select appropriate phages to include in cocktails, and possibly adapt cocktails to the MDR PA strains isolated during CF pulmonary exacerbations [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. Even though our review included only one study that specified the use of a phage from a certified bank [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] , and although phage banks lack international institutional regulations, our review identified no information or data on a promising new research direction for clinical phage therapy envisaging combining phage cocktails from individual researchers' phage banks or phage libraries, and a personalized approach using phages isolated on CF PA strains. An alternative solution to phage libraries for testing and selecting personalized phage-cocktails, taking into account the extremely high PA genome plasticity (Supplementary , might be to set up an international reference library based on clinical CF PA strains with various characteristics [bib_ref] Extensive genomic plasticity in Pseudomonas aeruginosa revealed by identification and distribution studies..., Shen [/bib_ref] [bib_ref] In vivo development of antimicrobial resistance in Pseudomonas aeruginosa strains isolated from..., Riou [/bib_ref] [bib_ref] Bacteriophages of Pseudomonas aeruginosa: long-term prospects for use in phage therapy, Krylov [/bib_ref]. Such presumptive phage selection on an internationally available PA reference set also lacks international regulations including well-combined and continuously updated newly-isolated lytic phages. An intriguing modular approach proposed in 2007 [bib_ref] Dispersing biofilms with engineered enzymatic bacteriophage, Lu [/bib_ref] and reported also by in 2016 entails formulating phage mixtures (mono-species or hetero-species), enhancing their activity by introducing newly characterized engineered phages to express the most effective EPS-degrading enzymes specific to the target biofilm, and combining them in a personalized way to treat PA infection in patients with CF. This modular approach and the mixtures included in phage libraries could help international regulatory agencies to address phage therapy in CF. For example, it could allow different research groups to cooperate in developing a central phage library, to ensure distributing phage mix on call, thus encouraging the use of the phage therapy in untreatable CF XDR PA pulmonary infections [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. Prompted by these promising results, to receive more information on their studies on phages used in clinical therapy in Eastern countries, including sequence information useful for determining whether some phages used in various studies belong to the same or related clusters, while collecting data for our review we contacted by email Krylov, and after receiving his advice, contacted two other authors (Shabalowa and Karpanov). Both answered in brief, specifying that they will report their findings in published papers, but failed to give more information. Hence, we were unable to compare phage efficacy across different studies. Although disseminating detailed unpublished research data could be a challenging matter, phage libraries are expensive, need to be continuously updated to account for variability in target bacteria, and can vary from region to region [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref]. Considering the known resistance to a three-phage cocktail in MDR PA and XDR PA [bib_ref] Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number..., Larché [/bib_ref] , another promising approach that helps in reducing MDR PA biofilms, as well as the PA planktonic cells responsible for exacerbations in CF, is to improve treatment effectiveness by combining phage therapy and antibiotics [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Although combined phage-antibiotic treatment started when the antibiotic era began [bib_ref] Combined action of penicillin and bacteriophage on Staphylococci, Himmelweit [/bib_ref] , questions remain on whether to use sequential treatment, optimal phage administration routes, dosing, suspended formulated products, and treatment duration. Surprisingly, only three studies in our review investigated phage therapy combined with tobramycin, streptomycin and colistin on laboratory PAO1 [bib_ref] Effect of bacteriophage infection in combination with tobramycin on the emergence of..., Coulter [/bib_ref] [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref] [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref]. The best strategy to ensure phage efficacy, allow a wide antibiotic choice, and minimize possible antibiotic antagonism and PA resistance, is to apply antibiotics at peak phage efficacy (within about 12 h) sequentially rather than simultaneously (Supplementary ; [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref] , as recently confirmed [bib_ref] Synergy and order effects of antibiotics and phages in killing Pseudomonas aeruginosa..., Chaudhry [/bib_ref]. We retrieved no studies testing other antibiotics (aminoglycosides, beta-lactams, quinolones, carbapenems or macrolides) combined with phage against CF and non-CF PA strains, and none investigated phage therapy combined with antibiotics for CF XDR PA strain infections in vitro or in vivo. We remain skeptical about why [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref] combined phage therapy with colistin, a drug known to alter the bacterial membrane thereby removing phage bacterial receptors [bib_ref] Colistin in multi-drug resistant Pseudomonas aeruginosa blood-stream infections: a narrative review for..., Martis [/bib_ref]. No comparative head-to-head studies reported antibiotic therapy and trained phages as alternative treatments in CF PA strains. Overall, the promising findings this review highlights on trained (man-guided) phages and on how certain antibiotics effectively combat CF MDR PA and new preliminary information suggesting that the amikacinphage combination could have potentially more benefits on PA biofilms than meropenem [bib_ref] Bacteriophage-antibiotic synergism to control planktonic and biofilm producing clinical isolates of Pseudomonas..., Nouraldin [/bib_ref] lead us to conclude that trained or even engineered phages combined with amikacin or meropenem at sub-inhibitory concentrations, might reduce PA resistance, thus boosting interest in phage therapy [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. The evolutionary rationale for combining phages with antibiotics provides support for designing clinical trials using combined phage-antibiotic therapy possibly in skin infections, such as diabetic ulcers, before applying the best results to systemic diseases in humans [bib_ref] Evolutionary rationale for phages as complements of antibiotics, Torres-Barceló [/bib_ref] [bib_ref] Synergy and order effects of antibiotics and phages in killing Pseudomonas aeruginosa..., Chaudhry [/bib_ref]. New evidence implies that genetically engineered phages that might act against PA biofilms better than newly-isolated phages, expand their host range and, by so far undefined co-evolutionary phagebacteria interactions, possibly sensitize MDR and XDR PA antibiotic efficacy [bib_ref] Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections, Pires [/bib_ref] [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref] [bib_ref] Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa, Chan [/bib_ref] [bib_ref] Evolutionary rationale for phages as complements of antibiotics, Torres-Barceló [/bib_ref]. Again underlining the need for combining phage with antibiotics in treating CF MDR PA, a future crucial research step, before undertaking in vivo therapy, must include pre-testing antibiotics to assess the standardized MIC for MDR PA isolates. Other essential stages include characterizing and selecting phages assessed for host range by EOP, by host receptor affinity, and by a personalized approach. Final steps include choosing phages also from phage libraries for formulating well-designed phage cocktails, and conducting research on antibiotic and phage dosing, timing, and administration routes [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. ## Future promises on phage therapy against cf pa Although clinical trials for phage therapy in patients with CF seem a long way off in Western countries [bib_ref] The role of regulated clinical trials in the development of bacteriophage therapeutics, Parracho [/bib_ref] [bib_ref] Phage therapy gets revitalized, Reardon [/bib_ref] , the alarming evidence on growing resistance to antibiotics can only encourage efforts to exploit the promising phage therapy for MDR and XDR PA in patients with CF. Researchers responsible for designing future clinical trials for phage therapy in humans will need to choose the correct experimental in vitro and in vivo models on biofilmproducing mucoid PA [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] The application of biofilm science to the study and control of chronic..., Costerton [/bib_ref]. As a first step, they should preferably test in vitro a mono-microbialbiofilm-forming mucoid PA model or a non-biofilm-forming PA incorporated in biofilm-producing isolates mimicking a CF lung condition [bib_ref] Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in..., Deligianni [/bib_ref]. These biofilm and non-biofilm forming PA often coexist in poly-microbial communities in CF lungs (Supplementary . Equally important, although phages focus their action specifically on the target pathogen (Loc-Carrillo and [bib_ref] Pros and cons of phage therapy, Loc-Carrillo [/bib_ref] , and although [bib_ref] Bacterial activity in cystic fibrosis lung infections, Rogers [/bib_ref] state that the various bacteria persist in an active form without interfering with the lung disease or influencing patients' prognosis, others observed that various microbial species present in the biofilm can alter phageinduced responses, influencing bacterial and phage densities, and determining how bacterial resistance evolves [bib_ref] Bacterial competition and quorumsensing signalling shape the eco-evolutionary outcomes of model in..., Mumford [/bib_ref]. After identifying the target and the strain to treat, the next step will be to select the most appropriate phages to combine in a cocktail, according to their EOP, their affinity for the various PA receptors, their ability to degrade the biofilm matrix, and their low burst size. Future research on phage administration routes will consider nebulizing phages, suspended in pre-specified material or giving them as a dry powder [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] In vitro lung delivery of bacteriophages KS4-M and KZ using dry powder..., Golshahi [/bib_ref] [bib_ref] Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections, Matinkhoo [/bib_ref] [bib_ref] Bacteriophage delivery by nebulization and efficacy against phenotypically diverse Pseudomonas aeruginosa from..., Sahota [/bib_ref]. Another crucial step is to characterize selected phages functionally (latency and burst size) and to detect those carrying undesirable genes coding for toxins and antibiotic resistance (Loc-Carrillo and [bib_ref] Pros and cons of phage therapy, Loc-Carrillo [/bib_ref]. Some evidence suggests that safety can be efficiently and reliably assessed by separating lytic phages from lysogenic (temperate) phages characterizing them by genome sequencing and analysis (Supplementary ; [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref] [bib_ref] On-demand isolation of bacteriophages against drug-resistant bacteria for personalized phage therapy, Mattila [/bib_ref] , and by using standardized control groups in in vivo studies [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Future directions should include research to investigate effective trained phages in cocktails [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref] or engineered phages for formulating new phage cocktails, even combined with antibiotics [bib_ref] Dispersing biofilms with engineered enzymatic bacteriophage, Lu [/bib_ref]. Phage cocktail efficacy and safety, and phages eventually combined with antibiotics, should then be validated in vivo in skin diseases before testing them in systemic diseases, and easily at a low cost in non-pulmonary models. Because human immunological responses to phages differ significantly during chronic infections [bib_ref] Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in..., Li [/bib_ref] [bib_ref] Phage therapy: an alternative to antibiotics in the age of multi-drug resistance, Lin [/bib_ref] [bib_ref] Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa, Waters [/bib_ref] , we also need to test outcomes in studies investigating long-term PA infections and biofilm forming PA. Hence our review stresses the challenging need to standardize current biofilm models. What we especially need are dynamic models closely resembling physiological conditions , or models mimicking PA growth in CF lungs including the mucus barrier, such as the airway surface liquid model used on epithelial cell lines [bib_ref] A proposed integrated approach for the preclinical evaluation of phage therapy in..., Danis-Wlodarczyk [/bib_ref] , artificial sputum medium [bib_ref] Characterization of JG024, a Pseudomonas aeruginosa PB1-like broad host range phage under..., Garbe [/bib_ref] or eventually patients' sputum [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] , and test in vivo specific animal candidate (pig and ferret) models. All experimental procedures should include standardized negative control groups [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref]. Future promises and perspectives in phage therapy for treating MDR and XDR PA lung infections in CF include models designed to apply antibiotics to phages at peak phage efficacy (within about 12 h) sequentially rather than simultaneously (Torres- [bib_ref] A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining..., Torres-Barceló [/bib_ref]. We envisage future head-to-head studies comparing antibiotics given at sub-inhibitory concentrations combined with trained phages and antibiotics given alone [bib_ref] Bacteriophage-antibiotic synergism to control planktonic and biofilm producing clinical isolates of Pseudomonas..., Nouraldin [/bib_ref] [bib_ref] Phage therapy: an alternative to antibiotics in the age of multi-drug resistance, Lin [/bib_ref]. These research advances might reduce PA resistance, and boost interest in phage therapy in patients with CF [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. # Conclusions In conclusion, our review results addressing practical in vivo models for advancing clinical research on phage therapy should help in designing protocols for pivotal clinical trials, to test phage therapy against CF PA strains in close coordination between microbiologists and clinicians caring for patients with CF [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. It also involves implementing dialogues between the medical community in consultation with CF community reference groups able to overcome complex ethical dilemmas [bib_ref] Morals, microbes, and methods, Caplan [/bib_ref] [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. The results from our review suggest using phages with a broad host range against specific infecting PA strains in CF lungs [bib_ref] Bacteriophages MR299-2 and NH-4 can eliminate Pseudomonas aeruginosa in the murine lung..., Alemayehu [/bib_ref] [bib_ref] Anti-pseudomonal bacteriophage reduces infective burden and inflammatory response in murine lung, Pabary [/bib_ref] , and avoiding polyvalent phage mixtures [bib_ref] Effectiveness of bacteriophages in the sputum of cystic fibrosis patients, Saussereau [/bib_ref] [bib_ref] Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model, Beeton [/bib_ref] [bib_ref] In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas..., Olszak [/bib_ref]. This approach promises clinical advantages including reducing costs, speeding phage cocktail delivery, analyzing dose-response curves, and "weeding out" phages that could show incompatibility [bib_ref] Phage therapy pharmacology: phage cocktails, Chan [/bib_ref]. Although no research has yet defined the kinetic mechanisms responsible for phage actions especially against CF PA biofilms, to simplify phage therapy development we agree with others [bib_ref] Phage therapy pharmacology: phage cocktails, Chan [/bib_ref] [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref] on the need to design clinical protocols investigating a minimum number of active phages (2-3 phages) via a nebulizer or by an inhaler delivered directly to the CF lungs [bib_ref] Anti-tuberculosis bacteriophage D29 delivery with a vibrating mesh nebulizer, jet nebulizer, and..., Carrigy [/bib_ref] [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. This standard way is effective and meets with patients' approval. Given the importance of simulating in vivo a condition resembling that in CF patients' lungs, overall the findings from our review suggest that research needs also to develop models in vivo able to detect a change from acute to chronic PA infections in CF, and to investigate the most important concerns on MDR and XDR PA phage co-evolutionary interactions and immunogenicity. Future studies on immunogenicity in animals that exhibit a high CFTR expression in lungs similar to that in CF (pigs and ferrets) will show whether mucus can stimulate phage adhesion to the lungs, prolong phage therapeutic activity, and how phage particles can overcome immune interference [bib_ref] Bacteriophage adhering to mucus provide a non-host-derived immunity, Barr [/bib_ref] [bib_ref] The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and..., Trend [/bib_ref]. To prevent therapeutic phages from spreading outside special clinical facilities designed for applying phage therapy, we agree also with those who recommend using phages propagated only on standard host strains (such as PAO1), and choosing therapeutic phages individually for each patient (personalized approach) [bib_ref] Modular approach to select bacteriophages targeting Pseudomonas aeruginosa for their application to..., Krylov [/bib_ref]. A crucial step is to use phage genome sequencing to identify possible virulence factors or prophages from host strains, so as to avoid transferring possible contamination in phage stocks [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref] ; [bib_ref] Information phage therapy research should report, Abedon [/bib_ref]. Convincing research from synthetic biology and nanotechnology techniques already exploits new engineered phages that display selected proteins binding specific CF PA targets isolated and re-amplified from commercially available phage libraries [bib_ref] Filamentous fusion phage: novel expression vectors that display cloned antigens on the..., Smith [/bib_ref] [bib_ref] Immunocompatibility of bacteriophages as nanomedicines, Kaur [/bib_ref] [bib_ref] Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine..., Henry [/bib_ref] [bib_ref] Bacteriophages and their immunological applications against infectious threats, Criscuolo [/bib_ref]. Using phages from certified libraries will help to overcome concerns requiring international regulatory agencies to approve and re-approve phage cocktails for each individual clinical trial [bib_ref] The role of regulated clinical trials in the development of bacteriophage therapeutics, Parracho [/bib_ref] [bib_ref] A century of the phage: past, present and future, Salmond [/bib_ref] [bib_ref] Phage approved in food not as a therapeutic?, Sarhan [/bib_ref] [bib_ref] Bacteriophages and their immunological applications against infectious threats, Criscuolo [/bib_ref]. Even though obtaining a phage set that effectively lyses all variants in a given pathogen is a challenging problem [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref] , our review findings suggest that successful phage therapy on a larger scale in patients with CF depends not only on involving the pharmaceutical industry in preclinical development and formulation before undertaking clinical trials. Preclinical industry-funded phage formulation could be developed also by creating the claimed first-in-human advisory system [bib_ref] Consider drug efficacy before first-inhuman trials, Kimmelman [/bib_ref]. Despite the high investment needed for creating an advisory board regulatory infrastructure, this regulatory board could critically match preclinical evidence and clinical scenarios for phage therapy in CF PA, so as to justify the first-in-human trials for phage therapy, thus meeting a great medical need in treating CF MDR and XDR PA. This new approach will help to improve laboratory techniques intended to develop phages combined with antibiotics able to combat MDR and XDR PA strains [bib_ref] Phage cocktails and the future of phage therapy, Chan [/bib_ref] [bib_ref] Back to the future: bacteriophages as promising therapeutic tools, Domingo-Calap [/bib_ref] [bib_ref] Evolutionary rationale for phages as complements of antibiotics, Torres-Barceló [/bib_ref] [bib_ref] Phage therapy: an alternative to antibiotics in the age of multi-drug resistance, Lin [/bib_ref]. Combining phage therapy with antibiotics might be more appealing to pharmaceutical companies because it gives them opportunities for new patents. Collectively, the findings from our review argue that, despite the continuing need to improve phage efficacy and safety in vivo and solve ethical and regulatory concerns, research into lytic phage therapy in CF PA strains holds promise. Progress over the past 20 years intended especially to combat increasing antibiotic resistance, including hospital studies, already suggests that lytic phage therapy of CF PA infections will become a useful, safe medical procedure. Though phage therapy will never completely replace antibiotics in patients with CF, pharmaceutical industries should eventually combine phages with one or two antibiotics scaled by MIC to pre-test their sensitivity to treat CF MDR and XDR PA. Advances that should encourage and simplify phage therapeutic use in CF MDR and XDR PA include regularly updating cocktail preparations in certified phage libraries for personalized phage therapy, preferably using genomic engineered phages, so as to ensure safety and possibly patentable engineered phages, thus providing certified phages for international regulatory agencies [fig_ref] FIGURE 2 |: Information from the 24 studies included in the review answering major open... [/fig_ref]. Researchers should release all available phage therapy results to the medical society so that procedures can be corrected and adjusted as necessary. Owing to easy phage isolation, sequence-specific targeting and replication on specific bacterial hosts, active penetration into bacterial biofilms, low production costs, safety, lack of known immunogenicity, and disinterest in producing new antibiotics, introducing phage therapy combined with existing PA-sensitive antibiotics in treating CF PA in clinical practice appears a promising development over time. # Author contributions EF and PR: searched and screened articles by title and abstract; PR and MR: mapped and coded information. All the three authors identified key results, limitations, advantages and disadvantages in study findings, combined the synthesis, wrote and revised the re-submitted paper, and approved the final version. # Acknowledgments [fig] FIGURE 1 |: A flow chart showing multiple search strategies and reasons for excluding and including publications in the review. *Four search strategies (November 2016): (((phage) OR bacteriophage) AND cystic fibrosis) AND Pseudomonas aeruginosa [TI] (92 publications); ((((phage therapy) AND bacteriophage therapy)) AND Pseudomonas aeruginosa) AND cystic fibrosis [TI] (16 publications); ((cystic fibrosis) AND ((phage therapy) AND bacteriophage therapy)) AND Pseudomonas aeruginosa biofilm [TI] (6 publications); and ((((phage therapy) AND bacteriophage therapy)) AND Pseudomonas aeruginosa) AND phage resistant* (11 publications). [/fig] [fig] FIGURE 2 |: Information from the 24 studies included in the review answering major open questions essential for planning future studies in vitro and in vivo on phage therapy to treat Pseudomonas aeruginosa (PA) lung infections in patients with cystic fibrosis (CF). [/fig]

Comparing the EQ-5D-3 L and EQ-5D-5 L: studying measurement and scores in Indonesian type 2 diabetes mellitus patients Background: The EuroQoL five-dimensional instrument (EQ-5D) is the favoured preference-based instrument to measure health-related quality of life (HRQoL) in several countries. Two versions of the EQ-5D are available: the 3-level version (EQ-5D-3 L) and the 5-level version (EQ-5D-5 L). This study aims to compare specific measurement properties and scoring of the EQ-5D-3 L (3 L) and EQ-5D-5 L (5 L) in Indonesian type 2 diabetes mellitus (T2DM) outpatients. Methods: A survey was conducted in a hospital and two primary healthcare centres on Sulawesi Island. Participants were asked to complete the two versions of the EQ-5D instruments. The 3 L and 5 L were compared in terms of distribution and ceiling, discriminative power and test-retest reliability. To determine the consistency of the participants' answers, we checked the redistribution pattern, i.e., the consistency of a participant's scores in both versions.Results: A total of 198 T2DM outpatients (mean age 59.90 ± 11.06) completed the 3 L and 5 L surveys. A total of 46 health states for 3 L and 90 health states for 5 L were reported. The '11121' health state was reported most often: 17% in the 3 L and 13% in the 5 L. The results suggested a lower ceiling effect for 5 L (11%) than for 3 L (15%). Regarding redistribution, only 6.1% of responses were found to be inconsistent in this study. The 5 L had higher discriminative power than the 3 L version. Reliability as reflected by the index score was 0.64 for 3 L and 0.74 for 5 L. Pain/discomfort was the dimension mostly affected, whereas the self-care dimension was the least affected.Conclusions: This study suggests that the 5 L-version of the EQ-5D instrument performs better than the 3 L-version in T2DM outpatients in Indonesia, regarding measurement and scoring properties. As such, our study supports the use of the 5 L as the preferred health-related quality of life measurement tool. # Introduction In 2011, the number of people suffering from diabetes mellitus (DM) in the world was reported at 366 million. Based on the latest data in 2017, this number has increased by almost 20% to reach 450 million. Worldwide, 90% of these suffer from type 2 diabetes mellitus (T2DM) . In Indonesia, in the same period mentioned, the number of people with T2DM even increased by 30%, i.e., from 7.3 million to 10.3 million. In this respect, the Indonesian Ministry of Health also reported that the national prevalence of T2DM in Indonesia had almost doubled from 1.1% in 2007 to 2.1% in 2013. Furthermore, the Ministry of Health's report stated that of the 34 provinces in Indonesia, 15 provinces had a higher prevalence of T2DM patients than the national average, inclusive Sulawesi island. Notably, the prevalence of T2DM amounts to 3.7% in Central Sulawesi province, 3.6% in North Sulawesi and 3.4% in South Sulawesi. The continued increase in the prevalence of T2DM patients in Indonesia requires serious attention, especially concerning control of T2DM costs and patients' health status and cost-effectiveness of interventions. In this respect, adequate measurement of health-related quality of life (HRQoL) reflects a core issue. The EuroQoL five-dimensional instrument (EQ-5D) is the recommended preference-based instrument to measure HRQoL in several countries. HRQoL is measured by this instrument in such a way that it generates a single index score or utility. This instrument consists of five items covering five health-state dimensions (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression), with each item originally having three levels of severity (EQ-5D-3 L). In 2011, the EuroQol Group expanded the number of severity levels for each dimension to five (EQ-5D-5 L). Both the EQ-5D-3 L (3 L) and EQ-5D-5 L (5 L) versions have been used in several studies, covering both clinical and methodological assessments. Several comparative studies of the 3 L and 5 L versions of EQ-5D have been conducted in the countries neighbouring Indonesia, notably Singapore and Thailand. Both studies reported that 5 L is the preferable version for T2DM patients considering its greater discriminative power and patients' preferences. Considering the 5 L and 3 L versions, it is noted that both versions have been used in several studies in Indonesia, already, but a structured, integrative and direct comparison is still lacking, however a structured integrative comparison is still missing, motivating the conduct of our study. Whereas such comparisons would be available for other countries, sociodemographic characteristics and cultural differences between Indonesia and other countries might differ potentially resulting in varying findings measurement properties of the two EQ-5D versions. Therefore, this study aims to directly compare specific measurement properties and scorings of the 3 L and 5 L versions in Indonesian type 2 diabetes mellitus (T2DM) outpatients. # Materials and methods ## Study design A cross-sectional study was conducted from July 2016 to April 2017. A secondary care setting in South Sulawesi and two primary care settings in Central Sulawesi were included. In particular, these were Jaury Academic Hospital in ## Participants Participants were T2DM outpatients with a minimum age of 18 years. The participants were informed of the study objectives and study procedure. The researcher or research assistants obtained signed informed consent forms from the participants. For the participants with disabilities or difficulties in reading, consent was based on confirmation from their caregiver who accompanied them during treatment at a health facility. The caregiver played a role in providing support to the participants as they filled in the instruments. It is important to note that all decisions on the exact health states chosen originated from the participants. In this study, all participants were treated by a consulting resident internal medicine who gave his/her consent to the data collection during the participant's T2DM consultation (in primary and secondary care). Instruments EQ-5D 3 L and 5 L consist of two parts: the EQ-5D descriptive system classification and the EQ visual analogue scale (EQ-VAS). The EQ-5D descriptive system comprises five items on its HRQoL dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension in the 3 L versionis completed with three response options: no problem, some problems, and confined to bed/unable/extreme problems, yielding a possible 243 (3 5 ) unique health states. A single digit expresses the level selected for that specific dimension. Therefore, the five-digit number for five dimensions describes a specific health state. For example, '11111' indicates 'no problems on any of the five dimensions', while '23231' indicates 'some problems walking, unable to wash or dress, some problems with performing usual activities, extreme pain/ discomfort, and no anxiety/depression'. The 5 Lhas five scale options to choose from: no problem, slight problems, moderate problems, severe problems, and extreme problems/unable. The 5 L instrument yields 3125 (5 5 ) unique health states. For example, '12345' indicates 'no problems walking, slight problems washing or dressing, moderate problems doing usual activities, severe pain/discomfort and extreme anxiety/depression'. The EQ-VAS presents the participants' self-rated health on a scale of 0 (worst imaginable health) to 100 (best imaginable health). The time frame for the EQ-VAS is 'today', meaning that participants were asked to describe their health state during the day they were interviewed. We used the 3 L and 5 L Bahasa Indonesia versions of the EQ-5D, produced by the EuroQol Group using a standardized translation protocoland having been proved as valid and reliable questionnaires in Indonesian patient groups. ## Data collection procedure and data sources After introducing the researchers and explaining the purpose of the study, a brief description to the participants was provided on how to use the EQ-5D instruments. An explanation of the concept of HRQoL as an aid on how they should describe their health state was presented. The participants were given the opportunity to ask questions throughout the data collection process. For EQ-VAS, we asked the participants to describe their health state and provide the most appropriate score to define their health state. Three research assistants were hired to collect the data. As a sequence, participants first classified their health state on the 5 L items, then provided their data (sociodemographic and clinical conditions), followed by the 3 L. According to socio-demographic data (gender, age, T2DM duration, occupation, level of education, and dependence on a caregiver) were obtained from selfreporting. In this study, participants were classified into two age categories based on the retirement age of Indonesian people (56 years): productive age (below 56 years) and retirement age (56 years and above). As for employment status, participants were defined as in active employment when they were still actively working, and unemployed if they reported not having a job. Those whose main responsibilities were for their family members and household chores were classified as housewives. Data on the clinical conditions, such as the type of therapy, T2DM-related complications, and comorbidities were obtained from treating physicians. Self-reported data from participants was used in the cases data could not be collected through the treating physicians. In this study, participants were defined as having comorbidities if they suffered from other diseases, such as asthma, gastritis and gout problems. Participants were defined as having complication and comorbidities if they suffered from other diseases and T2DM complications; for example, a participant with comorbid cancer and hypertension as a complication of diabetes. ## Test-retest reliability Test-retest reliability was analyzed using sequential measurements. Participants involved in this phase were those who visited the specific health facility twice. The time interval between the two measurement times was four weeks as the participants were scheduled to meet their consulting resident internal medicine each month. Notably, an additional question was asked before the participants completed the instruments the second round: 'Has there been any major change in your health state between the first time you completed the instruments last month and today? For example, have you been hospitalised, had an accident, experienced a natural disaster or have been bereaved'? Participants who answered 'yes' were excluded from the final sample. ## Analyses For self-reported health state profiles obtained from the two versions of EQ-5D, we calculated the percentage of participants who responded to each level of each dimension. To determine the consistency of the participants' answers, we checked the redistribution pattern, i.e., the consistency of individual participants' scores in both versions. A consistent response pair was defined as a 3 L response which is at most one level away from the 5 L response (e.g., a participant chose level 1 in 3 L and chose level 2 in 5 L). When the 5 L level was more than 1 level away from the 3 L level (e.g., a participant chose level 1 in 3 L and chose level 3 in 5), this was labelled inconsistent. Next, we converted their scores on 3 L to 5 L as follows: 1 in 3 L equals 1 in 5 L, 2 in 3 L equals 3 in 5 L, and 3 in 3 L equals 5 in 5 L. The ceiling effect was defined as the proportion of participants who reported not having problems in any of the five EQ-5D dimensions (health state '11111') for both 3 L and 5 L. This statistic is often used to assess the discriminatory power of health-state classification systems. As Indonesia only has the EQ-5D-5 L value set, not the 3 L, to obtain consistent 3 L and 5 L utility index scores, the UK 3 L and 5 L value setswere used. The test-retest reliability of the dimension scores was assessed using the weighted kappa. We applied Landis JR & Koch GG standardsto determine the strength of agreement of the kappa values as follows: < 0.00 = poor, 0.00-0.20 = slight, 0.21-0.40 = fair, 0.41-0.60 = moderate, 0.61-0.80 = substantial, and 0.81-1.00 = almost perfect. The test-retest reliability of the EQ-VAS and index scores were calculated using intra-class correlation coefficients (ICCs), two-way random effects and absolute agreements. The following reliability guideline was used for the strength of the ICC values: < 0.5 = poor, 0.5-0.75 = moderate, 0.75-0.90 = good and > 0.90 = excellent. The discriminative power was calculated using the Shannon index (H′) and Shannon's Evenness index (J'). The Shannon index combines the absolute information content as expressed by the number of categories with the extent to which the information is evenly spread over these categories. On the other hand, the J' expresses the relative information of a system or the evenness of the information distribution regardless of the number of categories. In case of an even distribution, when all levels are filled with the same frequency, J' is equal to 1. Larger H′ and J' values indicate more discriminatory performance. All the data were analysed using IBM SPSS Statistics for Windows version 23 (SPSS Inc., Cambridge, MA, USA), and statistical significance was set a priori at p < .05. # Results ## Descriptive A total of 198 participants were interviewed. The average age of the participants was almost 60 years, with 58% being female, and 70% of female participants reported being housewives as their main activity. Regarding the clinical conditions, more than 70% of participants were being treated with oral antidiabetic therapy (OAD), both monotherapy and OAD combinations, and 52% of participants reported T2DM-related complications. Furthermore, participants had various comorbidities, such as asthma (n = 6), gastritis (n = 5), and gout (n = 3). For test and re-test reliability, of the 198 participants who completed the first survey, 53 participants (62% female) completed the instruments twice. In this phase, only 12 participants had a university degree and most of the female participants were housewives (n = 20). Furthermore, of the almost 70% of participants treated with OADs, 40% reported T2DM without complications and 36% reported T2DM with at least one complication. There were no missing health state data. ## Scoring and ceiling Participants usually reported no problems (level 1) on both 3 L and 5 L, except for the pain/discomfort dimension with only 25 and 20% of participants reporting no problems on 3 L and 5 L, respectively. Therefore, pain/discomfort was more often reported at other 3 L and 5 L levels compared to the other EQ-5D dimensions . Regarding the ceiling effect, the 5 L version showed slightly fewer reports of absence of problems in all dimensions ('11111') compared to the 3 L version. The percentage of participants reporting the '11111' health state decreased from 15% in the 3 L to 11% in the 5 L. Nevertheless, no statistically significant difference was found (p-value = .178). Self-care reached the highest ceiling (82% for the 3 L, 78% for the 5 L) while pain/discomfort showed the lowest ceiling (as mentioned above, 25% for the 3 L, 20% for the 5 L). The anxiety/depression dimension showed the smallest reduction in the ceiling (3% less), whereas the mobility dimension showed the Participants were defined as having complication and comorbidities if they suffered from other diseases and T2DM complications largest reduction (7% reduction) when going from 3 L to 5 L. None of the ceiling reductions from 3 L to 5 L were statistically significant. The range of index scores was broader in the 3 L than in the 5 L version, especially for negative values . The lowest index score reported for the 3 L was − 0.349 (state '23333'), whereas this was − 0.263 (state '45554') for the 5 L. The most frequently reported health state was '11121' (slight problems in pain/discomfort and no problems in the other dimensions), i.e. 17% in the 3 L and 13% in the 5 L. There were 46 and 90, 3 L and 5 L health states reported in the study, respectively. ## Redistribution from 3 l to 5 l Of the participants who reported no problem (level 1) for a dimension on the 3 L, most (73-94%) reported the same on the 5 L, while 6-26% switched to slight problems (level 2) on the 5 L as shown in. The majority of the participants who reported moderate problems (level 2) on the 3 L indicated slight problems (level 2) on the 5 L (44-67%), while 20-28% switched to moderate problems (level 3) and 12-31% shifted to severe problems (level 4) on the 5 L. Most of the participants who indicated confined to bed/unable/extreme problems (level 3) on the 3 L indicated extreme problems (level 5) on the 5 L for the usual activities dimension, whereas most participants who reported extreme problems on 3 L redistributed into severe problems (level 4) for pain/discomfort and anxiety/depression. As for the self-care dimension, these percentages were equal. Redistribution occurred least frequently in the mobility dimension since no participant reported 'confined to bed' on the 3 L in that area. The inconsistent responses were ranging from 4% on self-care to 7.6% on the pain/discomfort and anxiety/depression dimensions. An example of such inconsistency was a participant choosing 'no problems walking' in 3 L (mobility level 1) and 'severe problems walking' in 5 L (mobility level 4). ## Discriminative power Compared to the 3 L version, the 5 L system had a substantial gain in classification efficiency for each dimension, indicated by higher H′ values of all the dimensions. The J' values were more similar among the two versions of EQ-5D as shown in, indicating that the degree of the potential use of the classification system was comparable between the two versions. ## Test-retest reliability Fifty-three participants (26.8%) completed the instruments twice. By inclusion criterion, all reported no major changes in their health between the first and second data completion point. The weighted kappa of the 5 L dimensions for the 3 L was judged as slightly in agreement for the self-care dimension at 0.14, while the other four dimensions fair agreement existed: mobility at 0.25, usual activities at 0.23, pain/discomfort at 0.25 and anxiety/depression at 0.40. For the 5 L, the pain/discomfort dimension was judged as slightly in agreement at 0.19, while the other four dimensions were in fair agreement: mobility at 0.35, self-care at 0.30, usual activities at 0.37 and anxiety/ depression at 0.39. The EQ-VAS ICCs were 0.35 and 0.32 for the 3 L and 5 L respectively. Moreover, the ICCs of the 3 L and 5 L index scores were 0.64 and 0.74 respectively, reflecting a moderate level of reproducibility. # Discussion We examined some important specific measurement properties of the 3 L and 5 L instruments in Indonesian T2DM outpatients. We found that the 5 L version had a lower ceiling effect, higher discriminative power, and in the majority of the dimensions a higher test-retest reliability coefficient compared to the 3 L. The 5 L classification system better represents the variety of patients' health states, showed by the more health states reported in the 5 L than the 3 L. With regards to the discriminative power, our Self-reported health on the EQ-5D-3 L and EQ-5D-5 L descriptive system, and the EQ-VAS EQ-5D-3 L EQ-5D-5 L results showed that 5 L was more discriminative compared to the 3 L, indicated by the gain of the Shannon H′ index from 3 L to 5 L. These results were similar to the findings from across the globe, as reviewed by Buchholz et al.. The J' index was also in line with the results of the aforementioned study. The 5 L version showed a lower ceiling effect (health state '11111') than the 3 L at 11 and 15%, respectively. Notably, a previous studysuggested that a ceiling effect of 15% and higher should be considered as 'serious' (as shown for the 3 L version) while relevantly below 15% is considered small (as shown by the 5 L version). Several studies suggested that other HRQoL instruments have shown lower ceiling effects than the EQ-5D while still strongly correlated with the EQ-5D scores, e.g. the SF-6D. Also, Round suggests to consider other HRQoL measures instead of EQ-5D. However, in several countries, including Indonesia, EQ-5D is the recommended preference-based instrument to measure HRQoL. Therefore, a lower ceiling effect as shown by the 5 L version supports the use of EQ-5D-5 L in Indonesia, especially in patients with T2DM. Next to better statistical properties, during discussions, also our participants stated that in the 5 L they could more accurately describe their own health state and the severity of T2DM. This is in line with studies in Thailand and Singapore which also stated in both studies that DM severity could be better described in 5 L compared to 3 L. Therefore, our study provides further support to advocate the use of 5 L in clinical, health policy and economic evaluation studies with EQ-5D index score assessments; in our case, notably for Indonesian T2DM outpatients. Another finding of our research concerns the fact that most participants reported problems on pain/discomfort dimension in the 3 L and 5 L. Notably, the '11121' was the most reported health state by the participants. Four previous studies in Asian populations with T2DM also reported similar findings. Also, a multicountry study stated that the Eastern European participants had three times higher mobility and usual activity problems and six times higher self-care problems compared to their Asian counterparts. In this study, the inconsistent responses were ranging from 4% (self-care) to 7.6% (pain/discomfort and anxiety/ depression). This was slightly higher than in the studies in China and Singapore at 0.7-1.4% and 2.5-4.1%, respectively. A similar study in Thailand resulted in no inconsistent response at all. It could be argued that higher education level, younger age, and more healthy DM patients (without complications or comorbidities) might play a role in this difference, which indeed seems the case in Thailand study. However, the age distributions and education levels of our participants were overall similar with those in the China and Singapore studies. A possible explanation offered is that the difficulties faced by our elderly participants in completing the 5 L produced these inconsistent responses, although we assisted with explanations. Notably, many elderly participants experienced decreased vision and hearing loss, especially participants in the secondary care facilities. Also, many Indonesian T2DM patients had low levels of Cumulative percentage of the EQ-5D-3 L and EQ-5D-5 L index scores education, so an explanation of the HRQoL concept and the EQ-5D instrument was a necessity. Our study has some limitations which should be considered. First, the participants were recruited from only two locations in Indonesia. Therefore, generalizing the findings nationally should be done with caution. Second, only outpatient participants were recruited for this study. These findings may not be generalizable to inpatients who probably experience more health difficulties: i.e. would report worse health states. Future investigations could include the inpatients to complement the analysis that we provide. Another limitation is that we did not randomize the order of the two versions of the EQ-5D instrument. One could argue that the presentation of 5 L first followed by the 3 L for all participants might produce some bias in the answers of the participants. Our reason was to limit the tendency to not use level 2 and 4 in 5 L. Also, this order was also used in other comparative studies, such as those in Thailand, Singaporeand one multi-country study Denmark, England, Italy, the Netherlands, Poland, and Scotland. Finally, it is noteworthy that, during our discussions, is seemed that participants with lower education levels and elderly participants preferred the 3 L version, often mentioning that the 3 L version was easier to understand, despite all explanations provided and the flexibility of the 5 L version to more precisely express the health state. Obviously, these patients' preferences come in as an additional important aspect and warrants further research in this area, inclusive options to even better convey the 5 L version to participants. Finally, further research should focus on other areas in Indonesia beyond our index area of Sulawesi; for example, a similar type of investigation on Java would be worthwhile, with the majority of the Indonesian population living there. *A consistent response pair was defined as a 3 L response which is at most one level away from the 5 L response (e.g., a participant chose level 1 in 3 L and chose level 2 in 5 L). When the 5 L level was more than 1 level away from the 3 L level (e.g., a participant chose level 1 in 3 L and chose level 3 in 5), this was labelled inconsistent # Conclusion This study suggests that the 5 L-version of EQ-5D performs better than the 3 L-version in T2DM outpatients in Indonesia. As such, our study supports the use of the 5 L as the preferred HRQoL tool to derive EQ-5D index scores, which are indispensable in pharmacoeconomic analyses and health economic evaluations of interventions in T2DM patients.

Framing of visual content shown on popular social media may affect viewers’ attitudes to threatened species Content published on social media may affect user's attitudes toward wildlife species. We evaluated viewers' responses to videos published on a popular social medium, focusing particularly on how the content was framed (i.e., the way an issue is conveyed to transmit a certain meaning). We analyzed videos posted on YouTube that showed vultures interacting with livestock. The videos were negatively or positively framed, and we evaluated viewers' opinions of these birds through the comments posted. We also analyzed negatively framed videos of mammalian predators interacting with livestock, to evaluate whether comments on this content were similar to those on vultures. We found that the framing of the information influenced the tone of the comments. Videos showing farmers talking about their livestock losses were more likely to provoke negative comments than videos not including farmer testimonies. The probability of negative comments being posted on videos about vultures was higher than for mammalian predators. Finally, negatively framed videos on vultures had more views over time than positive ones. Our results call for caution in the presentation of wildlife species online, and highlight the need for regulations to prevent the spread of misinformed videos that could magnify existing human-wildlife conflicts.Recent years have seen the explosive growth of social media 1-4 . These social networks offer an opportunity to produce and share content (e.g., pictures, tags, videos) that can be seen, commented on, and reproduced simultaneously by millions of people around the world. In fact, social media content may "go viral": information may spread very rapidly, in a way that can be compared with the dynamics of infectious diseases 5 . In response to this social media growth, a new conservation field named "conservation culturomics" has emerged; it aims to use digital sources to study human-nature interactions and provide new insights into how to deal with conservation problems 6 .Conservation culturomics offers a new opportunity to address certain aspects of the human-wildlife relationship, based on the gathering of knowledge (i.e., corpora) from web pages or video platforms. This enables the exploration of diverse issues related to conservation practices 6-8 . For instance, video platforms are an interesting data source, and can be used to evaluate what people do or think about wildlife 6 . Data shared on these platforms and other social media may provide information on cultural ecosystem services 9-11 , the preferences of tourists for observation of biodiversity, activities carried out in protected areas 12 , and the emotions these sites provoke 13 . In addition, these data can be used to evaluate illegal activities, such as hunting 7 , and to analyze people's tolerance and perceptions of wildlife species14,15. Evaluating the effect of this uploaded information on viewer's perceptions may help in planning more effective communication strategies for species conservation 16 .Unfortunately, certain user-generated content (USG) reproduced on social media about diverse wildlife species of conservation concern (e.g., obligate scavenging birds and carnivores) could negatively influence people's tolerance toward them 14,17 . In many cases, posted content is based on biased information without strong scientific evidence, or may even be fake news or illegal information (e.g., wildlife trading) 17,18 . This scenario can become more serious for wildlife conservation when these messages "go viral"18,19or are presented with misleading "frames" (a frame refers to the way an issue is described or how a problem is conceived and approached so that it conveys a certain meaning) 20 .People's attitudes and decisions can be influenced by the way a message is presented 16 . Messages can be strategically framed to emphasize what really matters to the sender and the audience 20 . For example, messages framed OPEN www.nature.com/scientificreports/ with close psychological distance tend to be highly effective; when an object is perceived as close it tends to be perceived in more concrete way, whereas when the object is perceived as distant it tends to be construed more abstractly [bib_ref] The future is now: Reducing psychological distance to increase public engagement with..., Jones [/bib_ref] [bib_ref] Personal experience and the 'psychological distance' of climate change: An integrative review, Mcdonald [/bib_ref]. In other words, psychological distance is reduced when the framing of a message emphasizes a problem which will affect people like the viewers themselves. However, this framing could have an unintentional effect, producing cognitive bias, such as cognitive dissonance [bib_ref] Five lessons to guide more effective biodiversity conservation message framing, Kusmanoff [/bib_ref]. Cognitive dissonance arises when people are exposed to information that is inconsistent with their own beliefs. If this dissonance is not resolved by the audience changing their beliefs, it can lead to misperception or even rejection of the information [bib_ref] An introduction to cognitive dissonance theory and an overview of current persepectives..., Harmon-Jones [/bib_ref]. In summary, the way information is presented could be a key aspect for wildlife species, since inappropriate messages may exacerbate existing human-wildlife conflict, with negative consequences for wildlife conservation. One of the most serious human-wildlife conflicts is produced by interaction between livestock producers and carnivorous and scavenger wildlife. This conflict has been studied in many parts of the world and for different species [bib_ref] The conflict between scavenging birds and farmers: Field observations do not support..., Ballejo [/bib_ref] [bib_ref] Characterization of puma-livestock conflicts in rangelands of central, Guerisoli [/bib_ref] [bib_ref] Conflicts between humans and terrestrial vertebrates: A global review, Torres [/bib_ref] [bib_ref] Human-felid conflict: A review of patterns and priorities worldwide, Inskip [/bib_ref]. Livestock producers usually argue that wildlife species such as obligate scavenging birds (hereafter, vultures) and mammalian predators are harmful, blaming them for most predation events and the associated economic loss [bib_ref] Characterization of puma-livestock conflicts in rangelands of central, Guerisoli [/bib_ref] [bib_ref] Conflicts between humans and terrestrial vertebrates: A global review, Torres [/bib_ref] [bib_ref] Education can improve the negative perception of a threatened long-lived scavenging bird,..., Arnulphi [/bib_ref]. In the case of vultures, however, scientific evidence shows that they rarely prey on livestock [bib_ref] The conflict between scavenging birds and farmers: Field observations do not support..., Ballejo [/bib_ref] [bib_ref] Vultures attacking livestock: A problem of vulture behavioural change or farmers' perception, Duriez [/bib_ref] [bib_ref] Vultures vs livestock: Conservation relationships in an emerging conflict between humans and..., Margalida [/bib_ref]. There is some evidence that social media could play an important negative role in this conflict, magnifying events and generating biased perceptions that may constitute an added hazard for these threatened birds [bib_ref] Fake news and vultures, Margalida [/bib_ref] [bib_ref] Presumed killers? Vultures, stakeholders, science and misperceptions, Lambertucci [/bib_ref]. However, the influence of social media as a potential threat (e.g., empowering existing conflicts) for vulture conservation has not yet been evaluated in depth. Here, we evaluate the influence of social media on people´s opinions towards wildlife by analyzing viewers' responses to content posted on a popular social medium (YouTube: http:// www. youtu be. com). We then propose ways to improve the presentation of information about threatened wildlife on social networks. We used vultures as a study case, as they are one of the most threatened species globally, and are currently being blamed for predation events in diverse areas of their distribution [bib_ref] Presumed killers? Vultures, stakeholders, science and misperceptions, Lambertucci [/bib_ref]. We evaluated the comments posted in response to YouTube videos showing vultures interacting with livestock. The videos were either positively or negatively framed (see details below). We also compared comments on negatively framed videos of mammalian predators interacting with livestock (e.g., felids, bears, foxes, etc.) with those on obligated scavenger vultures, to evaluate whether user perceptions vary between well-known mammalian predators and obligate scavenging birds blamed for predation events. In addition, we evaluated whether the video frame on vultures (negative-positive) could influence the number of views over time. We hypothesized that the way content was framed would influence viewer reactions, affecting their tolerance toward the species studied. We expected negatively framed videos on vultures to provoke non-empathetic reactions and negative perceptions, thus probably decreasing tolerance toward these birds. On the other hand, positively framed videos are likely to produce empathetic reactions toward vultures, and may therefore increase tolerance toward them. Moreover, although vultures rarely prey on livestock, we expected that comments with negative connotations posted in response to negatively framed videos of vultures (e.g., vultures involved in presumed predation events), and negatively framed videos of mammalian predators (e.g., bears or wolves hunting livestock) would be similar in frequency. Finally, we expected that negatively framed videos on vultures would have more views than positive ones. # Results Depending on the video framing (negative-positive) and the species involved (vultures-mammalian predators), the videos analyzed received different percentages of empathetic comments, consonant-dissonant comments and comments proposing lethal-nonlethal strategies to deal with the species involved-C, Supplementary [fig_ref] Table 2: Codification used for the comments posted in response to videos on vultures... [/fig_ref]. The percentage of empathetic comments posted in response to vultures tended to be higher in positively than in negatively framed videos. More empathetic comments were also posted for mammalian predators than for vultures when they were both shown in negatively framed videos. Dissonant comments were more frequently posted on negatively framed videos of vultures than positively framed videos of these birds and were also more common on negatively framed videos of vultures than negatively framed videos of mammalian predators. However, a considerable percentage of comments received (19%) were consonant with negatively framed videos of vultures where vultures were presented as predators. Finally, more comments proposing lethal strategies to deal with target species were posted on negatively framed videos of vultures and predators than positively framed videos on vultures. However, more of these comments were posted on negatively framed videos of vultures than negatively framed videos of mammalian predators. Our models showed that message framing (negative-positive), psychological distance, and species involved (vulture-mammalian predators) all influenced the probability of receiving comments proposing a lethal strategy and empathetic comments toward the species involved. Negatively framed videos on vultures had a higher probability of receiving comments proposing a lethal strategy than positively framed videos on these birds (Estimate (± SE) = − 3.53 ± 1.17; Z-value = − 3.00; P = 0.002). Moreover, negatively framed videos on vultures had a lower probability of receiving empathetic comments toward these birds than positively framed videos (Estimate (± SE) = 3.14 ± 0.59; Z-value = 5.28; P < 0.001). In negatively framed videos of vultures and mammalian predators, psychological distance influenced the probability of videos receiving comments proposing a lethal strategy to deal with these species. Videos with close psychological distance were more likely to generate comments proposing a lethal strategy for vultures and mammalian predators . Moreover, the species involved (vultures-mammalian predators) influenced the probability of generating comments showing empathy for wildlife. Videos on mammalian predators had a higher probability of generating empathetic comments than videos of vultures . Finally, the interaction between video frame (negative-positive) and the number of days a video was available online influenced the number of views of vulture videos (Estimate (± SE) = − 0.0008 ± 0.0000004; www.nature.com/scientificreports/ Z-value = − 1693.7; P < 0.001). Videos with a negative frame had more views over time than those with a positive frame [fig_ref] Figure 2: Predicted number of views of videos on vultures with negative or positive... [/fig_ref]. # Discussion We found that the way a video was framed influenced viewers' opinions of the species involved, and also the number of views. For vultures in particular, the title or video description and the way the images were presented influenced the tone of the comments posted. In addition, showing testimonies of people expressing their concerns www.nature.com/scientificreports/ about the damage their livestock stocks may suffer (close psychological distance) were more likely to receive comments with a negative connotation (e.g., expressions of desire to kill the animal involved) than videos showing only wildlife hurting livestock, with no personal testimonies shown (distant psychological distance) [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref]. Moreover, in negatively framed videos, comments on vulture species seemed to be more negative than comments on mammalian predators. Our results contribute to the discussion on communication strategies related to biodiversity conservation 20 , and are in agreement with other culturomic conservation studies suggesting that the characteristics of content posted on social media could have an influence on the tolerance of wildlife species [bib_ref] How do YouTube videos impact tolerance of wolves?, Casola [/bib_ref]. In addition, this culturomic approach can provide some useful tools for institutions and web platforms in terms of identification of problems and communication actions that can be taken with regard to conservation of threatened species as it does on many other social topics [bib_ref] Marketing challenges in the# MeToo era: Gaining business insights using an exploratory..., Reyes-Menendez [/bib_ref]. It is well known that communication approaches can influence people's attitudes, feelings and behavior 20 . Therefore, the way a message is presented is key to addressing conservation problems such as human-wildlife conflict [bib_ref] Five lessons to guide more effective biodiversity conservation message framing, Kusmanoff [/bib_ref]. In comparison with positively framed videos of vultures, we found that negatively framed videos of these birds had a lower percentage and probability of generating empathetic comments, but a higher percentage and probability of comments proposing a lethal strategy for dealing with them. This is of concern, considering that in most cases content posted on social media about vultures is based on anecdotic, biased information without scientific evidence to support it [bib_ref] Fake news and vultures, Margalida [/bib_ref] [bib_ref] Presumed killers? Vultures, stakeholders, science and misperceptions, Lambertucci [/bib_ref]. In fact, almost all the videos we analyzed that presented vultures as . Logistic regression mixed models to evaluate: (A) the influence of species and psychological distance on the probability of receiving comments proposing a lethal strategy to deal with species involved in negatively framed videos of vultures and mammalian predators, (B) the influence of species and psychological distance on the probability of receiving empathetic comments toward species involved in negatively framed videos of vultures and mammalian predators. Statistically significant results are indicated in bold. The estimates of the species and psychological distance variables correspond to "vultures" and "distant psychological distance". The best models containing both predictors (species and psychological distance) are reported in this table (for model comparisons see . www.nature.com/scientificreports/ . Importantly, some videos were later found to be fake, or misunderstandings, but were not removed from the platform or declared untrue. For instance, in a video we evaluated (number 11 in a farmer blames vultures for his livestock losses, even though feral dogs were actually responsible for this event (https:// agroi nform acion. com/ el-ataque-a-un-ganad ero-no-fue-culpa-de-los-buitr es-sino-de-canid os-los-buitr es-solo-llega ron-cuando-estab an-muert as/). Many more dissonant comments were received by negatively framed videos on vultures than by positively framed videos on these birds, or negatively framed videos on mammalian predators. This could be because information provided in videos where vultures are hurting livestock is dissonant for people who know that vultures are obligate scavenging birds and consume mainly dead animals. Despite this, a considerable percentage of comments received (19%) were consonant with negatively framed vulture videos, in which vultures are presented as predators, suggesting that many people agree that vultures are predators rather than obligate scavenger birds. Previous studies have shown that scavenger birds may hurt livestock only on rare occasions [bib_ref] The conflict between scavenging birds and farmers: Field observations do not support..., Ballejo [/bib_ref] [bib_ref] Education can improve the negative perception of a threatened long-lived scavenging bird,..., Arnulphi [/bib_ref] , but there are people who believe that these birds commonly attack and kill livestock [bib_ref] Education can improve the negative perception of a threatened long-lived scavenging bird,..., Arnulphi [/bib_ref] [bib_ref] A long and troublesome journey: People's perceptions and attitudes along the migratory..., Ballejo [/bib_ref]. In this context, a viral video showing these rare, isolated events supports or may intensify these misinformed beliefs, due to the effect of consonant cognition. In fact, this situation is observed in the tendency of Internet users to select information that is compatible with their previous beliefs (confirmation bias) [bib_ref] Debunking in a world of tribes, Zollo [/bib_ref] , and to form groups of like-minded people where points of view are polarized (echo chamber effect) [bib_ref] Users polarization on Facebook and Youtube, Bessi [/bib_ref]. Content showing exceptional behaviors that people may consider negative could have a strong influence on their perception, as our results show (negatively framed videos have more views over time than positive ones). Furthermore, negatively framed information has a stronger effect on final perception than positively framed information [bib_ref] Bad is stronger than good, Baumeister [/bib_ref]. Thus, a single negatively framed event about a threatened species, among others which are positively framed, will have longer lasting and more marked consequences, leading to misinformation about the species. We call for careful messaging when uploading videos on wildlife, particularly when a conflict exists and a threatened species is involved. Professional media play a primordial role and should be pioneers in this issue, reducing fake news and misinformation. The involvement of science journalists is essential, and they should be in close contact with researchers to corroborate facts and prevent the spread of misinformation. Our results suggest that psychological distance could influence responses to content posted on social media. The videos analyzed show how viewers´ comments are more negative toward vultures and mammalian predators when they see a person who is describing the loss of their livestock or claiming damages from the authorities for their losses (close psychological distance), than when they see only the interaction between these animals and livestock, without testimonies (distant psychological distance). The media probably find that these videos attract viewers, since they cause social alarm and curiosity [bib_ref] Fake news and vultures, Margalida [/bib_ref]. However, these messages may intensify conflicts that could impact negatively on threatened species such as vultures, increasing threats of action like poisoning or persecution [bib_ref] Fake news and vultures, Margalida [/bib_ref] [bib_ref] The perfect threat: Pesticides and vultures, Plaza [/bib_ref]. In this sense, the use of close psychological distance framing may be a good strategy for the implementation of conservation measures that promote positive perception of wildlife species. For instance, videos could be shown of people who have obtained benefits from wildlife (e.g., vultures cleaning dead animals produced by livestock production) instead of showing people who have lost livestock. Worryingly, and contrary to our predictions, empathetic comments were more likely to be posted in response to negatively framed videos about mammalian predators than negatively framed videos on vultures. Moreover, comments proposing a lethal strategy were more numerous for negatively framed videos of vultures than negatively framed videos of mammalian predators. This may be due to factors we cannot evaluate with our data, such as the fact that mammalian predators (e.g., wolves, bears or lions) are well known as potential predators of livestock, or because these species could be considered charismatic by viewers [bib_ref] Conservation inequality and the charismatic cat: Felis felicis, Macdonald [/bib_ref]. Predators such as large felines, bears and wolves could occasion livestock losses in different parts of the world 26,39-42 ; for example, losses generated by pumas (Puma concolor) in Argentina could reach 10% of the country's total sheep production [bib_ref] Characterization of puma-livestock conflicts in rangelands of central, Guerisoli [/bib_ref]. In contrast, vultures are rarely involved in predation events: predation events by obligate and facultative scavenger birds together represent only 0.1% of the total sheep production in northwestern Argentine Patagonia 43 . Despite this evidence, viewers' comments on vultures in response to videos uploaded onto the web generally seem to be more negative than for mammalian predators. This highlights the potential danger of showing obligate scavenging birds as predators on social media. While using posted data on social media can be an interesting approach to the evaluation of many issues, this kind of study may be subject to bias and limitations [bib_ref] Social media data for conservation science: A methodological overview, Toivonen [/bib_ref] [bib_ref] A decade of Google Trends-based Conservation culturomics research: A critical evaluation of..., Troumbis [/bib_ref]. For example, comments posted do not necessarily represent the thinking of all viewers; certain content may encourage individuals with a tendency to comment more often and in a particular direction [bib_ref] A decade of Google Trends-based Conservation culturomics research: A critical evaluation of..., Troumbis [/bib_ref]. While several videos showed vultures or mammalian carnivores interacting with diverse species, only a few were based on the target species or received the minimum number of comments allowing us to evaluate it. Culturomic studies are usually based on the Application Programming Interfaces (APIs) provided by platforms for analysis of the data, but these computational tools are limited for text analyses because of the complexity of the language itself; thus, ironic or referential comments, polysemous and synonymous words can lead to erroneous data interpretation [bib_ref] Social media data for conservation science: A methodological overview, Toivonen [/bib_ref] [bib_ref] A decade of Google Trends-based Conservation culturomics research: A critical evaluation of..., Troumbis [/bib_ref]. We solved this problem by analyzing and interpreting all comments individually. Therefore, while our study could have some limitations, it constitutes a first approach to understanding how people react to content about vultures presented on social media. Our results could thus be important in promoting conservation action, taking into account the influence social media may have on tolerance of the wildlife species included in this content, particularly in the case of threatened species. Further research can evaluate this issue on other social media such as Twitter, Instagram and Facebook, to increase sample size and identify the social characteristics (e.g., nationality, gender, age) that may influence people´s perception of wildlife. www.nature.com/scientificreports/ Vultures are among the most threatened avian groups in the world [bib_ref] The avian scavenger crisis: Looming extinctions, trophic cascades, and loss of critical..., Buechley [/bib_ref] [bib_ref] Dropping dead: Causes and consequences of vulture population declines worldwide, Ogada [/bib_ref] , and face not only threats such as poisoning with pesticides 37,48 and lead contamination [bib_ref] What do we know about lead contamination in wild vultures and condors?..., Plaza [/bib_ref] [bib_ref] Chronic lead exposure is epidemic in obligate scavenger populations in eastern North..., Behmke [/bib_ref] , but also persecution, trading of parts and food shortage [bib_ref] Dropping dead: Causes and consequences of vulture population declines worldwide, Ogada [/bib_ref]. This is a cause for concern given the important regulating ecosystem services that these species provide, removing organic material from the environment, which in turn could be important for public health and economy [bib_ref] Nature's clean-up crew: Quantifying ecosystem services offered by a migratory avian scavenger..., Grilli [/bib_ref] [bib_ref] Implications of bacterial, viral and mycotic microorganisms in vultures for wildlife conservation,..., Plaza [/bib_ref]. Most of the named threats are triggered by conflict between vultures and livestock producers [bib_ref] Presumed killers? Vultures, stakeholders, science and misperceptions, Lambertucci [/bib_ref] [bib_ref] Vultures attacking livestock: A problem of vulture behavioural change or farmers' perception, Duriez [/bib_ref] [bib_ref] The perfect threat: Pesticides and vultures, Plaza [/bib_ref]. Our results show that these conflicts could be exacerbated by social media, resulting in a growing problem that should be taken into account for these species. Greater caution is required in the presentation of information involving threatened species in conflict with humans, so as not to generate negative consequences. # Conclusions and recommendations Our results showed that the way video content is framed could influence tolerance toward wildlife species. Negatively framed content could affect the viewer's perception, encouraging aggressive reactions towards animals, some of which may be threatened species. Most of the videos analyzed show no clear evidence of livestock being attacked by scavenger birds; however, the video frame infers that the attack really happened, which makes the video potential fake news, and may generate negative consequences for species conservation. Social media are beginning to use more methods of control of fake news (https:// www. bbc. com/ news/ techn ology-47357 252). However, these control methods do not seem to be sufficiently effective, and it is necessary to extend the restrictions, without violating freedom of expression [bib_ref] The science of fake news, Lazer [/bib_ref]. YouTube allows their users to report videos that do not comply with a series of community norms (e.g., sexual, violent or terrorism content can be denounced). Unfortunately, these norms do not contemplate the effect wildlife videos can have on threatened species. We therefore propose the incorporation of a complaint category that enables social media users to report uploaded information or frames involving wildlife species that could negatively affect their conservation. Structural changes are also needed to prevent the exposure of individuals to fake news or misinformation. Platforms should provide consumers with some indication of the quality of the source; this could be incorporated into the algorithmic rankings of content. In addition, rather than favoring content similar to users' previous searches, platforms could present more diverse content, thus reducing the echo chamber effect [bib_ref] The science of fake news, Lazer [/bib_ref] and diluting the effect of negative frames [bib_ref] Bad is stronger than good, Baumeister [/bib_ref]. There is a need for greater care in the way messages about wildlife species are presented, especially messages about threatened species, since they could have negative consequences for their conservation. Taking regulatory action in the event of inaccurate messages about wildlife, without violating the freedom of expression, would help to improve conservation action for diverse wildlife species. # Methods Sample selection. We performed searches on YouTube (http:// www. youtu be. com) because it is one of the most popular social networks worldwide with millions of active users; it is specialized on video contents, where people can share, view, and comment all kind of videos. Moreover, this platform was used before to evaluate how social media consumption affects viewers' tolerance toward wildlife species e.g.14 . The searches were performed on the Google chrome incognito window during November 2020, filtering by relevance using the following key terms: "vultures", "carnivores" or "predators" combined with "livestock", "cow", and "lamb", both in English and Spanish. These keywords were selected because they encompass the groups of animals that are commonly involved in human-wildlife conflicts (either being attacked or blamed for attacking, producing human-wildlife conflict) [bib_ref] How do YouTube videos impact tolerance of wolves?, Casola [/bib_ref] [bib_ref] A long and troublesome journey: People's perceptions and attitudes along the migratory..., Ballejo [/bib_ref]. Moreover, these keywords are used widely to search for information about human-wildlife conflict on diverse Internet platforms, social media included [bib_ref] Puma-livestock conflicts in the Americas: A review of the evidence, Guerisoli [/bib_ref]. The aim of this search was to find videos showing obligate scavenging birds (vultures) or mammalian predators (e.g., wolves, bears, lions) from different parts of the world interacting with live or dead livestock (e.g., eating, flying around, hunting, injuring, walking around, etc.). Like other articles analyzing videos 7 , we evaluated up to 200 videos for each search we performed; these were generally the earliest videos we found related to the subject studied in this paper. We performed 18 searches combining the different keywords, resulting in 3600 videos. We excluded videos unrelated to the issue studied, videos with fewer than 10 comments that could be analyzed according to our aim, and videos not showing any species of interest or not contributing to the aims of this study. We obtained a subset of 56 videos involving vultures and mammalian predators. For each video, we recorded the following data: the title, URL, number of comments, number of views and days of permanence online since publication. We classified each video as positively or negatively framed by analyzing the title, description and content of the videos. We focused on detecting words, sentences or images with connotations denoting the message frame, as done in other studies (e.g., . Negatively framed videos of vultures and mammalian predators had titles or descriptions that included words such as "predation" or "killing" [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref]. In this category we also placed videos suggesting that vultures eat live animals (e.g., calves, lambs) instead of carrion, or videos edited to emphasize vultures injuring or intimidating livestock when still alive, in some cases without considering the entire story (e.g., not providing information on the previous events, the place, or the health status of the animal affected). Positively framed videos did not use words such as "predation" or "killing" in their titles or descriptions; they either referred to vultures eating carrion and not live animals, or were edited to emphasize the positive role of these birds in the ecosystem (e.g., cleaning carcasses) [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref]. In addition, we classified videos according to their psychological distance framing, as close or distant [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref] [bib_ref] The future is now: Reducing psychological distance to increase public engagement with..., Jones [/bib_ref]. Close psychological distance videos included contents showing farmers' testimonies about wildlife interaction with their livestock (e.g., farmers describing vultures or mammalian predators killing their livestock; [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref]. Distant psychological videos showed wildlife interacting with livestock but did not include farmer testimonies [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref]. We excluded videos without a clear frame (not classifiable in the categories described above). www.nature.com/scientificreports/ Comment codification. We evaluated user-generated content by using natural language processing approaches [bib_ref] Digital data sources and methods for conservation culturomics, Correia [/bib_ref] [bib_ref] Marketing challenges in the# MeToo era: Gaining business insights using an exploratory..., Reyes-Menendez [/bib_ref]. In this way we extracted quantitative information about the sentiments expressed in comment posted on YouTube videos. Then, we analyzed the tone of comments based on the words (particularly the adjectives) or written expressions (e.g., kill them all with machine guns, that poor calf) posted by users [fig_ref] Table 2: Codification used for the comments posted in response to videos on vultures... [/fig_ref]. First, one author (FB) codified the comments posted on each selected video, and this was then repeated by the other authors to verify the result; any dissidence was discussed. There were more than 95% of coincidences between authors, and the few dissidence were decided easily since at least 2 of the 3 authors supported one of the options. We evaluated up to the first 500 comments in each video, ordering them from the most recent to the oldest. Comments not related to the issue under study (e.g., political opinions, racist comments, etc.) and responses to the main comments were excluded. To standardize the data, we included only one comment from each user. Comments were grouped into the following categories: (1) empathetic comments, with the following nonexclusive subcategories: (a) toward wildlife (vulture or predators), (b) toward the livestock attacked or consumed, (c) neutral (comments non-classifiable in any previous category); [bib_ref] YouTube" telling or selling you something? Tobacco content on the YouTube video-sharing..., Freeman [/bib_ref] comments consonant with or dissonant to the opinions or images presented in the videos, with the following non-exclusive subcategories: (a) consonant comments: comments posted by people who believed that the videos were showing the natural behavior of the species involved, (b) dissonant comments: comments posted by people who believed that the video was showing uncommon or exceptional behavior for the species, (c) neutral (non-classifiable under any of the previous categories); [bib_ref] The power of "like": How brands reach (and influence) fans through social-media..., Lipsman [/bib_ref] comments proposing strategies to deal with species involved (vultures-mammalian predators), with the following non-exclusive subcategories: (a) lethal strategies (e.g., poisoning or shooting), (b) non-lethal strategies (e.g., livestock guardian dogs, surveillance), (c) neutral (non-classifiable under any of the previous categories) [fig_ref] Table 2: Codification used for the comments posted in response to videos on vultures... [/fig_ref]. We downloaded the comments of each video from the YouTube platform, and considered only videos with at least 10 comments that can be considered and analyzed under our criteria. After all exclusion criteria had been met, we obtained a definitive sample of 25 videos (11 negatively framed on vultures, 7 positively framed on vultures, and 7 negatively framed on predators). These videos received 1179 classifiable comments (494 belonging to negative frames on vultures, 132 comments belonging to positive frames on vultures, and 553 comments belonging to negative frames on predators) (Supplementary Tables S1, S2). # Statistical analysis. We first estimated the percentage of comments made in response to the videos analyzed, classifying them according to (i) comment category (empathetic comment, consonant-dissonant comment and strategy proposed to deal with species involved), (ii) video frame (negative-positive) and (iii) species involved (vultures-mammalian predators) [fig_ref] Figure 3: Scheme of the classification of videos according to frame [/fig_ref] ; [fig_ref] Table 2: Codification used for the comments posted in response to videos on vultures... [/fig_ref]. A set of four Generalized Linear Mixed Models (binomial distribution with Logit function) were then performed, with the video ID as a random effect. The first two models were used to evaluate a subset of videos, positively and negatively framed, showing vultures interacting with livestock. They were assessed for: (i) the influence of the predictor video framing (negative or positive) on the probability of a video receiving comments proposing a lethal strategy (e.g., shooting); (ii) the influence of the predictor video framing (negative or positive) on the probability of videos receiving empathetic comments toward vultures. We then performed two models with a subset of negatively framed videos of vultures and mammalian predators interacting with livestock, to assess the following: (i) the influence of predictor species (vulture or mammalian predators) and psychological distance (close-distant) on the probability of videos receiving comments proposing lethal strategies to deal with these species; (ii) the influence of predictor species and psychological distance on the probability of videos receiving empathetic comments toward the species involved. We performed model comparison based on AIC criteria, including both predictors (species and psychological distance) and their interactions . For all these models we assigned (1) to comments proposing a lethal strategy toward vultures or mammalian predators and for empathetic comments toward these species, and (0) for comments proposing non-lethal strategies toward vultures or mammalian predators and for empathetic comments toward the species consumed (e.g., sheep or cows). www.nature.com/scientificreports/ Finally, a Generalized Linear Model with a Poisson distribution (Log function) was used to evaluate whether the framing of the video, number of days available online, or interactions between these two factors influenced the number of views of videos on vultures (negative and positive frame). All statistical analyses were performed with R core team (2015), and we considered P values < 0.05 as significant. The glm and lme4 packages 57 were used to perform the Generalized Lineal Models and the Generalized Linear Mixed Models. www.nature.com/scientificreports/ [fig] Figure 1: (A) Percentage of empathetic comments toward vultures or mammalian predators and species consumed according to video framing. (B) Percentage of consonant-dissonant comments in videos according to framing of video and species involved (vultures-mammalian predators). (C) Percentage of comments proposing lethal and non-lethal strategies for vultures or mammalian predators, according to framing of video. Scientific Reports | (2021) 11:13512 | https://doi.org/10.1038/s41598-021-92815-7 [/fig] [fig] Figure 2: Predicted number of views of videos on vultures with negative or positive framing (seeFig. 3) in relation to the days since they were uploaded onto YouTube (based on a GLM with Poisson distribution, see "Methods"). [/fig] [fig] Figure 3: Scheme of the classification of videos according to frame. Negatively framed videos (1), including: (a) videos with a negative emphasis in titles or descriptions, (b) videos with a negative emphasis in their images. Positively framed videos (2), including (c) videos with a positive emphasis in titles or descriptions, (d) videos with a positive emphasis in their image. Videos with psychological distance frame (3), including (e) close psychological distance videos in which there are farmers' testimonies about wildlife animals killing their livestock, (f) distant psychological distance showing wildlife interacting with livestock but no human testimonies or presence (more details in the main text). [/fig] [table] Table 2: Codification used for the comments posted in response to videos on vultures and predators uploaded onto YouTube. Main awareness and affection for the animal consumed or injured (b) Main awareness and affection for vulture species or mammalian predator (c) Non-classifiable in this category (a) Poor sheep (b) Vultures are just doing their job (c) What on Earth?! Dissonant-consonant comments (a) Consonant: video shows the habitual behavior of a species (b) Dissonant: video shows exceptional behavior of a species (c) Neutral (a) People who believe the video shows the habitual behavior of a species (b) People who believe the video does not show the habitual behavior of a species, or shows exceptional behavior of a species (c) Non-classifiable in this category (a) They have done that since the planet was born! (b) These animals don't usually hurt anything alive (c) Our government in action. Oh, brother https://doi.org/10.1038/s41598-021-92815-7 [/table]

Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton ## Supporting information Table S1 Identification of chitinase genes in G. raimondii and their phylogenetic relationship in G. arboreum, G. hirsutum and G. ## Barbadense. Table S2 Information of PCR primers used in this study. . Silencing of the endogenous Cloroplastos alterados gene (GbCLA1) in cotton through VIGS. The leaf bleaching phenotype was observed two weeks after infiltration in TRV: GbCLA1 plants. . Infection of cotton seedlings with V. dahliae. Two cotton cultivars, G. barbadense cv. Hai7124 and G. hirsutum cv. Junmian 1, were used as controls that were resistant and susceptible to V. dahliae, respectively. The seedlings were grown in the same environment and inoculated with V991. We identified the phenotype 20d and 25d after inoculation. In Junmian 1, all the true leaves were defoliated 25 days after inoculation. [fig_ref] Figure S3: Real-time qRT-PCR analysis of the chitinase genes in response to Verticillium dahlia... [/fig_ref]. Real-time qRT-PCR analysis of the chitinase genes in response to Verticillium dahlia in G. hirsutum cv. Junmian 1. qRT-PCR expression analysis of chitinase genes to screen for differentially expressed genes after inoculation with V. dahliae strain V991. The error bars were calculated based on three biological replicates using standard deviation. "*": significant difference at P < 0.05. . Silencing of the endogenous Cloroplastos alterados gene (GbCLA1) in cotton through VIGS. The leaf bleaching phenotype was observed two weeks after infiltration in TRV: GbCLA1 plants. . Infection of cotton seedlings with V. dahliae. Two cotton cultivars, G. barbadense cv. Hai7124 and G. hirsutum cv. Junmian 1, were used as controls that were resistant and susceptible to V. dahliae, respectively. The seedlings were grown in the same environment and inoculated with V991. We identified the phenotype 20d and 25d after inoculation. In Junmian 1, all the true leaves were defoliated 25 days after inoculation. bars were calculated based on three biological replicates using standard deviation. "*": significant difference at P < 0.05. [fig] Figure S3: Real-time qRT-PCR analysis of the chitinase genes in response to Verticillium dahlia in G. hirsutum cv. Junmian 1. qRT-PCR expression analysis of chitinase genes to screen for differentially expressed genes after inoculation with V. dahliae strain V991. The error [/fig] [table] Table S2: Information of PCR primers used in this study [/table]

Perception of Upright: Multisensory Convergence and the Role of Temporo-Parietal Cortex We inherently maintain a stable perception of the world despite frequent changes in the head, eye, and body positions. Such "orientation constancy" is a prerequisite for coherent spatial perception and sensorimotor planning. As a multimodal sensory reference, perception of upright represents neural processes that subserve orientation constancy through integration of sensory information encoding the eye, head, and body positions. Although perception of upright is distinct from perception of body orientation, they share similar neural substrates within the cerebral cortical networks involved in perception of spatial orientation. These cortical networks, mainly within the temporo-parietal junction, are crucial for multisensory processing and integration that generate sensory reference frames for coherent perception of self-position and extrapersonal space transformations. In this review, we focus on these neural mechanisms and discuss (i) neurobehavioral aspects of orientation constancy, (ii) sensory models that address the neurophysiology underlying perception of upright, and (iii) the current evidence for the role of cerebral cortex in perception of upright and orientation constancy, including findings from the neurological disorders that affect cortical function.iNTRODUCTiON Spatial orientation refers to the perceptual awareness of the body position relative to the environment. While oriented to the surroundings, we maintain a stable perception of the world in upright orientation despite frequent changes in the eye, head, and body positions. Such "orientation constancy" is a key functional aspect of our spatial perception, and if disrupted the consequences can be quite debilitating due to ensuing dizziness, disorientation, and loss of balance. These symptoms are often triggered by motion or changes in the head or body positions, e.g., as in patients with vestibular dysfunction. Our perception of spatial orientation is possible because the position of the body is linked to the external environment through processing and integration of visual, vestibular, and proprioceptive information. In this process, the compensatory movement of the eyes through the vestibulo-ocular reflex is vital to maintain visual stability with changes in the head position. In frontal-eyed animals, in addition to the horizontal and vertical eye movements, lateral head tilts (i.e., with respect to gravity) lead to changes in the torsional eye position in the opposite direction of the head tilt. In humans, this ocular counter-roll (OCR) is a constrained, phylogenetically old vestibular reflex and does not match the magnitude of the head tilt (1). Such "visual-vestibular" mismatch, FigURe 2 | Subjective visual vertical (SVV) measurement with the line stimulus (solid orange) presented at a random orientation in each trial (A). In the forced-choice paradigm, the task is to report whether the line is tilted to the right or to the left of the perceived upright orientation (dashed orange) (B). SVV is then determined by fitting a psychometric curve to the responses from all trials and is calculated as the value on the curve at which the probability of left or right responses is 50% (point of subjective equality). In the active-adjustment paradigm, the line stimulus (solid orange) is adjusted (direction shown by arrow) to the perceived upright orientation (dashed orange) (C). In this paradigm, SVV is calculated as the average value from all trials. The true vertical is shown by the dashed white line (B,C). 3 Kheradmand and Winnick Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org October 2017 | Volume 8 | Article 552FigURe 3 | Systematic errors of subjective visual vertical (SVV): healthy individuals typically have SVV errors within 2° of earth vertical in upright position. At large tilt angles (usually greater than 60°), SVV errors are deviated toward the tilt direction, which reflect "underestimation" of upright orientation (known as the Aubert or A-effect). At smaller tilt angles (usually less than 60°), however, SVV errors are often opposite to the tilt direction, which reflect "overestimation" of upright orientation (known as the Entgegengesetzt or E-effect).4 Kheradmand and Winnick Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org October 2017 | Volume 8 | Article 552FigURe 4 | Schematic presentation of Mittelstaedt's idiotropic vector model: visual line orientation on the retina and head tilt are the two sensory inputs in this model. The visual signal is accurate, but the head tilt signal ( ) ρ  shows increasing errors with head tilt (a range of ±90° tilt is shown in black graphs). As part of the central neural processing, vectorial summation of the head tilt signal ( ) ρ  and the head-fixed idiotropic vector (MZ) yields the compensatory tilt signal (β). The compensatory tilt signal and the visual signal are then added to obtain an internal estimate of the upright orientation [i.e., subjective visual vertical (SVV)].5 Kheradmand and Winnick Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org A B C FigURe 1 | Perception of upright and sensory reference frames: The head, eye, and the world reference frames are all aligned in upright position along the gravitational vertical (A), but when the head is tilted, the ocular counter-roll only partially compensates for the amount of head tilt (gain about 10-25%), which results in a separation of the sensory reference frames that encode head-in-space and eye/retina-in-head orientations (B). Despite these differences, visual perception remains in upright orientation (C). Therefore, the brain-like any other sensorimotor system-must be able to integrate sensory inputs into a common reference frame to maintain a coherent perception of upright. although sounds counter-productive, may actually represent an evolutionary advantage, as it can provide the brain with pertinent cues to quickly deconstruct perceived tilts into changes in the body position and the visual world, thus facilitating interactions with the surrounding environment. In this scheme, however, to achieve orientation constancy, the brain must be able to generate a common reference frame based on the sensory inputs that are inevitably encoded in different reference frames. Let us examine a simple lateral head tilt more closely. In the upright position-where the vertical meridians of the eyes, head, body, and the visual world are all aligned with the gravitational vertical-maintaining upright perception is not challenging for the brain. However, as mentioned earlier, when the head is tilted and as the brain senses changes in the head position relative to gravity, OCR will only partially compensate for the amount of head tilt, typically with a low gain of about 10-25% in humans. Therefore, as a result of head tilt, the reference frames for the head, eye, and the visual world are no longer aligned along the gravitational vertical, and images become tilted on the retina . Despite separation of these individual sensory reference frames, our visual perception remains in upright orientation within a common reference frame. This perceptual constancy in upright orientation can be effectively studied by removing orienting visual cues, in which case the brain has to rely on information about the head and body positions in space and the eye position in the orbit to determine the orientation of external stimuli. A similar approach has been the basis of psychophysical experiments dating back to 1860. Around that time, Hermann Aubert, an expert in optics, used afterimages to investigate perception of vertical and horizontal line orientations in light and darkness. Using afterimages of a bright line, Aubert tilted his head with eyes closed until the afterimage was earth-horizontal. Upon opening the eyes, he found that the afterimage would deviate toward the side of the head tilt (2). George Elias Müller then investigated a range of smaller head tilts and found that the line would deviate away from the side of the head tilt (3). Müller also put forth theories to describe these perceptual errors, considering sensory contributions from the otoliths, semicircular canals, and proprioception (3). Later on, mathematical models were used to account for these findings. One of the initial quantitative models was put forth by , in which he proposed that the brain must generate an internal common reference to "stabilize man's confidence in the stability of his world" (4). From this perspective, he eloquently posited about discrepancies between the elements of our perception and the real world: … in this facet of his subjectivity, man appears as a creature, whose mind underrates the humble services of his bodily feelings while naively taking at face value what believes to see, unaware of being deceived, as it were, by the workings of a machinery which toils in the interest of survival but not in the service of truth… (4). In recent decades, contributions of various sensory modalities to perception of upright have been studied extensively. However, currently, less is known about the neural structures and functions involved in orientation constancy. In this review, we first focus on neurobehavioral aspects of orientation constancy and describe sensory models that address the neurophysiology underlying upright perception. We then review the current evidence for the role of cerebral cortex in perception of upright and orientation constancy. Finally, we outline findings from neurological disorders that impact cortical mechanisms underlying perception of upright. ## Neurobehavioral aspects of upright perception ## Measurement paradigms Upright perception is typically studied by means of a psychophysical task known as the subjective visual vertical (SVV). In this task, a visual line is used to report perceived earth-vertical orientation in the absence of visual cues. Various methods have been described for SVV measurement. Some paradigms use active adjustment of the visual line stimulus, and others are based on a forced-choice task, where in each trial a visual line orientation is reported with respect to the perceived upright orientation . Although the visual exposure in SVV paradigms is limited to a line stimulus without any other orienting cues, the line itself may affect SVV responses, especially during active adjustments (5-7). For example, the initial orientation of the line stimulus can bias upright perception in the direction of the starting line orientation, and in the opposite direction of the line movement (7-12). This bias, however, may reverse and occur as a "hysteresis" effect in the direction of the line movement when the line is presented in sequential angles in a forced-choice paradigm (6). Also, with active adjustment of the line, the upright estimate may gradually drift as a result of trial-to-trial dependency of upright adjustments and inter-correlation among consecutive SVV responses (13). In addition, the torsional position of the eyes can change in the direction of the visual line rotation, and such "torsional entrainment" may introduce biases when SVV is measured using the line rotation (14). Considering all these sources of error, a forced-choice task with a random line orientation in each trial would be the least biased method for SVV measurement, as it would remove the effects of line movement on SVV responses (15). The length of the line stimulus can also influence SVV responses, resulting in biases in the direction of the body tilt with longer lines and in the opposite direction of the body tilt with shorter lines (16, 17). Another factor that can affect magnitude of SVV errors is the viewing distance from the visual line stimulus (18). This effect has been attributed to ocular torsion induced by changes in the vergence angle of the eyes (i.e., cycloversion). The viewing eye (i.e., monocular or binocular viewing), on the other hand, does not significantly affect SVV errors, neither in upright position nor during head tilt (6, 18). ## Systematic errors Subjective visual vertical errors reflect challenges for the brain in maintaining a common reference frame based on sensory information encoding eye, head, and body positions. In upright position, SVV errors typically remain within 2° of earth vertical (4, 19-21). However, with lateral head or whole body tilts, there are systematic errors in the perceived upright orientation which do not correspond with the errors in perception of body tilt (4, 19, 22-25). Such inherent dissociation between the perceptions of body tilt and upright orientation is also seen with active body tilts (as opposed to passive tilts), even when the brain has access to additional proprioceptive cues or efference copy signals to encode the veridical position of the body (23). In general, SVV errors are biased toward the direction of the body position at tilt angles greater than 60°. This finding, which reflects underestimation of upright orientation, is known as the Aubert or A-effect (2, 4, 19, 20). At smaller tilt angles (e.g., less than 60°), however, SVV errors are often biased in the opposite direction of the body position. This finding, which reflects overestimation of upright orientation, is known as the Müller or E-effect (E for "Entgegengesetzt, " German for opposite) (3, 19-21). The peak underestimation error of the A-effect is usually around 130°, and beyond this tilt angle the E-effect usually occurs again which is attributed to switching of the internal upright reference frame from the head to the feet (19, 21, 24, 26-28). Overall, the E-effect presents less consistently and less often compared with the A-effect (21, 24, 29). The variability of SVV responses also increases with the body tilts up to 120-150°, and then decreases again with the tilt angles approaching 180° (21, 26, 29-34). This pattern of SVV variability has been attributed to a tilt-dependent noise in the otolith and proprioceptive inputs (4, 21). # Other measurement methods Some studies have used subjective visual horizontal (SVH) instead of visual vertical measurements. The results, however, show that SVV and SVH are not invariably orthogonal to one another, especially at larger tilt angles [bib_ref] How stable is perceived direction of gravity over extended periods in darkness?, Tarnutzer [/bib_ref]. In other words, errors of vertical and horizontal perception may not match at the same body tilt position and while SVV errors tend be larger in the direction of the tilt (i.e., SVV errors show larger A-effects), SVH errors tend to be larger in the opposite direction of the tilt (i.e., SVH errors show larger E-effects) (18). SVV and SVH errors have also been studied in the pitch plane, and-similar to the systematic errors in the roll plane-they reflect overestimations in the opposite direction of small pitch angles, and underestimations in the direction of large pitch angles (4, [bib_ref] Influence of whole-body pitch tilt and kinesthetic cues on the perceived gravity-referenced..., Bringoux [/bib_ref] [bib_ref] Difference in the perception of the horizon during true and simulated tilt..., Carriot [/bib_ref] [bib_ref] Perception of the vertical with body tilt in the median plane, Ebenholtz [/bib_ref]. Another common method for measuring upright perception is with a haptic stimulus. Similar to SVV, haptic upright responses become less precise at large body tilt angles, but in some individuals they can be more accurate compared with the visual vertical responses [bib_ref] The effect of body tilt on tactual-kinesthetic perception of verticality, Bauermeister [/bib_ref] [bib_ref] Reference frames and haptic perception of orientation: body and head tilt effects..., Luyat [/bib_ref] [bib_ref] Precision and accuracy of the subjective haptic vertical in the roll plane, Schuler [/bib_ref] [bib_ref] Egocentric and allocentric alignment tasks are affected by otolith input, Tarnutzer [/bib_ref]. Also, haptic measurements tend to produce larger E-effects at smaller tilt angles (i.e., less than 60°) and may become more accurate in the supine position compared with the upright position [bib_ref] Localization of the subjective vertical during roll, pitch, and recumbent yaw body..., Bortolami [/bib_ref] [bib_ref] The role of neck afferents in subjective orientation in the visual and..., Guerraz [/bib_ref] [bib_ref] Kinesthetic estimation of the main orientations from the upright and supine positions, Lejeune [/bib_ref]. More importantly, the perceived haptic or postural upright can be dissociated from the visual perception of upright [bib_ref] Dissociation between the perception of body verticality and the visual vertical in..., Anastasopoulos [/bib_ref] [bib_ref] The subjective visual vertical and the subjective haptic vertical access different gravity..., Fraser [/bib_ref]. For example, while patients with unilateral vestibular loss showed significant SVV errors, their postural vertical adjustments were not different from the healthy controls [bib_ref] Dissociation between the perception of body verticality and the visual vertical in..., Anastasopoulos [/bib_ref]. The disparity in the SVV and postural vertical responses in this study suggests different weights of sensory contributions to perception of upright depending on the method of measurement (e.g., haptic versus visual tasks) [bib_ref] The subjective visual vertical and the subjective haptic vertical access different gravity..., Fraser [/bib_ref] [bib_ref] The haptic perception of spatial orientations, Gentaz [/bib_ref]. However, only few patients were included here, and the postural vertical was measured while sitting in a motor-driven chair and adjusting its orientation to the perceived upright position. In keeping with such distinct sensory contributions, haptic upright responses, in contrast to SVV, were more biased by the whole body tilt than just the head-on-body tilt in a group of healthy individuals [bib_ref] The subjective visual vertical and the subjective haptic vertical access different gravity..., Fraser [/bib_ref]. ## Spatial perception models In recent years, several studies have addressed neural mechanisms underlying perception of upright and the systematic errors with changes in body tilt orientation. Mittelstaedt first put forward a model in 1983 that could account for the A-effect (4). He proposed that the brain implements a computational strategy based on an internal bias signal to correct for the noisy inputs from the otolith organs . This internal signal, referred to as "the idiotropic vector, " is a constant, body-fixed vector that is added to the estimated direction of gravity from the otolith inputs to determine upright orientation. At large body tilts, the effect of idiotropic vector results in a bias in upright estimates toward the body axis and thus the A-effect. According to this model, the computation of upright orientation does not influence the estimate of body tilt. Therefore, the idiotropic vector could be viewed as a computational strategy to reduce distortions in upright perception for commonly encountered small body tilts, at the expense of large A-effects for rarely encountered large body tilts. The effect of the idiotropic vector was later described within a Bayesian framework and was equated to the role of the Bayesian "prior" for processing noisy sensory signals (21, 29, 51-54). In this Bayesian spatial perception model, the upright estimate is determined by a weighted average of the existing knowledge of tilt position (i.e., the prior) and the likelihood of change in tilt position based on noisy sensory information . Since we spend most of our time in upright position, the prior for tilt position is a Gaussian distribution centered at 0° (i.e., upright position). Thus, the effect of prior could bias upright estimates and result in underestimation of true vertical at large tilt angles (i.e., the A-effect). According to the Bayesian model, the head estimate can be determined in the following relation [bib_ref] Accuracy-precision tradeoff in visual orientation constancy, Vrijer [/bib_ref] : [formula] H H S H H H S Sp Sp S = + ⋅ σ σ σ 2 2 2 . [/formula] (1) In Eq. 1,  HS represents the final head-in-space estimate by the brain (i.e., "the posterior" in Bayesian terms), Ĥ S the head orientation in space as measured by the head-in-space sensors, and HS the actual head-in-space position (i.e., measured head position with respect to the direction of gravity). Among the sensory signals in the model, the head-in-space input ( ) H S  is noisy (with a variance of σ HS  2 ), and thus the prior (with a small variance of σ HSp 2 ) is taken into account to estimate the final head position proprioceptors), and the relative position of the head and body (neck proprioceptors) (54) [fig_ref] FigURe 6 |: Schematic representation of the sensory integration model [/fig_ref]. In this model, based on the optimal observer theory, the body orientation in space can be determined either "directly" using proprioceptive information from the trunk graviceptors [bib_ref] Somatic versus vestibular gravity reception in man, Mittelstaedt [/bib_ref] [bib_ref] Interaction of eye-, head-, and trunk-bound information in spatial perception and control, Mittelstaedt [/bib_ref] [bib_ref] Origin and processing of postural information, Mittelstaedt [/bib_ref] or "indirectly" from subtracting the signals encoding head and neck positions. Likewise, the estimate of head-in-space orientation can be obtained directly from the head position or indirectly from the body and neck proprioceptive signals. Accordingly, the optimal estimate of upright orientation is determined by integrating (1) direct information from the head position sensors (i.e., otoliths), (2) indirect information from the body and neck proprioceptors, and (3) prior information about the head and body orientations in space. The indirect sensory signals require reference frame transformation before integration with other sensory information. Thus, altogether, the final error in upright perception is calculated based on the weights of the direct and indirect information and is given by the following relation: In this Eq. 5, WHD represents the weight of direct sensory information, and WHI represents the weight of indirect sensory information. Here, the weight of the prior (WHP) works through the weights of direct and indirect sensory information, as WHD + WHI + WHP = 1. Therefore, the narrower the prior distribution, the larger its relative weight compared with the weights of direct and indirect sensory information [for more details, see Ref. [bib_ref] Multisensory processing in spatial orientation: an inverse probabilistic approach, Clemens [/bib_ref] ]. In this scheme, the effect of the prior could be seen as the factor that reduces the variance of upright estimates, however, with an accuracy-precision trade-off especially at large tilt angles. Tarnutzer et al. have proposed a Bayesian model to account for the lower SVV precision at larger head tilts based on variability in the otolith inputs. In this model, the preferred directions of the otolith afferents represent different sensitivities to changes in the angle of the head tilt. Thus, an overall likelihood of head position estimate is obtained by combining the probability distributions from individual otolith afferents. In this scheme, the effectiveness of the otolith estimator-reflected by the width of the likelihood distribution-decreases at larger head tilt angles, and it is combined with the prior knowledge of the head orientation to derive the SVV estimate (21). # Multisensory contributions Various studies have addressed contributions of the head, neck, and trunk sensory signals to perception of upright. The findings from these studies indicate that the SVV errors are primarily processed in a head-in-space reference frame (30, [bib_ref] Influence of visual, vestibular, cervical, and somatosensory tilt information on ocular rotation..., De Graaf [/bib_ref] [bib_ref] Head orientation involvement in assessment of the subjective vertical during whole body..., Guerraz [/bib_ref] [bib_ref] Roll-dependent modulation of the subjective visual vertical: contributions of head-and trunk-based signals, Tarnutzer [/bib_ref] [bib_ref] Visual orientation during and after lateral head, body, and trunk tilt, Wade [/bib_ref] [bib_ref] Influence of head orientation on visually induced pitch and roll sensation, Young [/bib_ref]. On the other hand, perception of body orientation is largely modulated by the proprioceptive inputs encoding trunk position, with errors that are more accurate but less precise than SVV responses [bib_ref] Multisensory processing in spatial orientation: an inverse probabilistic approach, Clemens [/bib_ref] [bib_ref] Somatic versus vestibular gravity reception in man, Mittelstaedt [/bib_ref] [bib_ref] The perception of body verticality (subjective postural vertical) in peripheral and central..., Bisdorff [/bib_ref] [bib_ref] The role of the otoliths in perception of the vertical and in..., Mittelstaedt [/bib_ref] [bib_ref] Visually guided adjustments of body posture in the roll plane, Tarnutzer [/bib_ref]. In line with these findings, and consistent with distinct sensory contributions to perception of body orientation from perception of upright, SVV deviations induced by galvanic vestibular stimulation (GVS) were dissociated from the errors in perception of body orientation [bib_ref] Dissociation between subjective vertical and subjective body orientation elicited by galvanic vestibular..., Mars [/bib_ref]. In accordance with the multimodal sensory contributions to perception of upright, alterations in the neck, trunk, and interoceptive inputs have modulating effects on perceptual upright responses (30, 61, 67-75). For example, vibration of the neck muscles can shift SVV errors in the opposite direction of the head tilt and increase the E-effect [bib_ref] Neck muscle vibration alters visually perceived roll in normals, Mckenna [/bib_ref] [bib_ref] Neck muscle vibration alters visually-perceived roll after unilateral vestibular loss, Betts [/bib_ref] [bib_ref] Effects of neck muscle vibration on subjective visual vertical: comparative analysis with..., Kawase [/bib_ref]. Thus, the brain must be able to determine upright orientation either directly, by accessing the estimate of head-in-space orientation through the sensory inputs encoding head position (e.g., otolith signals), or indirectly, through the sensory inputs encoding neck and trunk positions [bib_ref] Multisensory processing in spatial orientation: an inverse probabilistic approach, Clemens [/bib_ref]. In this context, the sensory contributions to upright perception are modulated by the body tilt position, with likely a greater weight of the head position signals (e.g., from the otoliths) around the upright position, and a substantial weight of the trunk proprioceptive signals at larger tilt angles (30, 31). Such distinct patterns of sensory contributions to perception of upright are supported by the findings in patients with vestibular and proprioceptive loss (25, [bib_ref] Sensory substitution in bilateral vestibular a-reflexic patients, Alberts [/bib_ref] [bib_ref] Perception of slow pitch and roll body tilts in bilateral labyrinthinedefective subjects, Bringoux [/bib_ref] [bib_ref] Visually and posturally mediated tilt illusion in Parkinson's disease and in labyrinthine..., Bronstein [/bib_ref] [bib_ref] Linear acceleration perception in the roll plane before and after unilateral vestibular..., Dai [/bib_ref] [bib_ref] The effect of water immersion on perception of the oculogravic illusion in..., Graybiel [/bib_ref] [bib_ref] Changes of visual vertical perception: a long-term sign of unilateral and bilateral..., Lopez [/bib_ref] [bib_ref] How body position changes visual vertical perception after unilateral vestibular loss, Lopez [/bib_ref] [bib_ref] The peripheral nervous system and the perception of verticality, Mazibrada [/bib_ref] [bib_ref] Spatial orientation in patients with chronic unilateral vestibular hypofunction is ipsilesionally distorted, Müller [/bib_ref] [bib_ref] Influence of body laterality on recovery from subjective visual vertical tilt after..., Toupet [/bib_ref]. Patients with vestibular loss tend to have no E-effect at small tilt angles and more pronounced A-effects at larger tilt angles, consistent with reduced weight of head position signals and consequently relative underestimation of upright orientation (25, . Patients with proprioceptive loss, on the other hand, have decreased A-effect consistent with reduced weight of body proprioception, and consequently relative overestimation of upright orientation (25, [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref]. Perception of upright has been also studied with respect to changes in body position or posture [bib_ref] A Bayesian model of the disambiguation of gravitoinertial force by visual cues, Macneilage [/bib_ref] [bib_ref] How body position changes visual vertical perception after unilateral vestibular loss, Lopez [/bib_ref] [bib_ref] Gravity and observer's body orientation influence the visual perception of human body..., Lopez [/bib_ref]. Healthy participants lying supine had accurate SVV responses, but there were large errors in patients with vestibular loss in the supine position compared with the sitting and standing positions [bib_ref] How body position changes visual vertical perception after unilateral vestibular loss, Lopez [/bib_ref] [bib_ref] Subjective vertical and postural activity, Luyat [/bib_ref]. In general, SVV responses tend to be more accurate while maintaining precarious postures, where there is a risk of falling and thus a higher demand for balancing activity (e.g., standing on a beam) [bib_ref] We are most aware of our place in the world when about..., Bray [/bib_ref] [bib_ref] Interaction of posture and conscious perception of gravitational vertical and surface horizontal, Wright [/bib_ref]. Such findings underscore the ecological aspect of upright perception in which according to the task at hand the internal estimate of upright is modulated by available sensory cues. Systematic errors of upright perception also occur with body rotation in the roll plane, and-similar to the static rolltilts-these dynamic errors are dissociated from the perception of the body orientation (27, [bib_ref] Measuring dynamics of the subjective vertical and tilt using a joystick, Correia Grácio [/bib_ref] [bib_ref] Reciprocal error behavior in estimated body position and subjective visual vertical, Jaggi-Schwarz [/bib_ref] [bib_ref] Influence of dynamic tilts on the perception of earth-vertical, Jaggi-Schwarz [/bib_ref] [bib_ref] Dynamic effects on the subjective visual vertical after roll rotation, Lorincz [/bib_ref] [bib_ref] Body-tilt and visual verticality perception during multiple cycles of roll rotation, Vingerhoets [/bib_ref]. After constant-velocity roll rotations, SVV errors were transiently biased in the direction of the rotation [bib_ref] Reciprocal error behavior in estimated body position and subjective visual vertical, Jaggi-Schwarz [/bib_ref] [bib_ref] Influence of dynamic tilts on the perception of earth-vertical, Jaggi-Schwarz [/bib_ref] [bib_ref] Dynamic effects on the subjective visual vertical after roll rotation, Lorincz [/bib_ref] [bib_ref] Body-tilt and visual verticality perception during multiple cycles of roll rotation, Vingerhoets [/bib_ref]. This "dynamic" bias was dependent on the velocity of the rotation and the final tilt position at which SVV was FigURe 7 | Schematic representation of the periodic subjective visual vertical (SVV) modulation by the frame orientation (solid line) during head tilt: frame tilt orientations close to the subject's upright perception (dashed line) usually result in an "attractor bias" (i.e., toward the direction of the frame tilt), while there is a "detractor bias" at angles beyond 45° and up to 90° (i.e., away from the direction of the frame tilt). These biases caused by the frame orientation can either attenuate or accentuate SVV errors, depending on head tilt position (e.g., here 20° head tilt to the left). measured. For example, with clockwise rotations starting from the upright position, SVV errors showed a significant A-effect when the rotation stopped at large body tilt angles, whereas the errors were close to veridical when it stopped at smaller tilt angles. By contrast, with counterclockwise rotations passing through the upside-down position, SVV errors showed a significant E-effect when the rotation stopped at small tilt angles (i.e., close to the upright position), whereas the errors were close to veridical when it stopped at large tilt angles (i.e., close to the 90° tilt position) [bib_ref] Dynamic effects on the subjective visual vertical after roll rotation, Lorincz [/bib_ref]. This post-rotation "hysteresis" effect lasted about 1 min, suggesting that the transient bias in SVV errors was related to semicircular canal activation from the forces generated through deceleration. Perception of roll-tilt can also be induced during off-axis yaw rotation with the head upright or during on-axis yaw rotation with the head tilted on the body [bib_ref] Measuring unilateral otolith function via the otolith-ocular response and the subjective visual..., Clarke [/bib_ref] [bib_ref] The effect of ocular torsional position on perception of the roll-tilt of..., Wade [/bib_ref]. In these scenarios, rotational cues mainly from the horizontal semicircular canal stimulation affect the time course of tilt perception [bib_ref] Neural processing of gravito-inertial cues in humans. II. Influence of the semicircular..., Merfeld [/bib_ref] [bib_ref] Tilt perception during dynamic linear acceleration, Seidman [/bib_ref]. Moreover, SVV errors have been reported with the head pitched forward or backward during yaw-axis rotation. In this case, SVV errors were in the opposite direction of the rotation (same direction as the fast phase of the torsional nystagmus) and were more pronounced with the head pitched backward, consistent with a stronger effect from stimulation of the posterior semicircular canal [bib_ref] Effect of semicircular canal stimulation on the perception of the visual vertical, Pavlou [/bib_ref]. Perception of upright has been also studied with respect to the modulating effects of visual backgrounds. Our daily environment is rich with visual cues that indicate world-horizontal and vertical orientations. In general, various visual functions (e.g., orientation discrimination, contrast detection, or visual acuity) show superior performance along the horizontal and vertical axes compared with oblique angles (e.g., 45°), which is referred to as the oblique effect (13, 20). However, visual vertical cues can have a greater effect on one's perception of spatial orientation than the perceived orientation of objects [bib_ref] Perceived self-orientation in allocentric and egocentric space: effects of visual and physical..., Barnett-Cowan [/bib_ref] [bib_ref] The subjective visual vertical and the perceptual upright, Dyde [/bib_ref] [bib_ref] How different types of scenes affect the subjective visual vertical (SVV) and..., Haji-Khamneh [/bib_ref] [bib_ref] A tilt after-effect for images of buildings: evidence of selectivity for the..., Ismail [/bib_ref]. Strong effects of visual cues on upright perception have been shown in various settings, ranging from an entire tilted furnished room to more impoverished stimuli such as a simple square frame (20, 106, 108-119). Remarkably, even the addition of a single line in the SVV paradigm can induce a visual bias in upright responses [bib_ref] Fusion of visual and vestibular tilt cues in the perception of visual..., Vingerhoets [/bib_ref] [bib_ref] Differential effects of visual feedback on subjective visual vertical accuracy and precision, Bjasch [/bib_ref] [bib_ref] Visually perceived vertical (VPV): induced changes in orientation by 1-line and 2-line..., Li [/bib_ref]. In the case of a square frame, the visual vertical estimate is biased by the frame orientation, which is known as the rod-and-frame effect. The frame effect can be robust and, for example, significantly decrease SVV errors induced by rotating backgrounds [bib_ref] Framing visual roll-motion affects postural sway and the subjective visual vertical, Lubeck [/bib_ref] [bib_ref] The effect of repeated visual motion stimuli on visual dependence and postural..., Pavlou [/bib_ref]. This visual effect for the most part depends on the viewing distance and the head tilt position. It decreases with far viewing, indicating reduced reliability of the frame as a visual cue to upright orientation, and increases with head tilt, indicating reduced reliability of the vestibular cues to upright orientation [bib_ref] The role of frame size on vertical and horizontal observers in the..., Zoccolotti [/bib_ref]. Overall, changes in the frame tilt orientation can result in periodic modulation of SVV errors by the rod-and-frame effect. Usually, frame tilts close to the perceived upright orientation result in an "attractor bias" toward the frame orientation, whereas there is a "detractor bias" at frame tilts beyond 45° and up to 90°, and no bias at frame tilts close to 90° [bib_ref] Fusion of visual and vestibular tilt cues in the perception of visual..., Vingerhoets [/bib_ref] [bib_ref] Visually perceived vertical (VPV): induced changes in orientation by 1-line and 2-line..., Li [/bib_ref]. This modulating effect of the frame orientation is more pronounced at larger body tilts, and it can either enhance the E-effect or decrease the A-effect depending on the body tilt orientation [bib_ref] The subjective visual vertical and the perceptual upright, Dyde [/bib_ref] [bib_ref] Fusion of visual and vestibular tilt cues in the perception of visual..., Vingerhoets [/bib_ref] [bib_ref] Observer pitch and roll influence: the rod and frame illusion, Corbett [/bib_ref]. The rod-and-frame effect may also vary among individuals, as some exhibit a strong frame effect (i.e., visual dependence), while others may have a weaker effect (i.e., visual independence) [bib_ref] Role of gravitational versus egocentric cues for human spatial orientation, Bury [/bib_ref] [bib_ref] The role of head-centric spatial reference with a static and kinetic visual..., Guerraz [/bib_ref] [bib_ref] Selection of spatial frame of reference and postural control variability, Isableu [/bib_ref]. A similar pattern of variability with the rod-and-frame effect has been shown in patients with vestibular loss; however, the frame effect can be asymmetrical in these patients, with reduced or even abolished visual dependence when the frame is tilted toward the healthy side, as opposed to a significant frame effect when it is tilted toward the side of vestibular loss [bib_ref] Visual field dependence-independence before and after unilateral vestibular loss, Lopez [/bib_ref]. Background rotation in the roll plane (i.e., around the line of sight) can also affect upright perception and induce SVV errors in the direction of the rotation [bib_ref] Visually and posturally mediated tilt illusion in Parkinson's disease and in labyrinthine..., Bronstein [/bib_ref] [bib_ref] Optokinetic-graviceptive interaction in different head positions, Dichgans [/bib_ref] [bib_ref] Gravity dependence of the effect of optokinetic stimulation on the subject visual..., Ward [/bib_ref]. Similar to the rod-and-frame effect, this optokinetic effect is more pronounced at larger body tilt angles and can induce a larger bias toward the side of vestibular loss [bib_ref] Changes of visual vertical perception: a long-term sign of unilateral and bilateral..., Lopez [/bib_ref] [bib_ref] Moving visual scenes influence the apparent direction of gravity, Dichgans [/bib_ref] [bib_ref] Compensatory changes in static and dynamic subjective visual vertical in patients following..., Goto [/bib_ref] [bib_ref] Visual vertigo: symptom assessment, spatial orientation and postural control, Guerraz [/bib_ref]. Another important factor in perception of upright is the effect of gravity on sensory modalities that encode body position [bib_ref] Gravity and observer's body orientation influence the visual perception of human body..., Lopez [/bib_ref] [bib_ref] Modeling human perception of orientation in altered gravity, Clark [/bib_ref] [bib_ref] Influence of the gravitational vertical on geometric visual illusions, Clément [/bib_ref] [bib_ref] Shape-from-shading depends on visual, gravitational, and body-orientation cues, Jenkin [/bib_ref] [bib_ref] Two reference frames for visual perception in two gravity conditions, Lipshits [/bib_ref] [bib_ref] Human perception of verticality: psychophysical experiments on the centrifuge and their neuronal..., Mast [/bib_ref] [bib_ref] The oblique effect is both allocentric and egocentric, Mikellidou [/bib_ref]. As a fundamental reference for spatial orientation, the gravity vector plays a significant role in almost all aspects of our FigURe 8 | Subjective visual vertical (SVV) and torsional eye position measured simultaneously before, during, and after prolonged head tilts (~15 min) in 12 subjects (15): data points represent SVV or ocular torsion from 100 trials during 20° head tilts to the right and left. Error bars correspond with SEM across subjects. The SVV drift is in the same direction as the head tilt, and when the head returns to upright position there is an aftereffect, also in the same direction as the head tilt. Changes in ocular torsion do not correspond to the SVV drift or aftereffect. balance, perception, and action. In general, gravitoinertial forces can change perceived orientation of objects, an effect that has been described as the oculogravic illusion [bib_ref] Oculogravic illusion, Graybiel [/bib_ref]. Similarly, in microgravity and weightless conditions, space crews often report visual reorientation illusions such as difficulty distinguishing between spacecraft floors, walls, and ceiling surfaces [bib_ref] Inversion illusion in parabolic flight: its probable dependence on otolith function, Graybiel [/bib_ref] [bib_ref] Spatial orientation in weightless environments, Lackner [/bib_ref] [bib_ref] Human visual orientation in weightlessness, Oman [/bib_ref] [bib_ref] Spatial orientation in weightlessness and readaptation to earth's gravity, Young [/bib_ref]. With respect to upright perception, rotating rooms, parabolic flights, and human centrifuge have been used to study the effects of gravitoinertial forces [bib_ref] Difference in the perception of the horizon during true and simulated tilt..., Carriot [/bib_ref] [bib_ref] Neural processing of gravito-inertial cues in humans. II. Influence of the semicircular..., Merfeld [/bib_ref] [bib_ref] Tilt perception during dynamic linear acceleration, Seidman [/bib_ref] [bib_ref] Human perception of verticality: psychophysical experiments on the centrifuge and their neuronal..., Mast [/bib_ref] [bib_ref] Normative data for the subjective visual vertical test during centrifugation, Akin [/bib_ref] [bib_ref] Mechanisms of human static spatial orientation, Bortolami [/bib_ref] [bib_ref] Influence of gravitoinertial force level on the subjective vertical during recumbent yaw..., Bryan [/bib_ref] [bib_ref] Vertical frames of reference and control of body orientation, Carriot [/bib_ref] [bib_ref] Modification of unilateral otolith responses following spaceflight, Clarke [/bib_ref] [bib_ref] Perception of tilt (somatogravic illusion) in response to sustained linear acceleration during..., Clément [/bib_ref] [bib_ref] Unilateral centrifugation: utricular assessment and protocol comparison, Janky [/bib_ref]. For example, in a centrifuge experiment, perception of tilt significantly increased late in the spaceflight duration compared with the early flight and preflight results on earth [bib_ref] Perception of tilt (somatogravic illusion) in response to sustained linear acceleration during..., Clément [/bib_ref]. This exaggerated perception of tilt also persisted into the early post-flight days. Likewise, other studies using the rod-and-frame test, optokinetic stimulation, and unilateral centrifugations (i.e., stimulating only one labyrinth at a time) have shown significant visual dependency and asymmetry in SVV responses upon returning back to the earth [bib_ref] Spatial orientation in weightlessness and readaptation to earth's gravity, Young [/bib_ref] [bib_ref] Modification of unilateral otolith responses following spaceflight, Clarke [/bib_ref] [bib_ref] Optokinetic stimulation in microgravity: torsional eye movements and subjective visual vertical, Krafczyk [/bib_ref]. These results suggest that the multisensory contributions to the internal reference for upright orientation is reduced with adaptation to microgravity. The effect of gravity on this multisensory reference is shown with gravitational forces as little as 0.15 g (close to the force of gravity at the moon) and up to 1.5-2 g, resulting in significant deviations in perception of upright [bib_ref] Human perception of verticality: psychophysical experiments on the centrifuge and their neuronal..., Mast [/bib_ref] [bib_ref] Mechanisms of human static spatial orientation, Bortolami [/bib_ref] [bib_ref] The perception of verticality in lunar and Martian gravity conditions, De Winkel [/bib_ref] [bib_ref] How much gravity is needed to establish the perceptual upright, Harris [/bib_ref]. ## Upright perception and adaptation: drift during head tilt Upright perception may drift during prolonged tilts of the whole body or prolonged tilts of the head on body [fig_ref] FigURe 6 |: Schematic representation of the sensory integration model [/fig_ref]. The drift pattern is usually variable across individuals [bib_ref] Modulation of internal estimates of gravity during and after prolonged roll-tilts, Tarnutzer [/bib_ref] , but often there is a gradual change in the direction of the tilt, followed by a post-tilt bias referred to as the aftereffect [bib_ref] Visual orientation during and after lateral head, body, and trunk tilt, Wade [/bib_ref] [bib_ref] Modulation of internal estimates of gravity during and after prolonged roll-tilts, Tarnutzer [/bib_ref] [bib_ref] Effect of prolonged tilt on visual orientation, Wade [/bib_ref] [bib_ref] Effects of cervical muscle fatigue on the perception of the subjective vertical..., Gosselin [/bib_ref] [bib_ref] Apparent head position as a basis for a visual aftereffect of prolonged..., Wade [/bib_ref] [bib_ref] Development and dissipation of a visual spatial aftereffect from prolonged head tilt, Wade [/bib_ref]. When this aftereffect was studied across a wide range of body orientations, there was a "local" effect (as opposed to a "global" effect), where the post tilt bias was mainly seen in the tilt orientations adjacent to the initial, adapting position [bib_ref] Static roll-tilt over five minutes locally distorts the internal estimate of direction..., Tarnutzer [/bib_ref]. For example, if the subject was initially tilted at 90°, the SVV aftereffect was more pronounced at nearby tilt angles such as 60°. Based on this finding, it was proposed that maintaining a static tilt position could bias the internal upright reference toward this adopted position, thus resulting in an aftereffect at subsequent tilt positions [bib_ref] Static roll-tilt over five minutes locally distorts the internal estimate of direction..., Tarnutzer [/bib_ref]. As mentioned earlier, ocular torsion can be a significant source of SVV errors during head tilt, due to the low OCR gain and altered orientation of the images on the retina (15, 53, 100, 103). However, neither the drift in upright perception nor the aftereffect correlate with changes in ocular torsion (15) . These findings indicate that the torsional eye position-or its driving input from the otoliths-cannot be the source of the drift or the aftereffect in perception of upright. Similar drifts have been found with haptic measurements, which also confirms that the visual error induced by ocular torsion cannot be the source of drifts in upright perception during head tilt [bib_ref] Modulation of internal estimates of gravity during and after prolonged roll-tilts, Tarnutzer [/bib_ref] [bib_ref] Development and dissipation of a visual spatial aftereffect from prolonged head tilt, Wade [/bib_ref]. Overall, SVV drifts tend to be larger and more consistent across individuals with the head-on body tilts compared with the whole body tilts [fig_ref] FigURe 6 |: Schematic representation of the sensory integration model [/fig_ref]. These findings, along with predictions from the Bayesian spatial perception model, suggest that the adaptation of neck proprioceptive inputs is the primary source of SVV drift during head tilt (15). Thus, the SVV drift is likely modulated by the position of the head relative to the body rather than the position of the Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org October 2017 | Volume 8 | Article 552 head or trunk relative to gravity. Visual vertical responses may also drift in upright body position, but considerably less when compared with the drift during static body tilt (13, 37). This drift attenuated when upright visual cues were present, but did not completely disappear (13). ## Perception of upright and cerebral cortex ## Multimodal vestibular cortex Multisensory integration is a key functional aspect of neural processes involved in the perception of spatial orientation. In this context, vestibular inputs are often integrated with other sensory modalities that are incorporated into self-perception and extrapersonal spatial orientation to subserve high level cognitive and sensorimotor functions (e.g., visual and proprioceptive signals). Accordingly, graviception and orientation constancy can also be understood as functions mediated by multiple sensory modalities. Attempts to localize vestibular function to the cerebral cortex began with the ancient descriptions of vertigo and speculations about global cerebral function [bib_ref] Perspective on the vestibular cortex throughout history, Duque-Parra [/bib_ref]. In recent years, electrophysiological recordings in animal studies have identified multiple cortical sites sensitive to vestibular stimulation, thus laying the groundwork for comparisons with the human cortex. The findings reveal distinct areas within the parietal and temporal cortices that receive and process vestibular inputs. These cortical areas include the parieto-insular vestibular cortex (PIVC), parts of the somatosensory cortex, the lower tip of the intraparietal sulcus, the dorsal subdivision of the middle superior temporal cortex (MSTd), the visual posterior Sylvian area (VPS), and the ventral intraparietal cortex (VIP) [for comprehensive review, see Ref. [bib_ref] The thalamocortical vestibular system in animals and humans, Lopez [/bib_ref] ]. While these vestibular areas are interconnected, there is no clear evidence that they are organized in a hierarchy similar to other sensory regions such as visual and somatosensory cortices. Direct cortical recordings suggest that PIVC is involved in the integration of vestibular and somatosensory information into a concept of "head in space" [bib_ref] Macaque parieto-insular vestibular cortex: responses to self-motion and optic flow, Chen [/bib_ref] [bib_ref] Is there a vestibular cortex, Guldin [/bib_ref]. On the other hand, visual and vestibular signals have been recorded from MSTd, VPS, VIP, and caudal intraparietal area, with reference to heading perception or allocentric orientation in the earth-vertical direction [bib_ref] Clustering of self-motion selectivity and visual response properties in macaque area MSTd, Chen [/bib_ref] [bib_ref] Convergence of vestibular and visual self-motion signals in an area of the..., Chen [/bib_ref] [bib_ref] Diverse spatial reference frames of vestibular signals in parietal cortex, Chen [/bib_ref] [bib_ref] Functional specializations of the ventral intraparietal area for multisensory heading discrimination, Chen [/bib_ref] [bib_ref] Gravity influences the visual representation of object tilt in parietal cortex, Rosenberg [/bib_ref] [bib_ref] Representation of gravity-aligned scene structure in ventral pathway visual cortex, Vaziri [/bib_ref]. Note that despite the evidence for multimodal integration in these cortical areas, vestibular signals recorded from single neurons remain distinct, suggesting that sensory integration takes place through the function of a cortical network rather than individual neurons [bib_ref] Reference frames for representing visual and tactile locations in parietal cortex, Avillac [/bib_ref] [bib_ref] Efficient computation and cue integration with noisy population codes, Deneve [/bib_ref] [bib_ref] Spatial reference frames of visual, vestibular, and multimodal heading signals in the..., Fetsch [/bib_ref]. In human, as with primate studies, findings from cortical lesion analysis, functional imaging with caloric or galvanic stimulation (fMRI and PET), and also direct cortical stimulation point to a widely distributed multisensory vestibular system, mainly in the temporo-partieto-insular cortices [see Ref. [bib_ref] The thalamocortical vestibular system in animals and humans, Lopez [/bib_ref] for comprehensive review]. The vestibular or combined visualvestibular activations in these cortical regions are predominantly focused at the temporo-parietal junction (TPJ), and more specifically around the posterior parietal operculum, inferior parietal lobule, superior temporal gyrus (STG), and the junction of the intraparietal sulcus and the postcentral sulcus [bib_ref] Multisensory cortical signal increases and decreases during vestibular galvanic stimulation (fMRI), Bense [/bib_ref] [bib_ref] Cerebral representations for egocentric space: functional-anatomical evidence from caloric vestibular stimulation and..., Bottini [/bib_ref] [bib_ref] Dominance for vestibular cortical function in the non-dominant hemisphere, Dieterich [/bib_ref] [bib_ref] Identifying human parieto-insular vestibular cortex using fMRI and cytoarchitectonic mapping, Eickhoff [/bib_ref] [bib_ref] Cortical projection of peripheral vestibular signaling, Emri [/bib_ref] [bib_ref] Human vestibular cortex as identified with caloric stimulation in functional magnetic resonance..., Fasold [/bib_ref] [bib_ref] Performing allocentric visuospatial judgments with induced distortion of the egocentric reference frame:..., Fink [/bib_ref] [bib_ref] Focal increase of blood flow in the cerebral cortex of man during..., Friberg [/bib_ref] [bib_ref] Neural correlates of hemispheric dominance and ipsilaterality within the vestibular system, Janzen [/bib_ref] [bib_ref] Temporoparietal encoding of space and time during vestibular-guided orientation, Kaski [/bib_ref] [bib_ref] Visual gravitational motion and the vestibular system in humans, Lacquaniti [/bib_ref] [bib_ref] Functional MRI of galvanic vestibular stimulation, Lobel [/bib_ref] [bib_ref] Saccular stimulation of the human cortex: a functional magnetic resonance imaging study, Miyamoto [/bib_ref] [bib_ref] Neural basis of visually guided head movements studied with fMRI, Petit [/bib_ref] [bib_ref] Functional neuroimaging of visuo-vestibular interaction, Roberts [/bib_ref] [bib_ref] Cortical representation of saccular vestibular stimulation: VEMPs in fMRI, Schlindwein [/bib_ref] [bib_ref] Functional MRI of galvanic vestibular stimulation with alternating currents at different frequencies, Stephan [/bib_ref] [bib_ref] Cortical and subcortical vestibular response to caloric stimulation detected by functional magnetic..., Suzuki [/bib_ref] [bib_ref] A fronto-parietal system for computing the egocentric spatial frame of reference in..., Vallar [/bib_ref]. Overall, the patterns of cortical activity in these studies suggest that the posterior parietal operculum is the human homologue of PIVC area in monkey, and the human homologues of VPS, VIP, and MSTd areas are within or around the inferior parietal lobule [bib_ref] Identifying human parieto-insular vestibular cortex using fMRI and cytoarchitectonic mapping, Eickhoff [/bib_ref] [bib_ref] Meta-analytical definition and functional connectivity of the human vestibular cortex, Zu Eulenburg [/bib_ref]. Note, however, that a systematic mapping of TPJ is currently lacking, and we know little about the flow of sensory information among various areas within this cortical region, or how disruption in one sensory modality may affect multisensory integration and perception of spatial orientation. Although not addressed in animal studies, significant vestibular activation has been found in the non-dominant human cortex, i.e., the right hemisphere in right-handers and the left hemisphere in left-handers [bib_ref] Dominance for vestibular cortical function in the non-dominant hemisphere, Dieterich [/bib_ref]. Notably, the cortical mechanisms involved in spatial functions also modulate lowerlevel vestibular function, and a similar pattern of laterality has been shown for the cortical influence on the duration of the vestibulo-ocular reflex (i.e., the time constant) [bib_ref] Handedness-related cortical modulation of the vestibular-ocular reflex, Arshad [/bib_ref] [bib_ref] Mechanisms underlying visually induced body sway, Guerraz [/bib_ref] [bib_ref] The neuroanatomical correlates of training-related perceptuo-reflex uncoupling in dancers, Nigmatullina [/bib_ref]. With respect to the vestibular connections to the cerebral cortex, five distinct vestibular pathways have been identified based on functional and structural imaging analyses [bib_ref] Right-sided dominance of the bilateral vestibular system in the upper brainstem and..., Dieterich [/bib_ref] [bib_ref] Structural and functional connectivity mapping of the vestibular circuitry from human brainstem..., Kirsch [/bib_ref]. Three of these pathways run ipsilaterally, and two cross either within the pons or the midbrain. The ipsilateral pathways reach the inferior part of the insular cortex either directly or through the thalamus. Contralateral pathways run through the posterolateral thalamus to the parieto-insular cortex. In addition to connections with the brainstem, the parietal opercular regions also maintain communication with each other via an interhemispheric band of fibers passing through the antero-caudal splenium of the corpus callosum [bib_ref] Right-sided dominance of the bilateral vestibular system in the upper brainstem and..., Dieterich [/bib_ref] [bib_ref] Structural and functional connectivity mapping of the vestibular circuitry from human brainstem..., Kirsch [/bib_ref]. ## Temporo-parietal cortex and perception of upright The TPJ is a cortical hub for multiple sensory modalities, and it has been implicated in various aspects of spatial orientation including visuospatial attention, heading perception, visual gravitational motion perception, sense of embodiment, selflocalization, and egocentricity [bib_ref] Temporoparietal encoding of space and time during vestibular-guided orientation, Kaski [/bib_ref] [bib_ref] Visual gravitational motion and the vestibular system in humans, Lacquaniti [/bib_ref] [bib_ref] Functional neuroimaging of visuo-vestibular interaction, Roberts [/bib_ref] [bib_ref] Behavioral, neural, and computational principles of bodily self-consciousness, Blanke [/bib_ref] [bib_ref] Contributions of the human temporoparietal junction and MT/V5+ to the timing of..., Bosco [/bib_ref] [bib_ref] Distinct contributions of extrastriate body area and temporoparietal junction in perceiving one's..., Cazzato [/bib_ref] [bib_ref] Noninvasive stimulation of the temporoparietal junction: a systematic review, Donaldson [/bib_ref] [bib_ref] Spatial selectivity in the temporoparietal junction, inferior frontal sulcus, and inferior parietal..., Hansen [/bib_ref] [bib_ref] The inferior parietal lobule and temporoparietal junction: a network perspective, Igelström [/bib_ref] [bib_ref] Representation of visual gravitational motion in the human vestibular cortex, Indovina [/bib_ref] [bib_ref] Vestibular function and depersonalization/derealization symptoms, Renaud [/bib_ref] [bib_ref] Body ownership and embodiment: vestibular and multisensory mechanisms, Lopez [/bib_ref] [bib_ref] Functional neuro-anatomy of egocentric versus allocentric space representation, Saj [/bib_ref] [bib_ref] Right supramarginal gyrus is crucial to overcome emotional egocentricity bias in social..., Silani [/bib_ref] [bib_ref] Vestibular function in the temporal and parietal cortex: distinct velocity and inertial..., Ventre-Dominey [/bib_ref]. The role of TPJ in perception of spatial orientation is especially evident from the deficits in neglect syndrome as a result of lesions involving this cortical region. Patients with neglect are unable to attend to sensory stimuli in their contralesional hemispace and also show significant contraversive deviations of upright perception in both haptic and visual tasks [bib_ref] Integration of visual and haptic informations in the perception of the vertical..., Braem [/bib_ref] [bib_ref] Judgment of spatial orientation in patients with focal brain damage, De Renzi [/bib_ref] [bib_ref] Visual context modulates the subjective vertical in neglect: evidence for an increased..., Funk [/bib_ref] [bib_ref] The manual haptic perception of orientations and the oblique effect in patients..., Gentaz [/bib_ref] [bib_ref] Spatial neglect -a vestibular disorder?, Karnath [/bib_ref] [bib_ref] The anatomy of spatial neglect, Karnath [/bib_ref] [bib_ref] Multimodal spatial orientation deficits in left-sided visual neglect, Kerkhoff [/bib_ref] [bib_ref] Disorders of visuospatial orientation in the frontal plane in patients with visual..., Kerkhoff [/bib_ref] [bib_ref] Subjective visual vertical in pitch and roll in right hemispheric stroke, Saj [/bib_ref] [bib_ref] Multimodal and multispatial deficits of verticality perception in hemispatial neglect, Utz [/bib_ref]. These multimodal deficits in upright perception are often related to the severity of neglect symptoms and are also modulated by the head and body positions [bib_ref] The manual haptic perception of orientations and the oblique effect in patients..., Gentaz [/bib_ref] [bib_ref] Multimodal spatial orientation deficits in left-sided visual neglect, Kerkhoff [/bib_ref] [bib_ref] Effects of lateral head inclination on multimodal spatial orientation judgments in neglect:..., Funk [/bib_ref] [bib_ref] Systematic biases in the tactile perception of the subjective vertical in patients..., Funk [/bib_ref] [bib_ref] Neuroanatomy of space, body, and posture perception in patients with right hemisphere..., Rousseaux [/bib_ref] [bib_ref] Effect of posture on the perception of verticality in neglect patients, Saj [/bib_ref] [bib_ref] Perception of verticality after recent cerebral hemispheric stroke, Yelnik [/bib_ref]. In addition, abnormal visual modulation of upright perception has been reported in neglect patients. Using the rod-and-frame test, upright responses were more biased by the frame effect when it was tilted contralesionally, whereas the bias decreased when the frame was tilted toward the side of the lesion [bib_ref] Visual context modulates the subjective vertical in neglect: evidence for an increased..., Funk [/bib_ref]. Visuospatial deficits (i.e., visual extinction) have been also produced in healthy individuals by the inhibitory effect of transcranial magnetic stimulation (TMS) over the right TPJ. This transient effect, as with neglect patients, was dependent on the horizontal and vertical eccentricity of the visual stimulus [bib_ref] Horizontal and vertical dimensions of visual extinction: a theta burst stimulation study, Cazzoli [/bib_ref]. Taken together, these findings suggest that the perception of body orientation, visuospatial awareness, and upright orientation share the same cortical networks. In this scheme, sensory processing at the TPJ would be crucial for construction of the reference frames used for both self-position and extrapersonal space transformations. In line with the multisensory role of TPJ, cortical activations within this area during visual, tactile, and vestibular sensory conflicts correspond to the perception of self-location [bib_ref] Neural basis of embodiment: distinct contributions of temporoparietal junction and extrastriate body..., Arzy [/bib_ref] [bib_ref] Multisensory mechanisms in temporo-parietal cortex support self-location and first-person perspective, Ionta [/bib_ref] [bib_ref] The role of the right temporo-parietal junction in maintaining a coherent sense..., Tsakiris [/bib_ref]. Accordingly, TPJ lesions are also associated with symptoms such as out-of-body experience or room tilt illusion [bib_ref] Body ownership and embodiment: vestibular and multisensory mechanisms, Lopez [/bib_ref] [bib_ref] Multisensory mechanisms in temporo-parietal cortex support self-location and first-person perspective, Ionta [/bib_ref] [bib_ref] Neuropsychology: stimulating illusory own-body perceptions, Blanke [/bib_ref] [bib_ref] Out-of-body experience and autoscopy of neurological origin, Blanke [/bib_ref] [bib_ref] Towards a concept of disorders of "higher vestibular function, Brandt [/bib_ref] [bib_ref] Visualizing out-of-body experience in the brain, De Ridder [/bib_ref]. Overall, these lines of evidence indicate that TPJ is involved in generating the multisensory internal reference used by the brain to anchor "self " with respect to the surrounding environment and maintain orientation constancy especially with changes in the eye, head, and body positions. Studies focused on the effects of brain lesions on upright perception go back as far as 1948, where SVV errors exceeding 2° were described with fronto-parietal lesions, but not occipital lesions (for comparison, note the campanile of Pisa is currently at 4°) [bib_ref] Abweichungen der subjektiven optischen Vertikalen und Horizontalen bei Gesunden und Hirnverletzten, Bender [/bib_ref]. More recently, lesion studies have shown associations between cerebral cortex and abnormal upright perception in the context of hemispheric stroke [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref] [bib_ref] Disorders of visuospatial orientation in the frontal plane in patients with visual..., Kerkhoff [/bib_ref] [bib_ref] Neuroanatomy of space, body, and posture perception in patients with right hemisphere..., Rousseaux [/bib_ref] [bib_ref] Vestibular cortex lesions affect the perception of verticality, Brandt [/bib_ref] [bib_ref] Neural correlates of disturbed perception of verticality, Baier [/bib_ref] [bib_ref] Posterior insular cortex -a site of vestibular-somatosensory interaction, Baier [/bib_ref] [bib_ref] Inter-and intra-rater reliability of the visual vertical in subacute stroke, Piscicelli [/bib_ref] [bib_ref] An anatomical and psychophysical comparison of subjective verticals in patients with right..., Rousseaux [/bib_ref]. Note that these studies have recruited patients at different postlesion times which could affect the SVV results depending on the effect of brain adaptation following the stroke in these patients. While these studies indicate involvement of several cortical areas within and around TPJ, these lesions converge largely within the inferior parietal lobule and posterior aspect of the insular cortex [fig_ref] FigURe 9 |: Approximate projections of the cortical areas associated with subjective visual vertical deviation... [/fig_ref]. Isolated lesions within the posterior insula, however, are not associated with SVV deviations, which suggests that other cortical locations within TPJ are involved in perception of upright [bib_ref] Insular strokes cause no vestibular deficits, Baier [/bib_ref]. With respect to subcortical white matter regions, lesion extensions to the superior longitudinal fascicle, inferior longitudinal fascicle, inferior occipitofrontal fascicle, and superior occipitofrontal fascicle are shown in connection with SVV deviations [bib_ref] Neural correlates of disturbed perception of verticality, Baier [/bib_ref] [bib_ref] An anatomical and psychophysical comparison of subjective verticals in patients with right..., Rousseaux [/bib_ref]. In general, lesion studies have widely reported contralesional SVV deviations, whereas only about 10% of patients may have ipsilesional SVV deviations . This finding contrasts with to the SVV deviations seen with brainstem lesions, which more consistently are tilted toward the side of the lesion with caudal brainstem involvement, and away from the side of the lesion with rostral brainstem involvement [bib_ref] Vestibular syndromes in the roll plane: topographic diagnosis from brainstem to cortex, Brandt [/bib_ref] [bib_ref] Perceived vertical and lateropulsion: clinical syndromes, localization, and prognosis, Brandt [/bib_ref] [bib_ref] Ocular torsion and tilt of subjective visual vertical are sensitive brainstem signs, Dieterich [/bib_ref]. In addition, the extent of SVV deviations with cerebral cortical lesions is usually less than the SVV deviations with the brainstem or peripheral vestibular lesions [bib_ref] Ocular torsion and tilt of subjective visual vertical are sensitive brainstem signs, Dieterich [/bib_ref] [bib_ref] Idiosyncratic compensation of the subjective visual horizontal and vertical in 60 patients..., Hafström [/bib_ref]. These anatomical differences in SVV errors are likely related to the pathological changes in ocular torsion with low-level brain lesions. Such deviations in ocular torsion lead to SVV errors by directly affecting the orientation of the images on the retina. SVV errors at the level of cerebral cortex, on the other hand, are primarily linked to the neural sensory processes underlying spatial perception. Generally, SVV errors from the right hemispheric lesions tend to be larger, long lasting, and more often associated with contralesional deviations [bib_ref] Neural correlates of disturbed perception of verticality, Baier [/bib_ref] [bib_ref] Evolution of subjective visual vertical perturbation after stroke, Bonan [/bib_ref] [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] Assessing the visual vertical: how many trials are required?, Piscicelli [/bib_ref]. These findings are consistent with the dominance of the right hemisphere in processing spatial information. In addition, the magnitude of SVV deviations correlates with the extent of cortical lesions, highlighting the significance of a multisensory cortical network for coherent perception of upright [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref] [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref]. The contralesional SVV bias persists with small body tilts away from the side of the lesion, resulting in an A-effect toward the paretic side, instead of a normal E-effect in the opposite direction [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref] [bib_ref] Multimodal spatial orientation deficits in left-sided visual neglect, Kerkhoff [/bib_ref] [bib_ref] Perception of verticality after recent cerebral hemispheric stroke, Yelnik [/bib_ref] [bib_ref] Are rotations in perceived visual vertical and body axis after stroke caused..., Barra [/bib_ref]. Such bias, however, is not present when the body is tilted toward the side of the lesion (i.e., away from the paretic side), in which case the SVV errors are comparable to normal individuals [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref]. It is also shown that the errors of upright perception from cortical lesions could be dissociated from perception of body position or actual postural deviations. However, patients with concurrent errors in all these domains had lesions involving the right TPJ [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] The origin of contraversive pushing evidence for a second graviceptive system in..., Karnath [/bib_ref] [bib_ref] Influence of sensory loss on the perception of verticality in stroke patients, Saeys [/bib_ref]. When measured at different body tilts, SVV and perception of body position were correlated when the body was tilted toward the side of the lesion, but such correlation was not present while tilted away from the side of the lesion [bib_ref] Are rotations in perceived visual vertical and body axis after stroke caused..., Barra [/bib_ref] [bib_ref] Perception of longitudinal body axis in patients with stroke: a pilot study, Barra [/bib_ref]. There were also larger overestimation errors in perception of body position compared with SVV while the body was tilted away from the side of the lesion. Such dissociation between perceptions of upright and body position is consistent with different weights of sensory contributions for processing upright orientation versus body position. With respect to other axes of spatial perception, a significant backward deviation of upright responses in the pitch plane has been reported in patients with right hemisphere stroke in addition to the errors in the roll plane [bib_ref] Subjective visual vertical in pitch and roll in right hemispheric stroke, Saj [/bib_ref] [bib_ref] Multimodal and multispatial deficits of verticality perception in hemispatial neglect, Utz [/bib_ref]. The role of TPJ in perception of upright is also studied using non-invasive brain stimulation [bib_ref] Transcranial magnetic stimulation (TMS) of the supramarginal gyrus: a window to perception..., Kheradmand [/bib_ref] [bib_ref] The right temporoparietal junction plays a causal role in maintaining the internal..., Fiori [/bib_ref] [bib_ref] Polarity-dependent misperception of subjective visual vertical during and after transcranial direct current..., Santos-Pontelli [/bib_ref] [bib_ref] The role of the right superior parietal lobule in processing visual context..., Lester [/bib_ref]. We recently applied TMS in healthy participants at the right TPJ and probed its transient cortical effects on perception of upright using SVV measurements [bib_ref] Transcranial magnetic stimulation (TMS) of the supramarginal gyrus: a window to perception..., Kheradmand [/bib_ref]. The inhibitory effect of TMS at the posterior aspect of the right supramarginal gyrus (SMGp) resulted in a shift of SVV errors in the opposite direction of the head tilt [fig_ref] FigURe 10 |: Simultaneous subjective visual vertical [/fig_ref]. The direction of this error, induced by the focal cortical inhibition, is consistent with the "overestimation" errors reported by the cortical lesion studies [i.e., increase in E-effect; e.g., Ref. [bib_ref] Humans use internal models to construct and update a sense of verticality, Barra [/bib_ref] ]. On the other hand, when TMS was applied randomly at other cortical locations within or outside of the TPJ, there was no significant SVV deviation, suggesting a locationspecific effect at SMGp. In addition, there was no change in the torsional position of the eyes despite the SVV shift at SMGp, showing that the changes in perception of upright at the level of cerebral cortex were dissociated from the changes in ocular torsion [bib_ref] Transcranial magnetic stimulation of supramarginal gyrus alters perception of upright without changing..., Otero-Millan [/bib_ref] [fig_ref] FigURe 10 |: Simultaneous subjective visual vertical [/fig_ref]. Altogether, these findings suggest that unlike subcortical regions that have direct influence over ocular torsion, TPJ is primarily involved in sensory processing. Fiori et al. also investigated the role of TPJ in upright perception using the focal inhibitory effects of TMS [bib_ref] The right temporoparietal junction plays a causal role in maintaining the internal..., Fiori [/bib_ref]. They found that the effect of TMS at the right TPJ selectively increased SVV errors when no visual cue was provided (i.e., no visual frame during the SVV task). However, inhibition of V1-V3 and not TPJ disrupted the visual detection of a Gabor patch orientation. This functional distinction between TPJ and early visual cortex is in line with the role of TPJ in multisensory integration for perception of upright. A significant SVV shift has also been shown using transcranial direct current stimulation (tDCS) over TPJ [bib_ref] Polarity-dependent misperception of subjective visual vertical during and after transcranial direct current..., Santos-Pontelli [/bib_ref]. This shift was dependent on tDCS electrode placement, with SVV deviation toward the side of anode placement. There was also a rebound effect (i.e., reversal of the SVV shift) immediately after the stimulation, which lasted longer with the right cathode/left anode placement. Cortical involvement in perception of upright has also been investigated using EEG recordings [bib_ref] Electrophysiological evidence for a post-perceptual influence of global visual context on perceived..., Corbett [/bib_ref] [bib_ref] Spatiotemporal dynamics of visual vertical judgments: early and late brain mechanisms as..., Lopez [/bib_ref]. The results suggest that early cortical activity in the lateral temporooccipital cortex (around 100 ms post-stimulus) is important for extracting orientation features, whereas a later activation involving the temporo-occipital and parieto-occipital cortices (around 300 ms post-stimulus) reflects multisensory integration for perception of upright. Peripheral vestibular injuries can also provide clues to the mechanisms of recovery and multisensory compensation with respect to cortical function and upright perception. For example, it is reported that hemispheric dominance can affect the speed of recovery based on the side of peripheral vestibular injury. The recovery from the right-side vestibular loss was significantly slower than from the left-side vestibular loss in right-handers, while such difference was not found in left-handers [bib_ref] Influence of body laterality on recovery from subjective visual vertical tilt after..., Toupet [/bib_ref]. Based on this observation, it was hypothesized that the difference in the pattern of recovery in left-handers is related to a greater distribution of transcallosal connections between parietal cortices compared with right-handers (87). ## Cerebral cortical pathology and perception of upright Pathological perception of upright is widely reported with cerebral infarctions . SVV deviations in association with cortical strokes are typically found in the territory of the medial cerebral artery (MCA), mainly involving the temporal, parietal, and insular cortices. The absence of skew deviation of the eyes with these lesions suggests the affected cortical areas are primarily involved in processing sensory information [bib_ref] Vestibular cortex lesions affect the perception of verticality, Brandt [/bib_ref]. Notably, posterior cerebral artery infarctions, despite causing visual field defects, do not significantly alter perception of upright [bib_ref] Vestibular cortex lesions affect the perception of verticality, Brandt [/bib_ref]. In a sample of unilateral hemispheric infarction, the branches of the MCA resulting in SVV deviation were the temporal (mean SVV deviation about 6°), parietal (mean SVV deviation about 5°), and the deep cortical perforators (mean SVV deviation about 4°). Lesions affecting the anterior part of the internal capsule can also be associated with SVV tilt (mean SVV deviation about 3°), primarily via the lenticulostriate arteries and the anterior choroidal artery [bib_ref] Vestibular cortex lesions affect the perception of verticality, Brandt [/bib_ref]. In general, hemispheric infarcts more often result in contraversive SVV deviations, while about 10% of patients may show ipsiversive SVV deviations. Pathological SVV tilts can be as large as 15°, though usually they are 5-10° and deviated leftwards as a result of right hemispheric lesions (note again that the campanile of Pisa is currently at 4°) [bib_ref] Vestibular cortex lesions affect the perception of verticality, Brandt [/bib_ref] [bib_ref] Is perception of vertical impaired in individuals with chronic stroke with a..., Mansfield [/bib_ref] [bib_ref] Influence of subjective visual vertical misperception on balance recovery after stroke, Bonan [/bib_ref]. The range of SVV deviations in a sample of 40 patients with hemispheric stroke (time from lesions <13 weeks) was larger with the right hemispheric infarcts (−13.1° to 3.2°) compared with the left hemispheric infarcts (−3.6° to 9.3°) [bib_ref] Perception of verticality after recent cerebral hemispheric stroke, Yelnik [/bib_ref]. The asymmetric hemispheric contribution to upright perception has been also shown in stroke patients with the bottom-up effects of GVS [bib_ref] Subliminal galvanic-vestibular stimulation recalibrates the distorted visual and tactile subjective vertical in..., Oppenländer [/bib_ref] [bib_ref] Perception of the vertical in patients with right hemispheric lesion: effect of..., Saj [/bib_ref]. In these patients with right hemispheric infarcts and spatial neglect, left-cathodal but not right-cathodal galvanic stimulation significantly reduced SVV deviations, highlighting a significant cortical laterality for perception of upright. Another important factor affecting the extent and direction of SVV errors is the recovery time. Acute patients often have larger SVV errors compared with chronic patients, and such deviations often recover significantly within a few months [bib_ref] Neural correlates of disturbed perception of verticality, Baier [/bib_ref] [bib_ref] Evolution of subjective visual vertical perturbation after stroke, Bonan [/bib_ref] [bib_ref] Influence of subjective visual vertical misperception on balance recovery after stroke, Bonan [/bib_ref]. Patients with right hemispheric lesions also have higher variability (i.e., lower precision) in their SVV deviations [bib_ref] Influence of subjective visual vertical misperception on balance recovery after stroke, Bonan [/bib_ref]. Persistent SVV errors and low SVV precision are often linked to poor balance following stroke, especially in patients with the right hemispheric involvement [bib_ref] Verticality perceptions associate with postural control and functionality in stroke patients, Baggio [/bib_ref] [bib_ref] Influence of subjective visual vertical misperception on balance recovery after stroke, Bonan [/bib_ref] [bib_ref] Prevalence and length of recovery of pusher syndrome based on cerebral hemispheric..., Abe [/bib_ref] [bib_ref] Persistent pusher behavior after a stroke, Santos-Pontelli [/bib_ref]. However, perception of body orientation can be dissociated from SVV or from the actual postural deviations in these patients [bib_ref] The perception of body verticality (subjective postural vertical) in peripheral and central..., Bisdorff [/bib_ref] [bib_ref] Are rotations in perceived visual vertical and body axis after stroke caused..., Barra [/bib_ref] [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] Vestibular syndromes in the roll plane: topographic diagnosis from brainstem to cortex, Brandt [/bib_ref] [bib_ref] The origin of contraversive pushing evidence for a second graviceptive system in..., Karnath [/bib_ref] [bib_ref] Perception of longitudinal body axis in patients with stroke: a pilot study, Barra [/bib_ref] [bib_ref] Why acute unilateral vestibular cortex lesions mostly manifest without vertigo, Dieterich [/bib_ref] [bib_ref] Is perception of vertical impaired in individuals with chronic stroke with a..., Mansfield [/bib_ref] [bib_ref] The subjective visual vertical in patients with pusher behaviour: a pilot study..., Paci [/bib_ref] [bib_ref] Biased postural vertical in humans with hemispheric cerebral lesions, Pérennou [/bib_ref] [bib_ref] Measuring verticality perception after stroke: why and how, Pérennou [/bib_ref] [bib_ref] Visual verticality perception after stroke: a systematic review of methodological approaches and..., Piscicelli [/bib_ref] [bib_ref] Asymmetric standing posture after stroke is related to a biased egocentric coordinate..., Barra [/bib_ref] [bib_ref] Graviceptive misperception of the postural vertical after right hemisphere damage, Lafosse [/bib_ref] [bib_ref] Parietal-insular strokes are associated with impaired standing balance as assessed by computerized..., Miyai [/bib_ref] [bib_ref] The polymodal sensory cortex is crucial for controlling lateral postural stability: evidence..., Pérennou [/bib_ref] [bib_ref] The visual vertical in the pusher syndrome: influence of hemispace and body..., Saj [/bib_ref]. For example, in a sample of 80 stroke patients reported by Perrenou et al., 34 had abnormal contralesional postural vertical tilts (i.e., deviations in posture alignment with perceived upright orientation), 44 had contralesional SVV tilts, 26 had contralesional haptic vertical tilts, and none had ipsilesional haptic or postural vertical tilts [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref]. Forty-one patients (52%) showed deficits in more than one modality, and 18 (22%) had transmodal contraversive deviations (i.e., SVV, postural vertical, and haptic vertical were all tilted away from the side of the lesion). In general, postural deviations in stroke patients are more closely related to the errors of postural vertical perception than to the errors of upright perception [bib_ref] Multimodal spatial orientation deficits in left-sided visual neglect, Kerkhoff [/bib_ref] [bib_ref] Are rotations in perceived visual vertical and body axis after stroke caused..., Barra [/bib_ref] [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] Influence of sensory loss on the perception of verticality in stroke patients, Saeys [/bib_ref] [bib_ref] Influence of subjective visual vertical misperception on balance recovery after stroke, Bonan [/bib_ref]. A subset of patients with cortical infarctions and postural deviations exhibit robust SVV deviations and also actively resist attempts to correct their false postural orientation back to upright position [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] The origin of contraversive pushing evidence for a second graviceptive system in..., Karnath [/bib_ref] [bib_ref] Lateropulsion and imbalance in Wallenberg's syndrome, Bjerver [/bib_ref] [bib_ref] Ipsilateral pushing in stroke, Bohannon [/bib_ref] [bib_ref] Poststroke "pushing, Danells [/bib_ref] [bib_ref] Wallenberg's syndrome: lateropulsion, cyclorotation, and subjective visual vertical in thirty-six patients, Dieterich [/bib_ref] [bib_ref] Pusher syndrome" following cortical lesions that spare the thalamus, Johannsen [/bib_ref] [bib_ref] The subjective visual vertical in patients with pusher behaviour: a pilot study..., Paci [/bib_ref] [bib_ref] Measuring verticality perception after stroke: why and how, Pérennou [/bib_ref] [bib_ref] A topodiagnostic investigation on body lateropulsion in medullary infarcts, Thömke [/bib_ref] [bib_ref] The visual vertical in the pusher syndrome: influence of hemispace and body..., Saj [/bib_ref] [bib_ref] Remarks on paralysis of the movements of the trunk in hemiplegia, and..., Beevor [/bib_ref] [bib_ref] Subjective visual vertical (SVV) determined in a representative sample of 15 patients..., Johannsen [/bib_ref] [bib_ref] Understanding and treating "pusher syndrome, Karnath [/bib_ref] [bib_ref] Pusher behaviour: a critical review of controversial issues, Paci [/bib_ref] [bib_ref] Understanding the pusher behavior of some stroke patients with spatial deficits: a..., Pérennou [/bib_ref] [bib_ref] Towards a theoretical understanding of pushing behaviour in stroke patients, Punt [/bib_ref]. This phenomenon, referred to as "pusher syndrome" (also "listing," or "lateropulsion"), is typically toward the paretic side with an incidence of approximately 5-10% among acute stroke patients [bib_ref] Ipsilateral pushing in stroke, Bohannon [/bib_ref] [bib_ref] Ipsilateral pushing in stroke: incidence, relation to neuropsychological symptoms, and impact on..., Pedersen [/bib_ref] [bib_ref] Prevalence and length of recovery of pusher syndrome based on cerebral hemispheric..., Abe [/bib_ref]. In contrast to patients with Wallenberg Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org October 2017 | Volume 8 | Article 552 syndrome or thalamic astasia who pull themselves back toward upright to prevent an ipsilesional fall, pushers resist postural changes toward the non-paretic side. Patients with pusher behavior are often unable to learn to walk even with proper assistance, and their SVV errors or postural vertical deviations often last longer [bib_ref] Is perception of vertical impaired in individuals with chronic stroke with a..., Mansfield [/bib_ref]. Pushing behavior is also highly correlated with neglect symptoms and more often is associated with lesions involving the right posterior insula, STG, inferior parietal lobule, and postcentral gyrus [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] The origin of contraversive pushing evidence for a second graviceptive system in..., Karnath [/bib_ref] [bib_ref] Pusher syndrome" following cortical lesions that spare the thalamus, Johannsen [/bib_ref] [bib_ref] Ipsilateral pushing in stroke: incidence, relation to neuropsychological symptoms, and impact on..., Pedersen [/bib_ref] [bib_ref] Postural disorders and spatial neglect in stroke patients: a strong association, Pérennou [/bib_ref] [bib_ref] Measuring verticality perception after stroke: why and how, Pérennou [/bib_ref] [bib_ref] Prevalence and length of recovery of pusher syndrome based on cerebral hemispheric..., Abe [/bib_ref] [bib_ref] Understanding and treating "pusher syndrome, Karnath [/bib_ref] [bib_ref] Pusher syndrome: its cortical correlate, Baier [/bib_ref] [bib_ref] Postural abnormalities and contraversive pushing following right hemisphere brain damage, Lafosse [/bib_ref]. In the study of Perennou et al. mentioned earlier, the patients who showed lateropulsion and pusher behavior had contraversive transmodal tilt of postural vertical, haptic vertical, and SVV [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref]. This finding suggests that lateropulsion and pushing behavior lie on a continuum where pushers-as opposed to those with lateropulsion onlyactively align their body with their erroneous perception of upright [fig_ref] FigURe 11 |: Schematic showing the directions of subjective visual vertical [/fig_ref] [bib_ref] Lateropulsion, pushing and verticality perception in hemisphere stroke: a causal relationship?, Perennou [/bib_ref] [bib_ref] Biased postural vertical in humans with hemispheric cerebral lesions, Pérennou [/bib_ref] [bib_ref] Understanding the pusher behavior of some stroke patients with spatial deficits: a..., Pérennou [/bib_ref]. When postural vertical perception was measured while standing (as opposed to sitting in other studies), pushers had large uncertainty, and, on average, ipsilesional deviation in their responses [bib_ref] The subjective postural vertical determined in patients with pusher behavior during standing, Bergmann [/bib_ref] , showing that the postural vertical estimates can be altered by active pushing behavior while standing. Parkinson's disease (PD) is another pathology that can affect postural control and spatial perception, due to dysfunctions involving the cortical connections with basal ganglia [bib_ref] Management of postural sensory conflict and dynamic balance control in late-stage Parkinson's..., Colnat-Coulbois [/bib_ref] [bib_ref] A prospective study of alterations in balance among patients with Parkinson's disease, Rossi [/bib_ref]. On this premise, PD measures such as trunk flexion, stance, and gait parameters have been investigated in association with SVV deviation [bib_ref] Increased visual dependence in Parkinson's disease, Azulay [/bib_ref] [bib_ref] Judgment of the visual vertical and horizontal in patients with Parkinsonism, Danta [/bib_ref] [bib_ref] Subjective visual vertical in PD patients with lateral trunk flexion, Gandor [/bib_ref] [bib_ref] Judgment of visual and postural vertical by parkinsonian patients, Proctor [/bib_ref] [bib_ref] Subjective visual vertical in Pisa syndrome, Scocco [/bib_ref] [bib_ref] Vestibular impairment and adaptive postural imbalance in parkinsonian patients with lateral trunk..., Vitale [/bib_ref]. The postural instability in PD patients may correlate with SVV deviations, and both postural vertical perception and SVV show higher variability compared with age-matched, healthy controls [bib_ref] Multisensory determinants of orientation perception in Parkinson's disease, Barnett-Cowan [/bib_ref] [bib_ref] Correlation of impaired subjective visual vertical and postural instability in Parkinson's disease, Pereira [/bib_ref]. In these patients, however, visually induced postural sway cannot be linked to the deficits in perception of upright, which suggests that the postural instability is related to abnormalities in maintaining posture rather than perceptual errors [bib_ref] Visually and posturally mediated tilt illusion in Parkinson's disease and in labyrinthine..., Bronstein [/bib_ref] [bib_ref] Impaired vertical postural control and proprioceptive integration deficits in Parkinson's disease, Vaugoyeau [/bib_ref]. PD patients may also have trunk lateropulsion with the tendency for postural tilts in the direction opposite to the affected side of the body (once dubbed "scoliosis of Parkinsonism") [bib_ref] Note on the scoliosis of Parkinsonism, Duvoisin [/bib_ref]. The patients with lateral trunk deviation show significantly larger SVV errors toward the trunk tilt compared with those without trunk tilt [bib_ref] Subjective visual vertical in PD patients with lateral trunk flexion, Gandor [/bib_ref] [bib_ref] Subjective visual vertical in Pisa syndrome, Scocco [/bib_ref]. This lateral trunk tilt in PD has been attributed to vestibular hypofunction on the same side and described as postural imbalance syndrome with vestibular alterations or PISA [bib_ref] Vestibular impairment and adaptive postural imbalance in parkinsonian patients with lateral trunk..., Vitale [/bib_ref]. Patients with PISA have greater SVV deviations compared with those without the trunk tilt, either on or off of the effects of dopaminergic medications [bib_ref] Subjective visual vertical in Pisa syndrome, Scocco [/bib_ref]. Taken together, the above findings suggest that abnormal upright perception in PD patients can be linked to impaired sensorimotor processing related to corticobasal dysfunction. Migraine syndrome can also result in visuospatial symptoms due to dysfunctions affecting neural networks from the level of brainstem to the cerebral cortex. Migraine patients with these symptoms typically complain of vertigo, dizziness, disorientation, or sense of disequilibrium, often triggered or worsened with changes in the head or body positions. This type of migraine presentation accounts for the most common cause of episodic dizziness and is classified as vestibular migraine [bib_ref] Chapter 3 -Neurotransmitters in the vestibular system, Balaban [/bib_ref] [bib_ref] Vestibular migraine: the most frequent entity of episodic vertigo, Dieterich [/bib_ref] [bib_ref] Vestibular migraine: clinical aspects and pathophysiology, Furman [/bib_ref] [bib_ref] Vestibular migraine: diagnostic criteria, Lempert [/bib_ref]. Patients with vestibular migraine have more pronounced postural sway compared with other types of migraine or healthy controls [bib_ref] Vestibular function in migraine-related dizziness: a pilot study, Furman [/bib_ref] [bib_ref] Migrainous vertigo: results of caloric testing and stabilometric findings, Teggi [/bib_ref]. Consistent with the visuospatial symptoms in these patients, imaging analyses have found decreased gray matter volume within TPJ as well as metabolic changes in this cortical region during the attacks of vestibular migraine [bib_ref] Central vestibular system modulation in vestibular migraine, Obermann [/bib_ref] [bib_ref] Altered brain metabolism in vestibular migraine: comparison of interictal and ictal findings, Shin [/bib_ref]. With respect to upright perception, several studies have reported SVV measurements in migraine patients [bib_ref] Central vestibular system modulation in vestibular migraine, Obermann [/bib_ref] [bib_ref] Altered brain metabolism in vestibular migraine: comparison of interictal and ictal findings, Shin [/bib_ref] [bib_ref] Subclinical deviation of the subjective visual vertical in patients affected by a..., Asai [/bib_ref] [bib_ref] Comparison of linear motion perception thresholds in vestibular migraine and Menière's disease, Bremova [/bib_ref] [bib_ref] Migrainous vertigo: clinical, oculographic and posturographic findings, Çelebisoy [/bib_ref] [bib_ref] Patients with migraine correctly estimate the visual verticality, Crevits [/bib_ref]. According to these studies, patients with non-vestibular migraine correctly estimate upright orientation, while those with vestibular migraine show higher variability in SVV errors compared with other headache disorders or healthy controls [bib_ref] Vestibular migraine: diagnostic criteria, Lempert [/bib_ref] [bib_ref] Patients with migraine correctly estimate the visual verticality, Crevits [/bib_ref] [bib_ref] The prevalence of vestibular symptoms in migraine or tension-type headache, Akdal [/bib_ref] [bib_ref] Comorbidities in vestibular migraine, Eggers [/bib_ref] [bib_ref] Perception of verticality in patients with primary headache disorders, Kandemir [/bib_ref]. Patients with vestibular migraine also have reduced motion detection thresholds in the roll plane compared with non-vestibular migraine or healthy controls [bib_ref] Dynamic tilt thresholds are reduced in vestibular migraine, Lewis [/bib_ref]. However, currently, it is not known whether these patients with vestibular migraine also have altered perception of upright during static head or body tilts. # Summary and conclusion As a multimodal sensory reference, perception of upright represents neural processes that subserve orientation constancy. Consistent with the multisensory properties of these neural processes, several studies have described modulatory effects of gravity, visual cues, and position of the body on perception of upright. Also, various measurement paradigms have shown systematic errors of upright perception with tilting the head or body (i.e., underestimations of the true vertical orientation at large tilts and overestimations at small tilts). These errors reflect challenges for the brain in maintaining a common reference frame for upright orientation, based on the reliability of sensory signals that encode head, eye, and body positions. The computational mechanisms behind these systematic errors have been addressed using mathematical models that account for noisy sensory signals. In these models, the estimates of head, body, and ocular torsion that determine upright orientation are derived using frameworks such as Bayesian "prior" and relative weighting of sensory information. Concerning the role of cerebral cortex in various aspect of spatial perception, animal and human studies show a widely distributed cortical network, primarily within the temporal, insular, and parietal cortices. This is not surprising considering the vital role of the information about body orientation with respect to the surrounding environment while any motor action is being contemplated. With respect to upright perception, the higher-order neural mechanisms must solve the problem of different sensory reference frames in the process of integrating various sensory information. The evidence for cortical involvement in such neural processes comes from TMS and lesion studies. The inhibitory effect of TMS at the posterior aspect of the supramarginal gyrus results in overestimation of upright orientation in the opposite direction of the head tilt. Likewise, cortical lesions involving TPJ are associated with SVV deviations primarily away from the side of the lesion. Patients with these cortical lesions may also have neglect symptoms or out-of-body experiences. Altogether, these findings suggest that perception Upright Perception, Multisensory Convergence, and TPJ Frontiers in Neurology | www.frontiersin.org October 2017 | Volume 8 | Article 552 of body orientation, visuospatial awareness, and upright orientation share the same cortical networks in which an internal reference is generated to anchor "self " with respect to the outside world and maintain orientation constancy. Currently, little is known about the flow of sensory information within these cortical networks and how disruption of one sensory modality may affect processing or integration of other sensory modalities. Future studies will have to specifically address such sensory contributions with respect to cerebral cortical involvement in perception of upright. # Author contributions Both authors have contributed to the data gathering and writing of this manuscript. # Acknowledgments [fig] : HS ( ). Based on Eq. 1, the error in head estimate µ HS  ( ) is given by: et al. added another parameter to the Bayesian model to account for the error in estimating ocular torsion position by the brain µ EH  ( ) (Figure 5) (53). This "uncompensated" ocular torsion can explain the SVV error in the opposite direction of the head tilt at smaller tilt angles (i.e., the E-effect). The error in estimating ocular torsion µ EH  FigURe 5 | Schematic presentation of Bayesian spatial perception model: various sensory modalities are integrated into a common spatial reference frame to determine upright orientation. A vertical line (line in space) is presented in front of a tilted observer (head in space) (a range of ±90° tilt is shown in black graphs). Signal Ĥ S , encoding head orientation in space, is accurate but contaminated by Gaussian noise. Head tilt results in ocular counter-roll (OCR) and signal Ê H , encoding eye-in-head orientation, is also contaminated by independent noise. As part of central neural processing, the estimates of head-in-space HS ( ) and eye-in-head EH ( ) are generated separately from the likelihoods and priors of head tilt and torsional eye position (i.e., ocular torsion). These estimates are integrated to generate eye-in-space estimate ES ( ) , which is then integrated with retinal signal (line on retina) to obtain an internal estimate of the upright orientation [i.e., subjective visual vertical (SVV)]. [/fig] [fig] FigURe 6 |: Schematic representation of the sensory integration model: body sensors, neck sensors, and otoliths provide information about the body in-space, head-on-body, and head-in-space positions, respectively. As part of the central neural processing, the neck and body signals undergo coordinate transformation to indirectly encode head-in-space orientation. Overall, the optimal head-in-space estimate  HS ( ) is obtained by the relative weights of the otolith information (WHD, blue pathway), coordinate-transformed information from the body and neck sensors (WHI, green pathway), and the head prior information (WHP, gray pathway). The head-in-space estimate  HS ( ) is then integrated with eye-in-head estimate  EH ( ) and line orientation on the retina to obtain an internal estimate of the upright orientation [i.e., subjective visual vertical (SVV)]. [/fig] [fig] FigURe 9 |: Approximate projections of the cortical areas associated with subjective visual vertical deviation based on anatomical locations of the average lesion areas from seven studies. The color map shows the degree of overlap among cortical involvement in these studies with maximum convergence around the temporo-parietal junction. The average age of the patients in years and the average time from the stroke in days are included for each study. [/fig] [fig] FigURe 10 |: Simultaneous subjective visual vertical (SVV) and ocular torsion recordings during prolonged left head tilt of 20° in a single subject (500 trials ~15 min) [data from Ref.(260)]: SVV shift from transcranial magnetic stimulation (TMS) at SMGp (red) is shown along with the SVV shift from the sham stimulation (i.e., no TMS) (blue) (A). In both traces, there is a gradual drift over time toward the left (i.e., in the same direction as the head tilt), but the SVV shift from TMS is larger with a deviation opposite to the direction of the head tilt. Ocular torsion shift from TMS at SMGp (red) is not different form the sham stimulation (blue) (B). As opposed to SMGp, SVV shift from TMS at another cortical location outside of TPJ (orange) is smaller than the sham stimulation with a deviation in the same direction as the head tilt (C). PMC, primary motor cortex; SMG, supramarginal gyrus; AG, angular gyrus; STG, superior temporal gyrus; SF, Sylvian fissure. [/fig] [fig] FigURe 11 |: Schematic showing the directions of subjective visual vertical (SVV) tilt and postural deviation in pusher syndrome. In these patients, SVV and postural vertical perception deviate away from the side of the lesion (X), matching the direction of postural tilt (i.e., lateropulsion) as well as the pushing behavior toward the paretic side. Therefore, patients with pushing behavior seem to actively align their body with erroneous upright and postural estimates. [/fig]

Ballistic edge states in Bismuth nanowires revealed by SQUID interferometry ## Supplementary Supplementary . Simulation of critical current from short to long 1D ballistic SNS junctions. Symbols display the calculated critical current amplitude IJ, in units of /0, of ballistic one dimensional SNS junctions of varying lengths L, from the short to the long limit. The simulation uses a tight binding calculation described in (1) for a 1D periodic lattice. In the short junction limit L< we find IJ =  /0 and in the long junction limit the Josephson current is evF/L, where vF = 4ta is the Fermi velocity and the superconducting coherence length  = 2ta/ a is the lattice spacing and t the hoping energy. The red line corresponds to the long junction limit evF/L. . Comparison between field dependences of critical current of bismuth and silver nanowires. Differential resistance versus current (panels b and e) and field dependence of the critical current (panels c and f) of two Bi nanowires (segments s1JU and s3WH, whose SEM images are presented in panels a and d) and one Ag nanowire (whose SEM image is presented in panel g and critical current versus field curve is plotted in panel h) of similar aspect ratio connected to the same type of superconducting tungsten electrodes. The critical current of the Bi nanowires extends beyond several hundred flux quanta threading the nanowires, whereas the critical current of the Ag nanowire decays with a typical scale of a few flux quanta threading the wire. This points to many channel diffusive transport in the Ag nanowire, and very few narrow channels in the Bi nanowires. The critical current of the two Bi nanowires sections also exhibit strong field modulations (not shown), similar to our previous findings in Bi wires (with unknown crystalline orientations (2)), that are the signature of a lateral confinement of the carriers. . Periodicity of the critical current oscillations. a High-pass filtered CPR of the experiment over several SQUID periods, displaying periodic modulation of the critical current with a main period of 9.5 Gauss, corresponding to one flux quantum through the nanowire area. An amplitude modulation due to beating of the two edge paths (of unequal transmission, see main text) is also visible, with a beat period of 85 Gauss (indicated by hatched lines), corresponding to a flux quantum through the nanowire. b Modulation amplitude extracted from the experiment by integration of the CPR over the main (9.5 Gauss) sawtooth period. Here as well, the period of one flux quantum through the wire appears. c Theory for the maximum supercurrent of two topological edge states with different transmissions t1 and t2. ## Supplementary ## Supplementary ## Supplementary figure 8. a second bi(111) nanowire with a sawtooth current-phase relation. As in the device described in the main text, an asymmetric SQUID is fabricated with the focused-ion beam-assisted deposition of a superconducting W loop in parallel with a second Bi nanowire containing a (111) surface. A nanoconstriction is etched to generate the high critical current junction (See Scanning Electron Micrograph of Panel c). The asymmetry in this device is extremely large: the critical current of the W constriction was 173 µA and the modulation amplitude was 45 nA, as seen in panels a and b. Nevertheless, a periodic sawtooth signal in magnetic field with period of 8 G (corresponding to one  in the area of the loop) is clearly superimposed on a slowly varying background, panels a and b. The second path cannot be identified in the CPR. This could be due to the difficulty to detect it in this extremely asymmetrical SQUID configuration, or more likely because the lower channel is not connected to the tungsten electrode, in contrast to the sample presented in the main text. The SEM of panel c indeed suggests that the W electrode may not contact the bottom nanowire facet. Scale bar is 1 micrometer. : Characteristics of bismuth samples measured in Superconductor/Bi/Superconductor two wire configuration. Nine sections of three different Bi nanowires, labeled s1, s2 and s3, were investigated. Each sample corresponds to a different segment of the nanowire. Length is distance between superconducting contacts, RN is the normal state resistance at low temperature, determined by the resistance jump above the critical current Ic for the three shortest segments, which display a supercurrent [fig_ref] Supplementary, Figure 1: High resolution Transmission Electron Micrograph of a Bismuth nanowire [/fig_ref]. For longer segments that do not have a zero resistance at low bias, RN is the resistance measured with a bias current of 1 A. tr/m* is the transport time divided by the effective mass (in bare electron mass units), deduced from the magnetoresistance's parabolic dispersion shown in [fig_ref] Figure 4: Magnetoresistances of eight segments of three Bi wires [/fig_ref]. RN at RT is the sample resistance at room temperature. ## Nanowire Section Length (m) [formula] RN () [/formula] tr/m*( ps ) RN at RT ## Supplementary note 1: relative contributions from bulk and surfaces in the normal state Transport in the normal state is due to bulk and surface states (in addition to topological edge states), and we now characterize the properties of both. ARPES experiments (3) yield a surface carrier density ns ≈10 17 m -2 and a corresponding Fermi wavelength λs  8 nm. The effective mass is ms0.2 me where me is the free electron mass. The band structure of bulk Bi is quite complex, leading to several types of carriers (3). In a nanowire, quantum confinement prohibits light electrons whose Fermi wavelength is greater than the wire's transverse dimensions. Thus, it is likely that the majority bulk states are hole states with an effective mass mb 0.065 me and a Fermi wavelength of λb  60 nm. Those numbers suggest that in a 1 micron-long wire of width 200 nm and height 100 nm, the two non-topological surfaces contain roughly 100 times more carriers than the bulk. Thus we can assume that the greatest contribution to normal state conductance comes from the surfaces, GN = Gs + Gb ~ Gs. The mean free path on the surfaces is then deduced from the 300  resistance of a 1 micron by 200 nm surface, via σs = GQkFls=GL/W=1.7 10 -2  -1 , from which we deduce a surface-state mean free path ls 300 nm, and transport time s 3 ps. Interestingly, whereas the bulk states do not contribute much to the zero field conductance, we find that they yield an important contribution to the low field magnetoresistivity δρ(B) because their mobility beb/mb is greater than the mobility of the surface carriers, due to their smaller effective mass. Whereas elongated wires are not expected to exhibit sizable low field magnetoresistance, the situation is different when the conductivity =Ne results from 2 types of carriers with different mobilities (4): [formula] ( ) = 2 ( − ) 2 2 (1) [/formula] Since the surface conductivity is greater than the bulk conductivity, Supplementary Eq (1) becomes ( ) = 2 2 ( − ) [bib_ref] Magnetic field resistant quantum interferences in bismuth nanowires based Josephson junctions, Li [/bib_ref] (2). The magnetoresistivity coefficient extracted from the plot of Supplementary [fig_ref] Figure 4: Magnetoresistances of eight segments of three Bi wires [/fig_ref] is nearly sample independent, and yields: [formula] ( ) = (2 )². ( ) 2 . ²(3) [/formula] So that we find b 2 ps, and lb 200 nm. The bulk mobility µb is thus 2 times greater than the mobility of the surface states µs. ## Supplementary note 2: field dependence of this and other (111) bi nanowires' critical current We have measured several sections of three different (111) nanowires. The sections are described in . To induce superconductivity, we connect them to high-criticalfield-superconducting electrodes one to a few micrometers apart. This leads to a sizable supercurrent at subKelvin temperature for the three shortest sections, whose length is of the order of or smaller than 2 m. As shown in Supplementary for two samples s1JU and s3WH, we find that the critical current does not decay with magnetic field over the expected small range of one flux quantum through the wire's surface area, as in multichannel diffusive samples [bib_ref] Geometry-related magnetic interference patterns in long SNS Josephson junctions, Chiodi [/bib_ref]. Instead, the supercurrent persists up to unusually high fields, corresponding to several hundreds of flux quanta, persisting up to 10 Tesla in some samples. This is a clear indication of minimal orbital dephasing within Andreev pairs by the magnetic field, that is only possible if very few narrow conduction paths shuttle Andreev pairs across the wire, as suggested in previous experiments on Bi nanowires of unknown orientation [bib_ref] Magnetic field resistant quantum interferences in bismuth nanowires based Josephson junctions, Li [/bib_ref]. For comparison, we also show in Supplementary how the supercurrent induced in an Ag nanowire with the same aspect ratio as the Bi nanowire, connected to the same type of superconducting tungsten electrodes, is suppressed by a magnetic field above just a few 0/S, where S is the wire area perpendicular to the field direction. ## Supplementary note 3: self induction effects It is well known that self induction effects can strongly distort the current phase relation in SQUIDS [bib_ref] Bistability in superconducting rings containing an inhomogeneous Josephson junction, Gaass [/bib_ref]. In our experiments these effects were minimized by making the SQUID loops small enough. Screening of the flux into the loop of a SQUID is characterized by the parameter = 2LIc/0 where L is the inductance of the branch containing the small junction. This parameter is estimated to be respectively 0. In practice, we have evaluated  by adding to the geometrical inductance the (much greater) kinetic inductance of the W wire. The kinetic inductance is related to the normal state resistance RN via LK= RNh/2 (9), yielding 30 pH per micrometer of the FIB-deposited W wires, and 1 pH per micrometer of Al wire. The inductance used of the SIS reference SQUID thus amounts to 200 pH, yielding  Supplementary Note 4: Interference between two ballistic paths as seen in the CPR and as calculated for a quantum spin Hall state with partial transmissions. As mentioned in the main text, the supercurrent carried at the two (inner and outer) edges have a sawtooth-shaped CPR with slightly different periods, Bout=0/Sout=8.5 G, and Bin=0/Sin =9.5 G. This leads to an amplitude and phase modulation (a beating) with a beat frequency given by the difference of the two frequencies B -1 =Bout -1 -Bin -1 , i.e. a period of 85 Gauss. This period corresponds to a flux quantum through the wire, and the factor of 10 between the SQUID period and the wire period corresponds to the ratio of the SQUID loop area over the wire facet area Lsq/W. The 85 G period is visible both in the form of the beating in the CPR shown in and Supplementary , and (to within experimental uncertainly due to the position of the sample in the magnets in both experiments) in the period of the modulation of the critical current of the wire before insertion in the asymmetric SQUID configuration . Finally, this 85 G also corresponds to the inverse of the difference of the frequencies between the main Fourier peak and its satellite, in the Fourier transform of the CPR shown in of the main text. We have also performed simulations on the hexagonal lattice with second neighbor spin-orbit interactions following the Kane and Mele model for the quantum spin Hall state. As expected when the Fermi energy lies in the spin-orbit gap, the Andreev spectrum of a ribbon connected to superconducting reservoirs consists of two spin-degenerate levels, crossing at the Fermi energy for a phase difference of . They correspond to the counter-propagating 1D states along the edges of the ribbon. The maximum supercurrent, plotted in , is found to oscillate with the flux through the ribbon with an amplitude which depends on the relative transmission at the NS interfaces along these two edges, and a period corresponding to one flux quantum through the wire. Supplementary References [fig] Supplementary, Figure 1: High resolution Transmission Electron Micrograph of a Bismuth nanowire. The parallel atomic planes demonstrate the high cristallinity of the wire. A very thin amorphous oxide layer is also visible. Scale bar is 5 nm. Supplementary Figure 2. Bi nanowire with a top 111 facet. a Scanning Electron micrograph of a Bi nanowire with a top (111) facet, as demonstrated by the Electron Backscattered Diffraction pattern in the form of Kikuchi lines (b) and the orientation of the reconstructed unit cell (c). Scale bar is one micrometer. Supplementary Figure 3. Length dependence of Normal state resistance of three Bi nanowires. [/fig] [fig] Figure 4: Magnetoresistances of eight segments of three Bi wires. The magnetoresistances of the segments (described in Supplementary [/fig] [fig] 1: and 0.06 for the DC SQUIDs containing the SIS and Bi nanowire-based Josephson junctions. These values of  yield small corrections on the SIS SQUID, and negligible ones for the Bibased SQUID. [/fig]

From the Editors ## From the editors The issue begins with a letter from William Leiss about Yacov Haimes's [bib_ref] On the complex definition of risk: A systems based approach, Haimes [/bib_ref] recent risk and systems paper. Leiss emphasizes the importance of "systemic risk," and he refers to a conference in 2006. Professor Haimes responds that he presented a paper at that meeting, and he hopes that Leiss will prepare a paper to present his ideas. In this issue, Michael Siegrist comments on the review article published in our April issue by Timothy Earle on trust in risk management. Siegrist notes that while the review was thought-provoking and important for identifying research gaps, it may not have sufficiently clarified the distinction between trust and confidence measures. Earle responds that the issues pointed out by Siegrist are manageable with greater use of the consensus model in risk management settings. It is our intention that review articles such as Earle's will provoke interesting debates among our readers, and we welcome others to submit comments. Most of the articles in the issue are about risk assessment. In 2009, the U.S. National Research Council published Science and Decisions: Advancing Risk Assessment as part of an ongoing effort to improve the use of risks assessment.A perspective written by Eileen Abt, Joseph Rodricks, Jonathan Levy, Lauren Zeise, and Thomas Burke summarizes this interesting report, which considers issues that appear multiple times in every issue of this journal. We welcome comments on this article and the report. Chronic obstructive lung diseases (COPD) and asthma are serious problems, even without exacerbating factors. Chung-Min Liao, Nan-Hung Hsieh, Chia-Pin Chio, and Szu-Chieh Chen studied the impact of influenza viral infections on those with chronic occupational asthma. They found that these viral infections often increased morbidity, sometimes markedly so. In 2002, severe acute respiratory syndrome (SARS) commanded worldwide attention when a coronavirus (SARS-CoV) led to more than 8,000 cases and almost 80 deaths worldwide. Toru Watanabe, Timothy Bartrand, Mark Weir, Tatsuo Omura, and Charles Haas developed a dose-response model using mice-based and human epidemiological data. An exponential model provided the most effective fit to the data and led the authors to estimate a dose of SARS-CoV between 16 and 160 plagueforming units (PFU)/person for an apartment building in Hong Kong, depending on the floor. The authors note that this dose-response model helps us understand what would happen if SARS reemerged. Benzene is a ubiquitous hazard. C. Eric Hack, Lynne Haber, Andrew Maier, Paul Shulte, Bruce Fowler, W. Gregory Lotz, and Russell Savage Jr., all who work for or with U.S. government agencies, used a Bayesian network model in order to incorporate different data sets into an exposuredose-response assessment for benzene-induced acute myeloid leukemia (AML). The focus of their effort was to determine how to evaluate and compare individual biomarkers and quantitatively incorporate them into government policy. Estimating the likelihood of an adverse event is perhaps the most difficult part of risk assessment. Piero Baraldi and Enrico Zio compared probabilistic and Dempster-Shafer theory (DST) approaches to model uncertainty analysis in the performance assessment of radioactive waste repositories. The authors used expert opinion and demonstrated some advantage to the DST approach. Migration of contaminants into food packages is an important exposure assessment issue, as well as a recent public policy issue. Maria Pocas, Jorge Oliveira, Rainer Brandsch, and Timothy Hogg applied Monte Carlo simulation to the task of migration of Irgafos 168 (antioxidant in plastics) from polyethylene into isooctane. The authors report wide variability in migration due to length of migration period, temperature, and other micro environmental conditions. The outcome of earthquakes depends on a set of factors, most notably event characteristics, the structures impacted, and responses to the event. Katsuichiro Goda and Jiandong Ren developed a Editorial multivariate model to assess the outcomes of combinations of events and structures, applying it to an area of southwestern British Columbia. They found that their model form (Gumbel copula) was predictive of outcomes and recommend it as a way of more rapidly evaluating economic losses. The outcomes of natural disasters are affected by the type of event and by the host environment, including environmental and ecological conditions, as well as demographic and socioeconomic characteristics of the population. The interactions of these factors are complex and affected by random fluctuations. Lianfa Li, Jinfeng Wang, Hareton Leung, and Chengsheng Jiang built a Bayesian network (BN) that integrates multiple factors and quantification of uncertainties within their assessment system. The authors also used data mining and spatial data analysis to enhance the tool. They report that the model predicted more accurately than other options. Three papers examined risk perception and communications. Changes in risk perception should lead to changes in behavior, and vice versa. Stephen Brown tested the first part of this expectation with a telephone survey of 255 Australians. Drivers were asked questions about speeding perceptions and behavior, and then follow-ups were asked 6 and 14 weeks later. During weeks 2 through 5, an antispeeding mass media campaign was conducted. After controlling for baseline perceptions, the authors reported a lower frequency of self-reported speeding among those with stronger anti-speeding perceptions. Notably, decreases in risk perception about speeding did not lead to changes in self-reported speeding. Simone Dohle, Carmen Keller, and Michael Siegrist examined the influence of the affect heuristic and implicit associations. The latter are stored memories (e.g., cigarette smoking + bad). Using two experimental designs, the authors found both affect and implicit associations were strong predictors of perceptions, attitudes, and behaviors. Unraveling these associations is important to understanding public perceptions and behaviors, and likely responses to public policy. Branden Johnson and Mathew White examined public trust in risk managers. Using an experimental design to study drinking water standards and brownfields redevelopment, the authors found a complex pattern that demonstrates that the public trusts mangers to accomplish some tasks but not others. The implication is that multiple measures are required to assess competence, values, and other trust elements. Finally, Steven Gendel of the U.S. FDA reviews Science, Policy and the Value-Free Ideal (2009) by Heather E. Douglas. He finds the book very relevant and thought-provoking for risk assessors and analysts who contribute to public policy decisions. ## Michael greenberg, editor-in-chief Karen Lowrie, Managing Editor

Enterocin: Promising Biopreservative Produced by Enterococcus sp. Citation: Kasimin, M.E.; Shamsuddin, S.; Molujin, A.M.; Sabullah, M.K.; Gansau, J.A.; Jawan, R. Enterocin: Promising Biopreservative Produced by Enterococcus sp. Microorganisms 2022, 10, 684. https://doi. # Introduction Food preservation is synonymous with its use in the food industry. Food preservatives are applied in food to prolong the shelf life and improve the quality of food products. Failure in applying this technique will cause a huge impact on human health. Commonly, food made from animal sources is highly perishable as they have high nutrients, moisture, and a neutral pH [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref]. Various food preservation techniques have been used, such as traditional and modern preservation techniques. In this modern era, traditional preservation techniques have been less applied by the food industry as they often alter and eliminate the nutrients of the product. Consequently, modern techniques for food preservation have started to gain attention, such as the use of beneficial bacteria to maintain and improve the quality of the product. Such techniques are also capable to extend shelf life through the application of protective microbes known as lactic acid bacteria (LAB) [bib_ref] Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated..., Da Costa [/bib_ref]. LAB is a group of heterogeneous bacteria that play an important role in the food industry as a preservative agent in various food products, such as kimchi, bread, cheese, sausages, and fermented meat [bib_ref] Lactic acid bacteria: Their applications in foods, Bintsis [/bib_ref]. Biopreservation is a technique for extending the shelf life of food by using microbes or safe antimicrobials produced by LAB. These bacteria exhibit their antimicrobial properties, which retain the unique taste and texture of the food products. The main compounds produced by these bacteria are bacteriocin, organic acids, and hydrogen peroxide [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref]. main compounds produced by these bacteria are bacteriocin, organic acids, and hydrogen peroxide [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref]. The Food and Agriculture Organization of the United Nations has expressed concern over the production of agricultural food to accommodate the growing world population, which will have a detrimental impact on humans in terms of health, safety, and quality. If this problem is mismanaged, 30 to 40% of food manufacturing will be wiped out due to pests and several other factors that may lead to a paralyzed economy [bib_ref] Bioprotective culture: A new generation of food additives for the preservation of..., Ben Said [/bib_ref]. The control of pests and pathogens in food products nowadays has become more challenging due to the widespread use of artificial ingredients. The public is aware of the danger of using artificial ingredients in food, thus, demand for food products that are high in quality, safe, and use natural ingredients is increasing. Therefore, to curb the problem of foodborne illnesses from continuing to pose threat to human health, an initiative to study the ability of LAB against spoilage bacteria has started to gain more attention. LAB plays a crucial role in the agricultural and clinical sectors and has been widely used in the production of food products. Yusra and Effendi [bib_ref] Effect of the bacteriocin-producing Bacillus cereus strain HVR22 on the preservation of..., Yusra [/bib_ref] found that the LAB called fermenting bacteria have often been used as a preservative agent in commercial meat, vegetable and fruit, beverage, and dairy products. These food preservatives are intended to control the growth of pathogens, spoilage bacteria, yeast, and spoilage fungi. These bacteria are naturally found in the respiratory organs, intestines, and reproductive organs of humans and animals. The common genus of LAB includes Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus, while the genus of LAB that has been often found in food includes Aerococcus, Carnobacterium, Lactococcus, Onecoccus, Tetragenococcus, Veganococcus, and Weisella [bib_ref] Role of lactic acid bacteria (LAB) in food preservation, Hitendra [/bib_ref]. A study on the potential use of Enterococcus sp. as a food preservative has been actively pursued due to its ability to produce a bacteriocin known as enterocin [bib_ref] The genus Enterococcus: Between probiotic potential and safety concerns-An Update, Hanchi [/bib_ref]. Enterocin is a type of bacteriocin that consists of peptides [fig_ref] Figure 1: Chemical structure of enterocin [/fig_ref] with a small molecular weight and has specific antimicrobial activity that can inhibit the growth of Gram-positive and spoilage bacteria [bib_ref] The role of enterococci in food and health, Moreno [/bib_ref]. According to Hanchi et al. [bib_ref] The genus Enterococcus: Between probiotic potential and safety concerns-An Update, Hanchi [/bib_ref] , enterocin has a specific antimicrobial activity to inhibit the growth of species that are closely related to the producing bacteria and capable to inhibit the growth of Gram-positive pathogens, such as Listeria monocytogenes. Such ability has prompted its use as a starter and protective culture in the food preservation process [bib_ref] Between emerging pathogens and potential probiotics, Ben Braïek [/bib_ref]. ## Classification of enterocin Enterocin is a bacteriocin produced by Enterococcus sp., such as E. faecalis, E. faecium, E. durans, E. mundtii, E. lactis, and several others. The majority of these species are isolated from food, including dairy products, sausages, vegetables, and raw materials [bib_ref] Enterocins: Bacteriocins with applications in the food industry, Alvarez-Cisneros [/bib_ref]. The first successfully purified enterocin was enterocin As-48 produced by E. faecium, which has been classified as a cyclic peptide antibiotic. The finding has subsequently led to the discovery of a new enterocin-producing Enterococcus [bib_ref] The role of enterococci in food and health, Moreno [/bib_ref]. ## Classification of enterocin Enterocin is a bacteriocin produced by Enterococcus sp., such as E. faecalis, E. faecium, E. durans, E. mundtii, E. lactis, and several others. The majority of these species are isolated from food, including dairy products, sausages, vegetables, and raw materials [bib_ref] Enterocins: Bacteriocins with applications in the food industry, Alvarez-Cisneros [/bib_ref]. The first successfully purified enterocin was enterocin As-48 produced by E. faecium, which has been classified as a cyclic peptide antibiotic. The finding has subsequently led to the discovery of a new enterocin-producing Enterococcus [bib_ref] The role of enterococci in food and health, Moreno [/bib_ref]. Enterocin is a cationic peptide synthesized by ribosomes and has the characteristics of a compound with polar and hydrophobic regions due to the presence of excess lysyl and arginyl residues [bib_ref] Fermentation of non-digestible raffinose family oligosaccharides and galactomannans by probiotics, Zartl [/bib_ref]. It also has a heat-stable peptide with a molecular weight of about Microorganisms 2022, 10, 684 3 of 20 20-60 amino acids, insensitive to rennet and stable at varying pH values [bib_ref] Between emerging pathogens and potential probiotics, Ben Braïek [/bib_ref]. Enterocin can be classified into four classes namely the lantibiotic (Class I), non-lantibiotic (Class II), cyclic enterocin (Class III), and enterocin with high molecular weight (Class IV). Most enterocin produced by Enterococcus sp. are categorized as class II as shown in [fig_ref] Table 1: Enterocin classification [/fig_ref]. Enterocin in class I is a lantibiotic with peptides that undergo enzymatic modification during the process of biosynthesis, forming unusual amino acid molecular structures. The modification affects the properties of the enterocin, such as lantibiotics, Beta-methyl lanthionine, and dehydrated residues. The class I enterocin consists of peptide leaders that are important for enzyme recognition, transport, and ensuring that peptides remain in an inactive state which coalesces into core peptides [bib_ref] Enterocins: Bacteriocins with applications in the food industry, Alvarez-Cisneros [/bib_ref]. The structure of the peptide of class II enterocin is typically unmodified, conferring the ability to develop into a mature form without requiring enzyme, peptide leaders, or carriers. Class II enterocin consists of hydrophilic and cationic regions with common sequences of YGNGV (tyrosine, asparagine, glycine, and valine) at the N-terminal end and disulfide bridges formed by two cysteines at the N-terminal end [bib_ref] Classification of antimicrobial peptides bacteriocin, and the nature of some bacteriocin with..., Osama [/bib_ref] , which participate in the interaction with targeted bacteria. For instance, pediocin PA-1 is known as a bacteriocin of class II, whose properties are similar to that of enterocin class II. According to Ennahar et al. [bib_ref] Class IIa bacteriocins: Biosynthesis, structure and activity, Ennahar [/bib_ref] , the class II peptide has a strong inhibitory effect against L. monocytogenes in food. The presence of hydrophobic and cationic regions with an orderly sequence of YGNGV attach to the cytoplasmic membrane of the targeted bacteria, allowing pediocin molecules to penetrate the membrane and form pores that result in cell death [bib_ref] Classification of antimicrobial peptides bacteriocin, and the nature of some bacteriocin with..., Osama [/bib_ref]. Enterocin is produced by the majority of Enterococcus sp. in class II. According to Ben Braïek and Smaoui [bib_ref] Between emerging pathogens and potential probiotics, Ben Braïek [/bib_ref] , enterocin A is the most potent antimicrobial bacteriocin in the class, whereby it is also produced together with other bacteriocins, such as enterocin B, P, L50, or Q. This is proven with the ability of E. lactis to produce three types of enterocin namely enterocin A, B, and P. Class III enterocin is heat-labile and cyclic and includes enterocin As-48. Enterocin As-48 produced by E. faecalis contains a large number of basic amino acids as compared to acidic residues, with most of the amino acids being hydrophobic (Ala, Pro, Val, Met, Ile, Leu, and Phe) and hydrophilic (Ser, Gly, Thr, and Tyr). Enterocin As-48 has 70 amino acid residues in total and contains non-modified amino acid residues or disulfide bridges [bib_ref] The cyclic antibacterial peptide enterocin AS-48: Isolation, mode of Action, and possible..., Burgos [/bib_ref]. Class IV enterocin includes enterolysin A, which has a molecular weight between 34 and 501 amino acids, and is heat-labile, has a broad inhibitory spectrum and differs from other enterocins of other classes, which disrupt the cell wall of sensitive bacteria causing the cell to lyse. Not only that, the catalytic domains of various cell wall-degrading proteins with modular structures are homologous to the N terminus of enterolysin A [bib_ref] Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG 2333, Nilsen [/bib_ref]. Generally, the majority of the enterocin produced by Enterococcus sp. are categorized in class II because they have an N-terminal structure that influences the level of antimicrobial and capability of producing anti-listerial activity. II Class II-1: Consists of hydrophilic and cationic regions with a consensus sequence of YGNGV located at the end of N terminus and sulfide bridge formed by two cysteine residues at the extremity of N terminus Sub-group 1: Possess the ABC transport system for the secretion of enterocin Enterocin L50A, mundticin [bib_ref] The genus Enterococcus: Between probiotic potential and safety concerns-An Update, Hanchi [/bib_ref] [bib_ref] Between emerging pathogens and potential probiotics, Ben Braïek [/bib_ref] [bib_ref] The cyclic antibacterial peptide enterocin AS-48: Isolation, mode of Action, and possible..., Burgos [/bib_ref] [bib_ref] Bacteriocins from lactic acid bacteria and their applications in meat and meat..., Woraprayote [/bib_ref] Sub-group 2: Bacteriocin production through pre-mature proteins Enterocin P, Enterocin M, Enterocin B Class II-2: The synthesis process occurs without leader peptides, devoid of consensus sequence as well as the secretion of the ABC transport system. ## Preservative effects of bacteriocin produced by lactic acid bacteria on raw meat products LAB is known for its capability to produce a variety of antimicrobial agents that can inhibit the growth of pathogenic bacteria. In 1988, the FDA approved the use of nisin and pediocin, a bacteriocin produced from Lactococcus lactis and Pediococcus sp. as preservatives for application in the food industry. Nisin and pediocin have been successfully commercialized widely [bib_ref] The genus Enterococcus: Between probiotic potential and safety concerns-An Update, Hanchi [/bib_ref]. In addition to nisin and pediocin, a bacteriocin from Enterococcus sp. namely enterocin has also gained significant academic interest following the research conducted on the effectiveness of antimicrobial agents produced by this species for use in food as a preservative. Ben Braïek et al. [bib_ref] Genetic analysis with random amplified polymorphic DNA of the multiple enterocin-producing Enterococcus..., Ben Braïek [/bib_ref] stated that enterocin produced by Enterococcus sp. has high anti-listerial properties due to the bacteriocins produced by Enterococcus species being mostly classified as class III. It has a C-terminal disulfide bridge that stabilizes the posterior fold in the structure, which is crucial in enhancing the antimicrobial activity of the species [bib_ref] Enterococcal Bacteriocins and Antimicrobial Proteins that Contribute to Niche Control. In Enterococci:..., Ness [/bib_ref]. In a study conducted by Fathizadeh et al. [bib_ref] Evaluation of antibacterial activity of enterocin A-colicin E1 fusion peptide, Fathizadeh [/bib_ref] , recombinant bacteriocin, enterocin A and colicin E1 (ent A-col E1) exhibited antibacterial characteristics against both Gram positive and negative bacteria. Enterocin 12a produced by E. faecium was able to inhibit the growth of pathogens, such as Salmonella enterica, Shigella flexneri, Vibrio cholerae, E. coli and L. monocytogenes [bib_ref] Anticancer and antimicrobial potential of enterocin 12a from Enterococcus faecium, Sharma [/bib_ref]. Several studies have reported the effectiveness of bacteriocin produced by LAB in inhibiting the growth of L.monocytogenes as shown in . LAB are mainly from the genus of Enterococcus (E. lactis Q1, E. lactis 4CP3, E. faecalis), Lactobacillus (L. paracasei, L. plantarum, L. sakei, L. reuteri), and Pediococcus. Most of the bacteriocins produced by these LAB were able to inhibit the growth of L. monocytogenes. Based on , the treatment of E. lactis 4CP3 (enterocin A, B, P), and E. faecalis (enterocin AS-48) against L. monocytogenes resulted in growth inhibition activity as reported by Ben Braïek et al. [bib_ref] Genetic analysis with random amplified polymorphic DNA of the multiple enterocin-producing Enterococcus..., Ben Braïek [/bib_ref] [bib_ref] RAPD-PCR characterisation of two Enterococcus lactis strains and their potential on Listeria..., Ben Braïek [/bib_ref] and Sparo et al. [bib_ref] Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121, Sparo [/bib_ref]. Meanwhile, enterocin P produced by E. lactis Q1 reportedly exhibited antimicrobial activity, as observed in the stunted growth of L. monocytogenes after 7 days of treatment as compared to the untreated sample. Paracin C by Lactobacillus paracasei, Plantaricin (EF, W, JK, S) produced by Lactobacillus plantarum and bacteriocins produced by Lactobacillus sakei, L. reuteri, L. plantarum, L. fermentum inhibited the growth of L. monocytogenes while the treatment of Sakacin G produced by Lactobacillus sakei resulted in a decrease in the number of L. monocytogenes cells on roasted meat [bib_ref] Anti-listerial activity of bacteriocin-producing Lactobacillus curvatus CWBI-B28 and Lactobacillus sakei CWBI-B1365 on..., Dortu [/bib_ref]. In addition, pediocin produced by Pediococcus sp. was found to exert broad spectrum antimicrobial activity against L. monocytogenes [bib_ref] Antibacterial Activity of Pediocin and Pediocin-Producing Bacteria Against Listeria monocytogenes in Meat..., Khorshidian [/bib_ref]. . Bacteriocin produced by lactic acid bacteria tested on raw meat. ## Lactic acid bacteria bacteriocin inhibitory effect references Enterococcus lactis Q1 Enterocin P L. monocytogenes cell decreased to 6.47 ± 0.30 log unit after 7 days as compared to control that was not treated with E. lactis (7.25 ± 0.35 log unit after 14 days) and maintained until 28 days in the fridge. [23] Enterococcus lactis 4CP3 Enterocin A, B, and P The growth of listerial was completely inhibited from day 14 until 28. [17,21] The inhibition of L. monocytogenes growth on the rabbit meat during cold storage was detected on day 28. ## Enterococcus faecalis enterocin as-48 There was no detection of L. monocytogenes growth on the beef after 24 h treated with E. faecalis. ## [22] Lactobacillus paracasei Paracin C The growth of pathogenic bacteria was inhibited, and the color of the meat was retained until day 15. [26] Microorganisms 2022, 10, 684 6 of 20 . Cont. ## Lactic acid bacteria bacteriocin inhibitory effect references Lactobacillus plantarum Plantaricin EF, W, JK and S The growth of both spoilage bacteria was inhibited by L. plantarum until day 15 at 22 - C. [bib_ref] Inhibition of Listeria monocytogenes and Escherichia coli by bacterocin-producing Lactobacillus plantarum EC52..., Díaz-Ruiz [/bib_ref] Lactobacillus sakei Sakacin G The application of L.sakei takes on roasted meat resulted in a decrease in the number of L. monocytogenes cells. Meanwhile, for chicken breast, the inhibition effect depleted. [24] Lactobacillus sakei, L. reuteri, L. plantarum, L. fermentum Bacteriocins The formation of the inhibition zone after the treatment of bacteriocin demonstrated the growth inhibition of L. monocytogenes. [28] Pediococcus sp. Pediocin Pediocin and pediocin-like bacteriocins exerted a broad spectrum of activity against L. monocytogenes through the formation of pores in the cytoplasmic membrane and cell membrane dysfunction. [25] ## The application of enterocin on raw meat products Nowadays, the preservation methods in the food industry are evolving, with the use of bacteriocin aiding the process of preserving raw products. Bacteriocin is known for its capability to inhibit the growth of spoilage bacteria, such as L. monocytogenes, Salmonella sp., and E. coli in commercial food products so the quality can be maintained over a certain period. A newly reported byproduct rich in enterocin AS-48, and known to have a wide spectrum of antibacterial activity, might have good potential to be used as an additive since it achieved a good safety profile indicated by the negative result of the mutagenicity and genotoxicity assay test [bib_ref] Mutagenicity and genotoxicity assessment of a new biopreservative product rich in Enterocin..., Cascajosa-Lira [/bib_ref]. About 500 µL/animal/d of enterocin have been used as additives and were administrated in the drinking water of rabbits. As a result, the enterocin significantly affected the quality and mineral content of the rabbit meat, mainly iron and phosphorus. There are several species of Enterococcus used as preservatives in raw products. For instance, the cell-free supernatant of Enterococcus faecium TJUQ1 combined with the bacterial cellulose of Gluconabacter xylinus forms a composite film, BC-E, which shows antibacterial activity against L. monocytogenes after being soaked and applied on ground meat [bib_ref] Physical and antibacterial properties of bacterial cellulose films supplemented with cell-free supernatant..., Feng [/bib_ref]. Other examples were recorded as shown in [fig_ref] Table 3: The types of enterocin produced by Enterococcus sp [/fig_ref]. There are several techniques for incorporating bacteriocin into food products: (1) inoculation of bacteria producing bacteriocin directly onto the meat or meat products as a starter or protective culture, (2) the use of purified or semi-purified cell-free supernatant directly as a food preservative, and (3) incorporation of purified and semi-purified bacteriocin and in packaging material [bib_ref] Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated..., Da Costa [/bib_ref] [bib_ref] Bacteriocins from lactic acid bacteria and their applications in meat and meat..., Woraprayote [/bib_ref]. Abts et al. [bib_ref] Easy and rapid purification of highly active nisin, Abts [/bib_ref] stated that enterocin is used as a food preservative through two methods: (1) direct inoculation of bacteria producing enterocin directly as a starter or protective culture, and (2) the use of purified or semi-purified cell-free supernatant. However, enterocin is often widely applied as a starter culture. For example, E. faecium, E. mundtii, and E. classeliflavus have been used as a starter culture in the production of fermented sausage [bib_ref] The genus Enterococcus: Between probiotic potential and safety concerns-An Update, Hanchi [/bib_ref] [bib_ref] The role of enterococci in food and health, Moreno [/bib_ref] [bib_ref] Enterocins in food preservation, Khan [/bib_ref]. As a result, Enterococcus sp. competes partially during the meat fermentation process, inhibiting the growth of Listeria sp. in the product [bib_ref] The role of enterococci in food and health, Moreno [/bib_ref]. Enterocin is also associated with several biochemical activities that stimulate aroma development through glycolysis, proteolysis, and lipolysis activities. In addition, it also plays a role in reducing the activity of metmyoglobin (MetMbO), which is an important mechanism for maintaining meat color [bib_ref] Metmyoglobin reducing activity, Bekhit [/bib_ref]. Furthermore, enterocin also helps the degradation of stachyose and raffinose, the non-digestive oligosaccharides known as anti-nutrient factors [bib_ref] Fermentation of non-digestible raffinose family oligosaccharides and galactomannans by probiotics, Zartl [/bib_ref]. The use of purified or semi-purified cell-free supernatant is also one of the methods often used for raw products, conferring the same benefits as that of the inoculation method in terms of inhibiting the growth of L. monocytogenes. This method is particularly useful in stimulating the formation of compounds that give aroma and taste to the product. However, this preservation method also has several disadvantages. While bacteriocin can inhibit oxidative rancidity due to damage that occurs in fats or oils, the production of unwanted flavors may also occur as a result of fat hydrolysis by lipase enzymes or from contaminating microorganisms [bib_ref] Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated..., Da Costa [/bib_ref]. Several researchers have suggested that the use of purified or semi-purified cell-free supernatants is suitable for application in food products, as it is more effective than the direct inoculation of the bacteriocin-producing bacteria. The latter may cause damage to the food in hostile environments [bib_ref] Application of bacteriocins in food preservation and infectious disease treatment for human..., Zhang [/bib_ref]. During the purification process, all contaminants with low molecular weight are removed, leaving only the bacteriocin with a specific activity. The purification step allows for a more accurate determination of the biological activity of bacteriocin [bib_ref] Easy and rapid purification of highly active nisin, Abts [/bib_ref]. On the other hand, it has been reported in some cases that the use of cellfree supernatant on raw meat can potentially reduce the antimicrobial activity of bacteriocin due to the protein degradation that takes place when the supernatant is absorbed into the meat matrix [bib_ref] Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated..., Da Costa [/bib_ref]. Thus, Silva et al. [bib_ref] Application of bacteriocin and protective cultures in dairy food preservation, Silva [/bib_ref] and, Borges and Teixeir [bib_ref] Application of bacteriocins in food and health care, Borges [/bib_ref] have suggested an alternative method by incorporating the purified or semi-purified bacteriocin in packaging material to increase the activity and stability of the bacteriocin in complex food systems. Referring to [fig_ref] Table 3: The types of enterocin produced by Enterococcus sp [/fig_ref] , enterocin A and B from E. faecium were incorporated into an alginate film, which is one of the packaging techniques used for fermented dried sausages, minced pork, and ham [bib_ref] Bacteriocin: A Novel Approach for Preservation of Food, Bharti [/bib_ref]. ## Effects of enzyme, temperature, and ph on the activity of enterocin Characterization of bacteriocin is important to evaluate its effectiveness to be applied in the food industry. According to previous researchers, E. faecalis and E. faecium are the most commonly used bacteria from the genus Enterococcus, particularly in the food industry. The bacteria are used for the preservation of raw materials due to their high stability against extreme temperature and pH as compared to other species of Enterococcus as shown in [fig_ref] Table 4: Activity of bacteriocin produced by Enterococcus sp [/fig_ref]. The sensitivity of bacteriocin towards pH is diverse. The bacteriocin, known as enterocin As-48 produced by E. faecalis maintains its activity at pHs as high as 12 and temperatures of 121 - C for 15 min. Meanwhile, the activity of bacteriocin produced by E. lactis and E. durans was inhibited at 121 - C after 15 min. On the other hand, E. mundtii, which produces mundticin, can maintain its stability at 121 - C for 15 min; however, its activity is typically inhibited at pH 12. By referring to [fig_ref] Table 4: Activity of bacteriocin produced by Enterococcus sp [/fig_ref] , E. faecalis and E. faecium are suitable for application on food products, such as raw meats and vegetables since they are stable at high temperatures and pH. Bacteriocin produced by Enterococcus sp. as listed in [fig_ref] Table 4: Activity of bacteriocin produced by Enterococcus sp [/fig_ref] is typically sensitive to proteolytic enzymes, such as protease K and trypsin, which demonstrated the proteinaceous properties of the bacteria. Meanwhile, chymotrypsin, lipase, and catalase do not exert any effect on enterocin activity, indicating that the inhibition of bacterial growth is not due to the production of hydrogen peroxide [bib_ref] An antilisterial bacteriocin BacFL31 produced by Enterococcus faecium FL31 with a novel..., Chakchouk-Mtibaa [/bib_ref]. Application of this proteolytic enzyme leads to protein degradation, and therefore, is safe for human consumption [bib_ref] Fermentation of non-digestible raffinose family oligosaccharides and galactomannans by probiotics, Zartl [/bib_ref]. In the meantime, the loss of activity of bacteriocin depends on the formation of peptides and amino acid sequences. According to Gao et al. [bib_ref] Inhibitory effect of lactic acid bacteria on foodborne pathogens: A review, Gao [/bib_ref] lowering the pH will gradually deactivate the growth of microorganisms. Most cationic bacteria will undergo cell lysis as a result of stimuli formed by negatively charged molecules found on the bacterial cell surface, such as lipopolysaccharide (LPS), lipoteichoic and teichoic acids. The findings demonstrate that the bacteriocin produced by Enterococcus sp. has a high resistance to extreme pH ranges and has the potential to be used in acidic and alkaline processed foods [bib_ref] Effect of pH and heat treatment on bacteriocin activity of Pediococcus pentosaceus..., Adesina [/bib_ref]. Temperature is crucial in ensuring the stability of bacteriocin activity. Based on [fig_ref] Table 4: Activity of bacteriocin produced by Enterococcus sp [/fig_ref] , the activity of enterocin from E. faecalis, and E. mundtii is stable at a maximum temperature of 121 - C for 15 min while E. faecium, E. durans, and E. lactis could only withstand temperatures up to 100 - C for 30 min. Enterocin produced by Enterococcus is a heat-tolerant bacteriocin. The activity performed differs according to the species and molecular structure of the respective bacteriocin [bib_ref] Antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11 against some..., Abanoz [/bib_ref] [bib_ref] Effect of pH and heat treatment on bacteriocin activity of Pediococcus pentosaceus..., Adesina [/bib_ref]. Some highly heat-sensitive bacteriocin lose their activity at 50 - C due to the loss of their original secondary and tertiary structure as a result of denaturation [bib_ref] A study on characterization of new bacteriocin produced from a novel strain..., Gautam [/bib_ref]. The resistance of Enterococcus at pasteurization temperature and its adaptability to substrate and growth conditions demonstrates its potential application in food products. ## Various methods used in raw meat preservation Preservation of raw meat is crucial to ensure that the quality of the product is maintained throughout the long period of transportation and commercialization without damaging the texture, color, and nutritional value of the food. Currently, there is high demand for food that is free from synthetic ingredients. Consequently, biopreservation techniques have started to gain significant interest for application in the food industry. One of the challenges faced by the butchers and sellers is to maintain the quality of raw products while ensuring that the products are free from unwanted microbial growth. This is particularly critical for global suppliers who import raw materials to be commercialized around the globe [bib_ref] Influence of storage period and packaging methods on quality attributes of fresh..., Bagdatli [/bib_ref]. Therefore, various methods have been invented to solve the problem. These methods are categorized into four categories based on the use of (1) natural biopreservatives and chemicals, (2) refrigeration technique, (3) innovative packaging, and (4) non-thermal processing [fig_ref] Table 5: Various methods used in preserving meat [/fig_ref]. ## Application of chemical and organic ## Biopreservation The application of bacteriocin produced by lactic acid bacteria with a specific antimicrobial activity Helps in extending the shelf life of the product and subsequently, improves the unique taste and texture of the product Sensitive to food enzyme, low level of solubility, and easy to be absorbed [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref] [bib_ref] Production of bacteriocin and their application in food products, Udhayashree [/bib_ref] [bib_ref] The current approaches and challenges of biopreservation, Oluk [/bib_ref] Chemical preservation The process of controlling spoilage microorganisms using antimicrobial compounds, such as chlorides, nitrites, sulfides, and organic acid Adds flavor and extends the shelf life of the product Causes change in color, bad odor, and long-term harmful effects on human health [bib_ref] Food: Its preservatives, additives and applications, Abdulmumeen [/bib_ref] [bib_ref] Artificial preservative and their harmful effects: Looking toward nature for safer alternatives...., Anand [/bib_ref] [bib_ref] Application of various techniques for meat preservation, Pal [/bib_ref] Refrigeration Chilling Reducing the microbial reproduction at temperatures below its optimal temperature for growth by 2-5 - C. Ensures the cleanliness and safety of the meat, prolongs the shelf life, and retains the nutritional quality An increase in air velocity or a decrease in temperature affects the cooling time, as well as the slow heat-release throughout the meat tissues [bib_ref] Preservation technologies for fresh meat-A review, Zhou [/bib_ref] Super Chilling The process of freezing the water content in a particular product at 1-2 - C. Prevents the growth of microbes, reduces labor cost, and maintains the product weight Complex calculations are required to determine effective heat transfer as well as temperature control [bib_ref] Preservation technologies for fresh meat-A review, Zhou [/bib_ref] [bib_ref] Advanced meat preservation methods: A mini review, Ur Rahman [/bib_ref] Freezing The temperature of −55 - C is the ideal storage condition to freeze the product to ensure that the product quality is maintained Helps in inhibiting microbial growth and stops the enzyme activity Deformation of product occurs due to the cryogenic process, affecting the commercialization process [bib_ref] Preservation technologies for fresh meat-A review, Zhou [/bib_ref] [bib_ref] Application of various techniques for meat preservation, Pal [/bib_ref] [bib_ref] Effect of chilling, freezing and thawing on meat quality: A review, Aidani [/bib_ref] Packaging Vacuum packaging The use of packaging for raw meat includes the use of air-permeable packaging, low oxygen vacuum, low MAP oxygen with anoxic gas, and high MAP oxygen This process can prevent the product from being contaminated and reduced in weight, increase the tenderness of the meat, and maintain the color of oxymyoglobin in meat The active compound is unstable during the process of spraying the packaging material and the mass is too small to be transferred to the product Biopreservation is a method of preserving food by using microorganisms as a protective culture. This method is performed by inoculating the food with selected lactic acid bacteria to inhibit the growth of spoilage bacteria [bib_ref] The current approaches and challenges of biopreservation, Oluk [/bib_ref]. The demand for high-quality processed foods that are safe to eat has encouraged the application of this method in the food industry. For example, bacteriocin known as nisin is one of the food preservatives produced from lactic acid bacteria, which have been commercialized and approved by the FDA [bib_ref] Bio-preservation challenge for shelf-life and safety improvement of minced beef, Salem [/bib_ref]. According to Singh [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref] bacteriocin is considered a good preservative substance as it does not stimulate the immune response (non-immunogenic) and has high thermal resistance, as well as extensive antimicrobial activity. In the meat-based industry, bacteriocins, such as nisin, enterocin As-48, enterocin A and B, sakacin, leucocin, and pediocin are highly effective at inhibiting the growth of L. monocytogenes and other pathogenic bacteria [bib_ref] Biopreservation, promising strategies to improve the safety and shelf-life of food: A..., Bekuma [/bib_ref]. However, the antimicrobial activity of nisin is very limited when applied to meat products because of its low solubility rate, besides the potential destruction of the enzyme activity related to inhibition of pathogenic bacteria [bib_ref] Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated..., Da Costa [/bib_ref]. Thus, enterocin As-48, enterocin A and B, sakacin, leucocin, and pediocin are more effective for meat preservation as the majority of these bacteriocins belong to class II, known to inhibit L. monocytogenes [bib_ref] Antibacterial efficacy of nisin, pediocin 34 and enterocin FH99 against L. monocytogenes,..., Kaur [/bib_ref]. The ability of bacteriocin to inhibit the growth of pathogenic bacteria in the food is attributable to its ability to form electrostatic interaction with the negatively charged phosphate groups in the cellular membrane of the targeted bacteria. This interaction occurs with initial bonds formed on the cell membrane, followed by the formation of pores that induces cell lysis [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref]. ## Chemical preservation The chemicals that are commonly added to the food to maintain the nutritional value and quality of the food include chlorides, nitrites, sulfides, and organic acids. The combination of this method with freezing techniques often provides the best protection to the meat product. The storage methods alone are not effective in preventing oxidative disorders and inhibiting microbial or enzyme activity. On the other hand, the combined methods can increase the stability, maintain the freshness and nutritional value as well as the quality of the product [bib_ref] Application of various techniques for meat preservation, Pal [/bib_ref]. Nitrites are often used in the process of meat and cheese production as an additive to increase the stability of the product. European Union under Commission Regulation (EU) No 1129/2011 has allowed the use of nitrites, such as sodium nitrite, potassium nitrite, and sodium nitrate in food, However, the use of nitrate is limited to 150 mg Kg −1 for meat processing and 100 mg Kg −1 for meat sterilization processes [bib_ref] Nitrates/nitrites in food-risk for nitrosative stress and benefits, Karwowska [/bib_ref]. Nitrate can maintain the quality of raw meat through the formation of bonds between the nitrate oxide (NO) in the nitrate and the ferum ion (Fe 2+ ) at the center of the myoglobin porphyrin ring system, resulting in the formation of unstable nitrosomyoglobin. The nitrosomyoglobin may be converted into a characteristic red pigment in meat products called nitroso-myochromogen through a heating process in an acidic state (fermented meat) [bib_ref] Nitrates and Nitrites in meat products, Govari [/bib_ref]. However, this method has become a concern among buyers in terms of the potential negative impact on human health and worse, it can be fatal. For example, nitrite is known to potentially inhibit the growth of Clostridia sp. in meat products but may also react with the secondary amines to form carcinogenic nitrosamine, which is harmful to the fetus [bib_ref] Application of bacteriocins in food and health care, Borges [/bib_ref]. As for organic acids, they act as antioxidants and prevent damage to meat products. This acid is easy to find and typically non-toxic, making it suitable for application in raw meat processing [bib_ref] Use of organic acids for preservation and safety of traditional meat products, Mir [/bib_ref]. Thus, FDA has classified organic acids as a Generally Recognized as Safe (GRAS) for application in meat products, with no known side effects reported [bib_ref] Application of various techniques for meat preservation, Pal [/bib_ref]. ## Refrigerator ## Chilling A traditional cooling temperature is usually between 0 - C and 7 - C. This method is used to reduce microbial growth in food. Chilling is very important in maintaining meat hygiene, safety, quality, and appearance. Chilling by air can lower the temperature on the surface of the carcass and increase its drying rate. As a result, the microbial growth in meat products can be reduced [bib_ref] Preservation technologies for fresh meat-A review, Zhou [/bib_ref]. ## Super chilling Super chilling is a process that involves a change in temperature between conventional cooling and freezing. This method occurs through two stages namely (1) initial freezing, and (2) heat absorption. At the initial freezing, ice of approximately 1-33 mm in size is formed when the temperature of the food is within 1-2 - C. Thereafter, the heat on the outer layer of the food will be absorbed, promoting equilibration in the temperature between its inner and outer layers during the storage and distribution process [bib_ref] Superchilling of muscle foods: Potential alternative for chilling and freezing, Banerjee [/bib_ref] [bib_ref] A histological study of the microstructure sizes of the red and white..., Lilian [/bib_ref]. There are several benefits in applying this method that include the inhibition of unwanted microbial growth and extension in the shelf life of the product by at least 1.4 to 4 times as compared to that of traditional methods, However, this method was found effective only on seafood and non-meat products as it often results in the loss of water content in meat [bib_ref] Superchilling of muscle foods: Potential alternative for chilling and freezing, Banerjee [/bib_ref]. ## Freezing Freezing is one of the methods commonly used in ensuring that perishable food, such as raw food, lasts longer. The ideal storage temperature for frozen meat is −55 - C, which maintains the quality of the food. This freezing process allows the temperature of the meat to drop to the desired temperature causing the formation of ice crystals in the meat. The formation of these ice crystals prevents the changes in the texture and taste of the meat [bib_ref] Advanced meat preservation methods: A mini review, Ur Rahman [/bib_ref] since the low temperature can minimize enzyme reaction processes, reduce oxidative rigidity, ice recrystallization, and reduce the damage rate. Note that some products may still be damaged during storage [bib_ref] Preservation technologies for fresh meat-A review, Zhou [/bib_ref] [bib_ref] Recent research on factor influencing the quality of frozen seafood, Nakazawa [/bib_ref]. ## Packaging ## Vacuum packaging Vacuum packaging is a method of modified atmosphere packaging (MAP), which involves the removal of air from gas-tight packaging before an immediate sealing. Air is an important factor in the regulation of microbial growth in food products. Therefore, air should be removed from the packaging as it supports the growth of lactic acid-producing bacteria, which are capable of producing carbon dioxide which is important in preventing the growth of food spoilage bacteria. As a result, the shelf life of the product can be increased [bib_ref] Influence of skin packaging on raw beef quality: A review, Stella [/bib_ref]. The carbon dioxide emission will increase rapidly by 10 to 20% within the first 4 h of the process, and subsequently, reach a maximum level of up to 30%. In the meantime, the oxygen level will be reduced to 1 to 3% as a result of the enzyme activity that occurs in the meat [bib_ref] Packaging and storage practices of meat, Mathew [/bib_ref]. According to Stasiewicz et al. [bib_ref] Quality of meat products packaged and stored under vacuum and modified atmosphere..., Stasiewicz [/bib_ref] , sausages that were subjected to this process have a more palatable taste compared to that of the MAP. However, there are some negative opinions from researchers regarding the use of this packaging method. The use of vacuum packaging for food products is constrained by the fact that the red pigment of the fresh meat will change into dark purple after the packaging process (Deoxymyoglobin), which influences the buyer's decision in buying the product [bib_ref] Fresh meat packaging: Trends for retail and food service, Nassu [/bib_ref]. Furthermore, this method also affects the water content in meat as compared to the MAP method due to the high pressure and long storage period. ## Modified atmosphere packaging (map) This process involves the removal of gas in the environment and replacing it with other gases, such as oxygen, carbon dioxide, and nitrogen. Oxygen acts as an inhibitor for the growth of anaerobic bacteria and can maintain the myoglobin form. Meanwhile, carbon dioxide can inhibit the growth of aerobic spoilage bacteria and nitrogen can maintain the shape of the package [bib_ref] Influence of storage period and packaging methods on quality attributes of fresh..., Bagdatli [/bib_ref]. These gases can be used separately or in combination to achieve optimal effect. In addition, several parameters influencing the effectiveness of MAP have been identified, such as the film permeability to oxygen, carbon dioxide, water vapor, film thickness, the surface area of the packaging, and free volume in the package [bib_ref] Modified atmosphere packaging technology of fresh and fresh-cut produce and the microbial..., Caleb [/bib_ref]. ## Active packaging Active packaging is a type of food packaging method that has an additional function other than inhibiting microbial growth. This method involves absorbing the chemicals derived from food or the environment surrounding the package and secreting substances that include the preservative, antioxidants, and flavoring compounds into the food or its surroundings [bib_ref] Active and intelligent packaging in meat industry, Fang [/bib_ref]. There are two types of active packaging, namely the active absorbent system (absorber) and active release system (release). Examples include oxygen absorption and the use of carbon dioxide emitters and absorbers, moisture absorbers, and antimicrobials. Compounds, such as carbon dioxide, oxygen, ethylene, moisture, or odors present in the product and its surrounding will be released. Then, other compounds will be mixed into the packaging of the product. Antimicrobial packaging is a system in which the growth of microorganisms is inhibited so the shelf life of the food can be extended by using natural antimicrobials. Most antimicrobial packaging is invented in the form of edible films or coatings [bib_ref] Antimicrobial packaging for meat products, Rawdkuen [/bib_ref]. Various antimicrobial compounds are incorporated in this system, such as organic acids, chitosan, bacteriocin, EDTA, lysozyme, essential oil, cinnamon, and many others as they are volatile and have great antimicrobial activity [bib_ref] Antimicrobial packaging for meat products, Rawdkuen [/bib_ref] [bib_ref] A comprehensive review on the application of active packaging technologies to muscle..., Ahmed [/bib_ref]. These antimicrobial compounds are often used by associating them with the film coating and various antimicrobial agents to further enhance the effectiveness of the antimicrobial activity of the agents that are already present in the matrix. The compounds will be released to the food surface either through migration or evaporation headspace [bib_ref] A comprehensive review on the application of active packaging technologies to muscle..., Ahmed [/bib_ref]. Through this combination, the antimicrobial agents can be released gradually to control the level of antimicrobial activity and sensory changes in food products [bib_ref] Antimicrobial edible films and coatings for meat and meat products preservation, Sánchez-Ortega [/bib_ref]. ## Non-thermal processing ## Ionizing radiation Radiation is a preservation process using radiation, such as gamma, infrared, and UV on the product. It is also known as cold sterilization. As a result of this radiation energy, the chemical bonds in the microbial DNA molecules are broken, stunting the growth of the microorganism [bib_ref] Factor influencing the microbial safety of fresh produce: A review, Olaimat [/bib_ref]. The food processed using ionizing rays have distinct qualities compared to those of traditional methods. This process is carried out by subjecting the product to ionizing radiation. The chemical bonds present in the product will absorb the energy generated from the radiation, prompting some of the bonds to break down, which leads to the production of reactive and unstable free radicals. Subsequently, the free radicals will form a new bond from the adjacent compound, leading to the formation of radiolysis compounds [bib_ref] Irradiation and its potential to food preservation, Yousefi [/bib_ref]. ## High hydrostatic pressure High hydrostatic pressure is another example of the non-thermal process, which is used to deactivate microbes and reduce the chemical reaction in food at a pressure of 100 MPa (986.9 atm/1019.7 kgf/cm 2 ) [bib_ref] Microbiological food safety assessment of high hydrostatic pressure processing: A review, Rendueles [/bib_ref]. This process is called batch processing in which the packaged food is treated in a chamber surrounded by a pressure transmitter fluid. A semi-continuous system has also been invented for pumpable food, whereby the food is compressed without the use of a container and packaged aseptically. This method allows for microbial inactivation in a food product through disruption of the cell morphology and some susceptible cellular components, such as plasma membranes, ribosomes, and enzymes, including those that are essential for DNA replication and transcription [bib_ref] Application of various techniques for meat preservation, Pal [/bib_ref]. The study conducted by Bover-Cid et al. [bib_ref] Model for Listeria monocytogenes inactivation on dry-cured ham by high hydrostatic pressuring, Bover-Cid [/bib_ref] showed that at a high hydrostatic pressure of 600 MPa, the growth of L. monocytogenes was reduced by 5 logs in dry-cured ham after a 10-min treatment. Through this study, they confirmed that pressure, time, and temperature are interrelated with each other in inhibiting the growth of L. monocytogenes. Apart from inhibiting microbial growth, high hydrostatic pressure also increases the tenderness of the meat and the thermal gel capacity or actomyosin in the meat. Such effects are achieved due to the changes in the tertiary structure of myosin, causing hydrophobic and sulfhydryl bonds to be exposed, which strengthen the gel, especially at low salt concentrations [bib_ref] The effect of high pressure and subzero temperature on gelation of washed..., Malinowska-Pańczyk [/bib_ref]. ## Comparison of preservation method by biopreservation and commercial techniques Generally, each technique used for food preservation has its advantages in terms of inhibiting microbial growth and maintaining the quality of the food product. Similarly, each method also has its drawbacks as shown in [fig_ref] Table 5: Various methods used in preserving meat [/fig_ref]. [fig_ref] Table 6: Comparison of preservation method by biopreservation and commercial techniques [/fig_ref] shows a comparison between biopreservation and commercial techniques used in the preservation of raw meat in terms of cost, effectiveness, control range, type of food products, and methods and materials used. Overall, biopreservation techniques are known for their low cost and effectiveness in maintaining the intrinsic characteristics of the product, although the application is quite limited. Meanwhile, commercial preservation has been widely applied despite the high operating cost and the potential changes in the intrinsic characteristics of the food product. Biopreservation is known as a preservation technique applied to help extend the shelf life of food products by using beneficial bacteria namely LAB, which produces a broadspectrum antimicrobial agent called bacteriocin [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref]. Meanwhile, commercial techniques often involve the use of advanced technologies, such as high hydrostatic pressure, ionizing rays, vacuum and active packaging, modified atmosphere packaging (MAP), and freezing technology. Therefore, the cost of commercial techniques is generally higher than that of the biopreservation technique and typically requires skilled manpower to operate. For example, Yordanov and Angelova [bib_ref] High Pressure Processing for Foods Preserving, Yordanov [/bib_ref] stated that high-pressure technology is currently more expensive than other processing technology because it involves the use of highpressure vessel components and covers, temperature control devices, and material handling systems. On the other hand, the application of biopreservatives in food products does not affect their intrinsic properties [bib_ref] Bioprotective culture: A new generation of food additives for the preservation of..., Ben Said [/bib_ref] [bib_ref] Role of biopreservation in improving food safety and storage, Nath [/bib_ref]. Intrinsic properties are divided into several categories namely water content, nutrients, pH, redox potential, and biological structure [bib_ref] A growth of different types of microorganism, intrinsic and extrinsic factors of..., Moral [/bib_ref]. The use of commercial techniques in preserving food is less effective because of the potential effect on the intrinsic properties of food products. This is supported by Lilian et al. [bib_ref] A histological study of the microstructure sizes of the red and white..., Lilian [/bib_ref] who stated that supercooling from freezing technology led to water loss from the food product, causing changes in texture on the products. Additionally, Scetar et al. [bib_ref] Trends in meat and meat products packaging-A review, Scetar [/bib_ref] , and Mathew and Jaganathan [bib_ref] Packaging and storage practices of meat, Mathew [/bib_ref] have asserted that the lipid and protein oxidation that takes place in a vacuum and active packaging techniques often result in a significant change in the color of the food, particularly that of raw meat. Bacteriocin is known to exhibit extensive antimicrobial activity to inhibit the growth of pathogens. Nowadays, the commercial use of bacteriocin produced by Gram-positive bacteria, such as LAB is gaining popularity because of its inhibitiory activity against the growth of foodborne pathogens, especially Gram-positive pathogens [bib_ref] Recent approaches in food bio-preservation-A review, Singh [/bib_ref] [bib_ref] Fermentation of non-digestible raffinose family oligosaccharides and galactomannans by probiotics, Zartl [/bib_ref]. However, Gram-negative pathogens are less sensitive to bacteriocin produced by Gram-positive bacteria due to the presence of an outer layer membrane that effectively protects the pathogens [bib_ref] Strategies for the use of bacteriocin in Gram-negative bacteria: Relevance in food..., Prudêncio [/bib_ref]. Commercial techniques, such as high hydrostatic pressure and ionizing rays can disrupt the stability of the outer membrane layer of Gram-negative bacteria and inhibit their growth. However, this method is not applicable for Gram-positive bacteria as it requires higher pressure to be inactivated. Therefore, the combination of biopreservative application with other commercial techniques can enhance inhibitory activities against foodborne pathogens. For instance, the ability of biopreservatives to inhibit the pathogen of Gram-positive bacteria can be exploited via the use of high hydrostatic pressure [bib_ref] Strategies for the use of bacteriocin in Gram-negative bacteria: Relevance in food..., Prudêncio [/bib_ref]. Moreover, based on the study of Babich et al. [bib_ref] Effect of biopreserv-atives combined with modified atmosphere packaging on the quality of..., Babich [/bib_ref] fruit samples that had been treated with biopreservatives and packaged using modified atmosphere packaging (MAP) produced relatively good microbiological results after storage for 25 days compared to not combining the techniques. Biopreservatives are commonly applied in food, such as dairy products, meat, and vegetables. For example, Lactococcus lactis, which produces bacteriocin known as nisin, is used as a starter culture in cheese making to inhibit the growth of L. monocytogenes [bib_ref] Application of bacteriocin and protective cultures in dairy food preservation, Silva [/bib_ref]. Meanwhile, E. faecium is often used in meat-based products, such as fermented sausages, minced pork, and ham [bib_ref] Bacteriocin: A Novel Approach for Preservation of Food, Bharti [/bib_ref]. The products that are often subjected to preservation using commercial techniques include dairy products, meat, vegetables and fruits, fresh juices, vegetable purees, and jams. The high hydrostatic pressure technique is typically used to improve the coagulation properties of milk and retain the moisture in fresh cheese. It is also used to preserve and retain the color and stability of fruit juices during the storage process [bib_ref] High pressure processing of foods: A review, Srinivas [/bib_ref]. On the other hand, freezing technology allows for perishable foods, such as meat to last longer [bib_ref] Advanced meat preservation methods: A mini review, Ur Rahman [/bib_ref]. # Conclusions The preservation effects of bacteriocin produced by lactic acid bacteria of the genus Enterococcus are discussed in this review. The use of natural food preservation aids in the reduction of the use of chemical ingredients that may be damaging to consumer health. This review compiles various methods of food preservation techniques that can be applied at multiple scales. The application of biopreservatives combined with other techniques should be investigated further in future research to determine the most effective way in preserving food using via biopreservation techniques. The findings will benefit the food industry, in addition to enhancing the safety and quality of food products that are delivered to consumers. [fig] Figure 1: Chemical structure of enterocin. [/fig] [table] Table 1: Enterocin classification. [/table] [table] Table 3: The types of enterocin produced by Enterococcus sp. used in raw meat products. [/table] [table] Table 4: Activity of bacteriocin produced by Enterococcus sp. against enzymes, temperature, and pH. [/table] [table] Table 5: Various methods used in preserving meat. [/table] [table] Table 6: Comparison of preservation method by biopreservation and commercial techniques. Cost Low in cost as there is no need for advanced equipment High in cost due to the use of advanced technology and skilled manpower Effectiveness More effective as it does not affect the intrinsic properties of the product Less effective as it affects the intrinsic properties of the product Range of control of food pathogens More efficient against food pathogen of Gram-positive than Gram-negative Less efficient against food pathogen of Gram-positive than Gram-negative High hydrostatic pressure, ionizing radiation, vacuum and active packaging, modified atmosphere packaging (MAP), and refrigeration. [/table] [bib_ref] Enterocins: Bacteriocins with applications in the food industry, Alvarez-Cisneros [/bib_ref]

Acute Hydrocephalus Following a Spontaneous Ventriculoperitoneal Shunt Catheter Fracture With Scrotal Migration # Introduction The vast majority of complications involving ventriculoperitoneal (VP) shunt procedures are presented at an average of three to four months of surgery, mainly in the pediatric population and somehow related to the distal catheter functioning. Although distal catheter migration usually produces mild and benign implications, it might result in VP shunt dysfunction (and ultimately acute hydrocephalus as reported), testicle torsion, or perforations, which are disabling and possibly lethal conditions. The distal catheter's scrotal migration is a poorly characterized, rare condition with no consensus regarding management due to its irregular and variable presentation. To date, there are less than 30 cases reported involving distal catheter migration to the scrotum. To the best of our knowledge, none of them involved non-traumatic system fractures and distal catheter migration. Henceforth, the elucidation of the underlying mechanisms involving distal catheter migration is still unclear in medical literature and might be aided by the reports of series and cases with variable presentations of this condition. This study reports the first case of a young adult patient that presented a scrotal migration of non-traumatic distal catheter fracture, with no inguinal hernia history upon admission. ## Case presentation A 22-year-old male was presented to the emergency department with a seven-day medical history of headache, reduced consciousness level, and vomiting. The patient was admitted with a Glasgow Coma Scale score of 13 (E:3 V: 4 M:6), photoreactive pupils, and no other significant deficits. The family companion referred to a unilateral scrotal protruding and painful mass noticed by the patient two days following the first symptom. A slow filling of the occipital ventriculoperitoneal shunt valve was detected during physical examination. The subject was otherwise healthy. Admission head CT scan showed hydrocephalus and an appropriate proximal catheter positioning. Distal CT scan tridimensional reconstruction showed catheter discontinuation around the thoracoabdominal transition level. Further evaluation showed a hypodense collection in the scrotal region with an intrinsic filamentary coiled hyperdensity within the mass, suggesting the catheter placement within the scrotum. General surgery assessment performed on the same anesthetic procedure suggested an indirect inguinal hernia containing a peritoneal fluid-filled fold, with no intestinal loop involved. The patient was submitted to a VP shunt revision surgery and further open inguinal hernia surgical repairment. He was discharged on day 2 of in-hospital admission with no complaints or other complications. The patient authorized all the information and images provided in this report through an official personal statement in agreement with our ethics committee. # Discussion Until this article's literature research, there was a total of 29 case reports involving scrotal complications of VP shunting procedures. A case series on this condition involving 25 patients has reported a 6.8 months interval between surgery and symptom onset, in contrast with another study, published in 2017, reporting a three-to four-month interval. In contrast, our patient presented with an atraumatic fracture of the distal catheter leading to its migration at eight years following the procedure. In consensus with other reports, shunt migration was more common on the right side, as in the presented case, which is partially explained by the incidence of hernias in the pediatric age group. Despite the scarcity of information regarding management, etiology, and risk factors of the distal catheter migration to the scrotum, the literature reveals an incidence of inguinal hernias following shunting as high as 16.8% in the pediatric population, and mostly being the first clinical repercussion of a patent vaginal process, present in up to 90% of male newborns and 15% of male adults. Although distal catheter migration usually produces mild and benign implications, it might result in VP shunt dysfunction (and ultimately acute hydrocephalus as reported) and acute scrotum/testicle torsion perforations, which are disabling and possibly lethal conditions. Multifactorial mechanisms underlying the condition and the risk factors involved hypothetical but somehow consensual among the available literature. There are several hypotheses around the mechanism and predisposing factors of scrotal migration. The most widely accepted theory is based on the fact that shunting leads to increased intra-abdominal pressure due to the drainage of cerebral-spinal fluid into the abdominal cavity, resulting in dissection and re-opening (or prevention of the closure) of the vaginal process, known to be the leading cause of inguinal hernias in the pediatric population. Furthermore, peritoneal cavities volume is known to be related to the body surface, in the proportion of 80mL/m 2. A small peritoneal cavity, such as present in children, might predispose this downward migration of the distal catheter in increased abdominal pressure situations. In this specific case, the patient presented an asymptomatic patent vaginal process seen in the surgical correction. Considering the scarce information on this rare condition and the lack of guidelines in these situations, the VP shunt revision followed by surgical closure of the patent vaginal process seems consensual in the reports and series available in the literature. Our patient was submitted to a VP shunt revision, followed by inguinal and abdominal cavity exploration for the patent vaginal process closure in the same anesthetic period. As the proximal catheter and valve function and patency could be proven during the revision surgery, only the distal catheter was changed and connected to the system. Despite the absence of proven modifiable factors involving this condition in the literature, some deductible factors are hypothesized to be related to distal catheter migration when considering non-fractured systems. Low, fixed-pressure valves and the extra-lengthy catheters employed to avoid reoperation (especially when considering growing individuals) are the more widely accepted factors involved. Conversely, this report presents the first documented case of a distal catheter fracture with a scrotal migration to the best of our knowledge, resulting in a non-functioning VP shunt. Some of the factors potentially related to the atraumatic system fracture are the first surgery age onset, taken during the individual growth spurt, and the extra-tight peritoneal-end sutures, resulting in a traction pressure of the distal catheter. Our experience proposes the loose suture sealing of the abdominal catheter to avoid excessive traction of the distal catheter, especially in individuals expected to present a significant stature increasing in the future. Another essential option to consider in these situations, when available, is the laparoscopic placement of the distal catheter in the subdiaphragmatic space, which is associated with a significant reduction of valve dysfunction (including fractures). # Conclusions This article reports a rare presentation of VP shunt dysfunction being the first to describe an acute hydrocephalus presentation following non-traumatic distal catheter fracture and scrotal migration. Even though distal catheter migration is not an avoidable complication, some surgical strategies are hypothesized to reduce its incidence, as reported in this study. Increasing awareness of such complications may provide means to avoid them and provide better care to the patients. # Additional information disclosures Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

A Matter of Taste: Lineage-Specific Loss of Function of Taste Receptor Genes in Vertebrates Vertebrates can perceive at least five different taste qualities, each of which is thought to have a specific role in the evolution of different species. The avoidance of potentially poisonous foods, which are generally bitter or sour tasting, and the search for more nutritious ones, those with high-fat and high-sugar content, are two of the most well-known examples. The study of taste genes encoding receptors that recognize ligands triggering taste sensations has helped to reconstruct several evolutionary adaptations to dietary changes. In addition, an increasing number of studies have focused on pseudogenes, genomic DNA sequences that have traditionally been considered defunct relatives of functional genes mostly because of the presence of deleterious mutations interrupting their open reading frames. The study of taste receptor pseudogenes has helped to shed light on how the evolutionary history of taste in vertebrates has been the result of a succession of gene gain and loss processes. This dynamic role in evolution has been explained by the "less-is-more" hypothesis, suggesting gene loss as a mechanism of evolutionary change in response to a dietary shift. This mini-review aims at depicting the major lineage-specific loss of function of taste receptor genes in vertebrates, stressing their evolutionary importance and recapitulating signatures of natural selection and their correlations with food habits. # Introduction Pseudogenes have historically been considered genomic fossils and junk DNA, because of their classic definition of non-functional sequences of genomic DNA, originally derived from functional genes, but containing mutations or lacking promoter sequences precluding their expression [bib_ref] Pseudogenes: are they "junk" or functional DNA?, Balakirev [/bib_ref]. However, an increasing number of studies have shown how some pseudogenes have a function regulating gene expression [bib_ref] Pseudogenes: pseudo-functional or key regulators in health and disease, Pink [/bib_ref] , or being transcribed into RNA [bib_ref] GENCODE pseudogenes, Frankish [/bib_ref]. Multiple studies also highlighted how pseudogenes have had a remarkable functional plasticity and a dynamic role in species' evolution [bib_ref] Death and resurrection of the human IRGM gene, Bekpen [/bib_ref] [bib_ref] Not so pseudo: the evolutionary history of protein phosphatase 1 regulatory subunit..., Korrodi-Gregório [/bib_ref] [bib_ref] Genetic variation in taste receptor pseudogenes provides evidence for a dynamic role..., Risso [/bib_ref] : chemosensory genes, in particular, have been characterized by a succession of birth-and-death processes in different lineages . The aim of this mini-review is to provide an integrative view of the evolutionary history of taste receptor genes, with a focus on the main gene losses that have occurred in different lineages, pinpointing the genetic and biological factors driving these episodes. ## Evolution of taste receptors Vertebrates can perceive at least five different taste qualities, each of which is thought to have evolved to face a challenge or play a specific role in species' evolution. Bitter taste, for instance, likely occurred as a defensive and protective mechanism to prevent species from ingesting potentially toxic and dangerous foods. Conversely, sweet foods are naturally attractive and accepted, since sugars serve as the main energy source for animals. Umami senses amino acids in proteins representing the taste of monosodium glutamate, naturally-occurring in meats, vegetables and fermented products. Salty and sour taste are additional protective mechanisms aimed at assuring internal sodium or acid-base balance, respectively. Genes encoding taste receptors for different tastes, as depicted below, are highly diversified in terms of both inter-and intra-specific conservation and differences [bib_ref] Genetics of taste receptors, Bachmanov [/bib_ref]. Although most authors primarily focused on vertebrates, a few studies highlighted the presence of a large GPCR gene family in Drosophila named Gustatory Receptor (GR) which shows a differential expression in feeding-related tissues and confers a fine taste sensitivity to a broad range of alkaloids and other bitter compounds [bib_ref] Sensory coding for feeding deterrence in the grasshopper Schistocerca americana, Chapman [/bib_ref] [bib_ref] Electrophysiological evidence for two transduction pathways within a bitter-sensitive taste receptor, Glendinning [/bib_ref] [bib_ref] Candidate taste receptors in Drosophila, Clyne [/bib_ref]. ## Bitter taste Bitter taste is recognized by receptors encoded by the TAS2R gene family [bib_ref] A novel family of mammalian taste receptors, Adler [/bib_ref]. These genes are expressed in type II taste bud cells, lack introns, are relatively small (∼1,000 bp) and have a short extracellular N-terminus. TAS2R products consist of G protein-coupled receptors (GPCRs), seven-transmembranedomains-proteins that detect and signal neurotransmitters and hormones, among other stimuli. In particular, the binding of bitter compounds to TAS2R receptors activates heterometric GTP-binding proteins, consisting of a taste-selective Gα subunit (i.e., α-gustducin) and its βγ and β3γ13 partners [bib_ref] Gustducin is a taste-cell-specific G protein closely related to the transducins, Mclaughlin [/bib_ref] [bib_ref] Gγ13 colocalizes with gustducin in taste receptor cells and mediates IP 3..., Huang [/bib_ref]. This initiates a phosphoinositide pathway that elevates cytoplasmic Ca 2+ and depolarizes the membrane via cation channels, resulting in the intracellular release of the taste bud transmitter ATP, which conveys signals through gustatory nerves to the brain [bib_ref] The cell biology of taste, Chaudhari [/bib_ref]. Studies on bitter perception highlighted a differential ability to detect the bitter molecule phenylthiocarbamide showing that this trait is linked to two haplotypes on TAS2R38 producing the two phenotypes "taster" and "nontaster" [bib_ref] Positional cloning of the human quantitative trait locus underlying taste sensitivity to..., Kim [/bib_ref]. Further studies on the 3D structure of TAS2R38 showed how PTC and its analogous molecule 6-npropylthiouracile (PROP) can form hydrogen bounds in the transmembrane domain of the "taster" form while this bond is not formed in the "nontaster" one [bib_ref] 3D structure prediction of TAS2R38 bitter receptors bound to agonists phenylthiocarbamide (PTC)..., Tan [/bib_ref]. The number of TAS2R genes and pseudogenes varies greatly among different vertebrate species, spanning from 0 in the bottlenose dolphin to 52 in the western clawed frog [fig_ref] FIGURE 1 |: Distribution of bitter taste receptor genes [/fig_ref]. A notable interspecific variation can be observed when comparing different classes of animals: mammals (n = 3-34) and reptiles (n = 3-10) for example, have a higher number of pseudogenes when compared to birds (n = 0-3) and fishes (n = 0). The limited amount of studies analyzing the TAS2R genes repertoire in amphibians precludes the possibility of making general considerations on this group. Further studies analyzing multiple amphibian species are therefore necessary to better comprehend the evolutionary history of these genes. Similarly, the number of functional TAS2Rs differs among different classes: n = 0-37 in mammals, n = 0-19 in birds, n = 52 in frogs, n = 1-6 in fishes and n = 11-39 in reptiles [bib_ref] Evolution of olfactory receptor genes in primates dominated by birth-and-death process, Dong [/bib_ref] [bib_ref] Extraordinary diversity of chemosensory receptor gene repertoires among vertebrates, Shi [/bib_ref] [bib_ref] Major taste loss in carnivorous mammals, Jiang [/bib_ref] [bib_ref] Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire, Li [/bib_ref] [bib_ref] Evolution of sweet taste perception in hummingbirds by transformation of the ancestral..., Baldwin [/bib_ref] [bib_ref] Birds generally carry a small repertoire of bitter taste receptor genes, Wang [/bib_ref] [bib_ref] Dietary specialization drives multiple independent losses and gains in the bitter taste..., Liu [/bib_ref]. Humans occupy an intermediate position, possessing 25 functional TAS2Rs and 11 pseudogenes mapping to chromosomes 5, 7, and 12 [bib_ref] A novel family of mammalian taste receptors, Adler [/bib_ref] [bib_ref] Lineage-specific loss of function of bitter taste receptor genes in humans and..., Go [/bib_ref]. This remarkable variation of TAS2R repertoires among vertebrates has been explained by a birth-and-death model involving a complex evolution of this family, made of gene expansions, contractions, deletions, and duplications in different lineages in response to environmental changes [bib_ref] Adaptive diversification of bitter taste receptor genes in mammalian evolution, Shi [/bib_ref] [bib_ref] Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor..., Shi [/bib_ref] [bib_ref] Lineage-specific expansions and contractions of the bitter taste receptor gene repertoire in..., Go [/bib_ref] [bib_ref] Copy number variation in TAS2R bitter taste receptor genes: structure, origin, and..., Roudnitzky [/bib_ref]. ## Sweet and umami taste Sweet and umami tastes are also recognized by GPCRs that activate a signal transduction cascade through α-gustducin. However, sweet and umami GPCRs are encoded by the TAS1R gene family, with genes bigger in size, containing introns and a large N-terminal extracellular domain [bib_ref] Perceptual variation in umami taste and polymorphisms in TAS1R taste receptor genes, Chen [/bib_ref]. This large extracellular domain is composed of two additional domains: a Venus Flytrap Domain and a cysteine-rich domain. The first is named after the carnivorous plant (i.e., Dionaea muscipula) as its structure can switch between open and closed conformations. The cysteine-rich domain links the first domain to the transmembrane ones and could facilitate the binding of allosteric proteins [bib_ref] Evolution. structure, and activation mechanism of family 3/C G-protein-coupled receptors, Pin [/bib_ref]. Both sweet and umami receptors are heterodimers formed by two members of the TAS1Rs gene family: the receptor encoded by the combination of TAS1R1+TAS1R3 genes senses umami compounds, while the one encoded by TAS1R2+TAS1R3 is activated by sweet substances [bib_ref] Mammalian sweet taste receptors, Nelson [/bib_ref]. A thorough research of the heterodimer TAS1R2+TAS1R3, showed how its combinations of closed-open conformations could host sweet molecules. The authors concluded that, in order to bind low molecularweighting sweeteners, at least one monomer has to be in the open configuration. The simultaneous binding on both monomers, instead, can take place only with a combination of a closed and an open monomer, bounded to a small and a large sweetener respectively [bib_ref] From small sweeteners to sweet proteins: anatomy of the binding sites of..., Morini [/bib_ref]. This latter observation is consistent with sweetener synergy, where the action of a sweetener upon the receptor is augmented in the presence a second sweetener; moreover, the authors found that sweet proteins can bind an external portion of the receptor other than the Venus Flytrap. Additionally, sweet and umami receptors are highly conserved among different species: most vertebrates have only three receptors sensing sweet and umami tastes (i.e., TAS1R1, TAS1R2, and TAS1R3) and this configuration rarely changes, dating the divergence of these genes before the separation of teleost fishes and tetrapods, around 400 million years ago [bib_ref] Two families of candidate taste receptors in fishes, Ishimaru [/bib_ref] [bib_ref] Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor..., Shi [/bib_ref]. However, some notable exceptions represented by lineage-specific changes in number of TAS1R genes, exist and will be discussed in a separate section of this mini-review. (2013); [bib_ref] Birds generally carry a small repertoire of bitter taste receptor genes, Wang [/bib_ref] , and [bib_ref] Dietary specialization drives multiple independent losses and gains in the bitter taste..., Liu [/bib_ref]. When different references indicated different numbers of TAS2R genes, the highest estimate was considered. Frontiers in Molecular Biosciences | www.frontiersin.org ## Salty and sour taste Unlike bitter, sweet, and umami, the receptors responsible for salt and sour perception are not well-known. However, candidates have been identified for both tastes: studies have shown how salt and sour taste sensations are presumably not detected through GPCRs like the other tastes, but through ion channels. The amiloride-specific and sodium-specific epithelial sodium channel (ENaC) has been proposed as a candidate for salt taste perception in vertebrates [bib_ref] Salt taste transduction occurs through an amiloride-sensitive sodium transport pathway, Heck [/bib_ref]. This membranebound ion-channel permeable to Na + is a heteromer constituted by four subunits named α, β, γ, and δ and encoded by the closely-linked SCNN1A, SCNN1B, SCNN1G, and SCNN1D genes. These subunits are organized in two transmembrane domains, forming the pore through which Na + can enter the intracellular environment. Modeling studies propose ligand binding-induced rotational movements of the pore altering the position of residues and enabling cation entrance [bib_ref] ENaC structure and function in the wake of a resolved structure of..., Kashlan [/bib_ref]. Genes encoding for them were found in both tetrapods and the coelacanth, indicating their ancient evolutionary origin [bib_ref] The epithelial sodium channel δ-subunit: new notes for an old song, Giraldez [/bib_ref]. Many ion channels have also been proposed as sour taste perception mediators, even though the genetic basis of the perception of this taste are still poorly known. However, two transient receptor potential (TRP) channels, PKD1L1 and PKD2L3 (polycystic kidney disease-1-and −3-like), have been identified as potential candidates for sour transduction [bib_ref] Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour..., Ishimaru [/bib_ref]. TRP structure comprises six transmembrane domains with a pore loop situated between the fifth and the sixth domains [bib_ref] TRP channels, Venkatachalam [/bib_ref]. PKD1 and PKD2 possess a domain with an apical pore at the top of such TRP structure, surrounded by negatively charged residues possibly drawing cations to this molecular entrance. Movements of the transmembrane helices are involved in the opening of the pore loop and thus in the activation of the receptor [bib_ref] Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2), Grieben [/bib_ref]. Although these remarkable efforts have been made to identify potential candidates for the transduction of salty and sour taste, a deep understanding of the molecular basis of these tastes is still lacking and further studies are therefore necessary to better support the physiological and genetic evidence on such candidate genes. ## Lineage-specific gene losses Although most vertebrates can detect the same five basic tastes, some have lost particular tastes along their evolutionary paths. For most cases, these lineage-specific losses have occurred in response to specific dietary changes, feeding ecologies or changes in the environment. This complex network of coevolution between organisms and niches is explored by the niche construction theory, where modifications of species' niches cause diverse evolutionary constraints and ecological inheritances [bib_ref] Niche construction, Odling-Smee [/bib_ref]. [fig_ref] TABLE 1 |: Correlation of pseudogene and taste losses in different lineages [/fig_ref] recapitulates the major lineage-specific taste losses occurred in different vertebrate lineages, together with their genetic signatures, as a consequence of the intimate correlation between taste behavior, feeding ecology and taste receptor function. Most of the taste losses regard sweet or umami tastes, associated with the pseudogenization of Tas1r2 and either Tas1r1 or Tas1r3 respectively: in particular, the loss of sweet taste perception for obligate carnivores is hypothesized to have been the result of their meat-only diets. Other animals have lost more than one taste quality, being adapted to swallowing their food whole: this has been associated with the loss of bitter Tas2rs and sour Pkd2l1 genes [bib_ref] Molecular evidence for the loss of three basic tastes in penguins, Zhao [/bib_ref]. In this paragraph, we explore some of the most exemplifying cases of taste losses, focusing on the genetic basis of these episodes. ## Aquatic mammals Several terrestrial mammals have returned to the water for a fully aquatic lifestyle: among these, Cetacea and Carnivores represent two orders that returned to the sea around 50 and 35 million years ago, respectively [bib_ref] Evolution of marine mammals: back to the sea after 300 million years, Uhen [/bib_ref] [bib_ref] Major taste loss in carnivorous mammals, Jiang [/bib_ref]. This change in lifestyle and habitat inevitably caused more or less dramatic changes in the anatomy and behavior of these creatures, depending on the lineage. One change in common to all these lineages is their feeding behavior: this dietary switch introducing meat in their diets reduced the importance of some tastes (i.e., bitter and sweet), because of the presence of little or no bitter and sweet compounds in meat. Many species of these orders also swallow their prey whole without mastication, reducing the importance of tasting their food. In addition, the high sodium concentration in oceans masks other tastes, further decreasing the need for taste in the feeding behaviors of these animals. These behavioral observations are consistent with anatomical evidence, showing how some aquatic mammals have atrophied taste systems with few or no taste buds [bib_ref] A comparative morphological study on the tongue and the lingual papillae of..., Yoshimura [/bib_ref] [bib_ref] Scanning electron microscopy study of the tongue and lingual papillae of the..., Yoshimura [/bib_ref]. A survey of taste receptor genes analyzed these loci in 12 species of toothed and baleen whales and compared it to various other species (i.e., rat, dog, human). The study better defined the repertoire of taste genes in the examined species, highlighting a relaxation of selection forces since the common ancestor of whales (between 36 and 53 Ma) and showing how all members of bitter and sweet/umami gene families have been pseudogenized because of shared premature stop codons. Moreover, the authors noted how the candidate gene for sour perception (i.e., Pkd2l1) is a pseudogene as well, while the candidate salty taste receptor genes (Scnn1a, Scnn1b, and Scnn1g) are intact and have experienced strong purifying selection [bib_ref] Massive losses of taste receptor genes in toothed and baleen whales, Feng [/bib_ref]. Another study showed how Tas1r1, Tas1r2, and Tas1r3 are also pseudogenized in the sea lion, because of deletions or nonsense mutations [bib_ref] Major taste loss in carnivorous mammals, Jiang [/bib_ref]. ## Terrestrial carnivores Carnivores that remained on mainland also experienced taste losses during their evolutionary history. Remarkably, sweet taste was independently lost in several lineages: dietary specializations, rather than dietary switches, were likely the drivers of these events. A study on the Carnivore order with different dietary habits (i.e., obligate carnivores, omnivorous and herbivores), for example, showed how the pseudogenization of the sweet taste receptor gene, Tas1r2, was widespread but independent in many lineages, because of mutations disrupting its openreading-frame. This pseudogenization is also related to animals' diets: exclusive meat eaters are in fact lacking an intact form of this gene. In addition, [bib_ref] Major taste loss in carnivorous mammals, Jiang [/bib_ref] analyzed Tas1r2 sequences in the order Carnivora: the authors calculated the nonsynonymous-to-synonymous substitution ratio (dN/dS) finding a ratio significantly lower than one. This led to the conclusion that purifying selection may have been acting on these genes of this order. However, despite their carnivorous diet, both the Canadian otter and the ferret have an intact Tas1r2 (sweet) receptor sequence, suggesting that additional factors may have been involved in shaping feeding strategies and gene loss/retention. Nonetheless, further studies showed how cats and felids' (i.e., tigers, cheetahs and lions) indifference toward sugar can be accounted by the pseudogenization of Tas1r2, reflecting their eating behaviors. As obligate carnivores, cats and other felids possess a high-protein diet, with little carbohydrates and no simple sugars contained in plants [bib_ref] Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar, Li [/bib_ref] [bib_ref] Analyses of sweet receptor gene (Tas1r2) and preference for sweet stimuli in..., Li [/bib_ref]. Conversely, despite their herbivorous diet, horses miss Tas1r2 [bib_ref] Evolution of the sweet taste receptor gene Tas1r2 in bats, Zhao [/bib_ref]. Thus, the correlation between diet and disrupting events upon this sequence may not always be the best explanation. Nonetheless, domestic cats with a carnivore diet still retain functional bitter taste receptor genes [bib_ref] A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43..., Sandau [/bib_ref]. Considering that bitter receptors are expressed in extra oral tissues , selective forces could have also acted on these compartments. Further studies investigating these extra-oral bitter taste receptors will be crucial in understanding the selective forces acting in these gain-andloss mechanisms. Another example comes from the giant panda and red panda, with specialized herbivore bamboo-based diets, have a functional copy of Tas1r2 but a pseudogenized Tas1r1 (encoding the umami receptor) because of insertions or deletions interrupting its open-reading-frame. This was interpreted as convergent evolution in response to adaptation to a specialized diet [bib_ref] The sequence and de novo assembly of the giant panda genome, Li [/bib_ref] [bib_ref] Pseudogenization of the umami taste receptor gene Tas1r1 in the giant panda..., Zhao [/bib_ref] [bib_ref] Comparative genomics reveals convergent evolution between the bambooeating giant and red pandas, Hu [/bib_ref]. Unlike panda, horse and cow intriguingly retain a functional Tas1r1 and have an herbivorous diet [bib_ref] Pseudogenization of the umami taste receptor gene Tas1r1 in the giant panda..., Zhao [/bib_ref]. Similarly, vampire bats lack a proper Tas1r2 even though sugars are present in blood, their sole food source [bib_ref] Mismatches between feeding ecology and taste receptor evolution: an inconvenient truth, Zhao [/bib_ref]. ## Birds An additional example of how evolution accounts for taste comes from birds, descendants from theropod dinosaurs with a carnivorous diet [bib_ref] The evolution of dinosaurs, Sereno [/bib_ref]. A study analyzing Tas1r genes in 10 bird species with different diets (i.e., insectivorous, nectarivorous, and frugivores) demonstrated how birds were lacking Tas1r2, while the two other Tas1r genes (i.e., Tas1r1 and Tas1r3) were present and intact. It was also suggested that the Tas1r2 loss likely occurred within Dinosauria, before the non-avian reptile and bird lineages split. However, the observation that hummingbirds, differently from other birds, showed a distinctive preference for nectar and sugar solutions, initially represented a paradox. This was solved by the striking finding that hummingbirds' Tas1r1 and Tas1r3, responsible for umami sensitivity in other species, were repurposed through the appearance of mutations leading to the acquisition of sugar responsiveness. In vitro reconstructions of chicken and hummingbird chimeric T1R1/T1R3 receptors, highlighted in fact how 19 amino acids in the hummingbird's T1R3 Venus Flytrap domain are responsible of modulating sucrose and sucralose perception. Intriguingly, a response to sucrose was only detected when all 19 amino acids were tested in the same chimeric receptor. These mutations were also found to be under positive selection, suggesting a new energy source unavailable to other birds [bib_ref] Evolution of sweet taste perception in hummingbirds by transformation of the ancestral..., Baldwin [/bib_ref]. In addition, other birds have lost more than one taste quality (similar to aquatic mammals): penguins, for example, have lost three basic tastes along their evolutionary path. A study analyzing genomes of 16 bird species has shown how penguins lack functional Tas1r1, Tas1r2, and Tas1r3. Further analyses showed that the pseudogenization of Tas1r1 happened in the common ancestor of all penguins, since its separation from tubenose seabirds . These taste losses correlate well with penguins' feeding behaviors of swallowing food whole and their tongue structure and function, suggesting that these animals don't have a real need of perceiving the taste of their food. ## Primates The evolutionary role of bitter taste receptors (i.e., TAS2Rs) for avoiding potentially toxic and harmful compounds is of particular importance when considering that even small changes in the TAS2R gene repertoire could affect feeding habits of different animals, considering ligand-receptor specificity [bib_ref] Genetics of taste receptors, Bachmanov [/bib_ref]. Analysis of primates showed a higher level of pseudogenes accumulation after the separation from the common ancestor of other species, such as rodents [bib_ref] Lineage-specific loss of function of bitter taste receptor genes in humans and..., Go [/bib_ref]. Nonhuman primates differ significantly in the TAS2R repertoire in respect to humans, showing higher pseudogenization rates. However, a relaxation of selective constraint on human TAS2Rs occurred instead of positive selection [bib_ref] Evolution of bitter taste receptors in humans and apes, Fischer [/bib_ref] [bib_ref] Relaxation of selective constraint and loss of function in the evolution of..., Wang [/bib_ref] [bib_ref] Lineage-specific loss of function of bitter taste receptor genes in humans and..., Go [/bib_ref] [bib_ref] Global diversity in the TAS2R38 bitter taste receptor: revisiting a classic evolutionary, Risso [/bib_ref]. In primates, lineage-specific gene duplication and pseudogenization events played a major role in shaping the TAS2R gene repertoire. In particular, only eight pseudogenes are in common to more than one primate species, while the majority (n = 23) of these pseudogenization events occurred specifically in different lineages: five in prosimians and tupais, and 19 in anthropoidea, respectively. Interestingly, three of these pseudogenes (TAS2R2, TAS2R62, and TAS2R64) are uniquely confined to Homo sapiens because of a two-base deletion at codon position 160, two fixed nonsense mutations at codon positions 235 and 292 and one fixed nonsense mutation at codon position 280, respectively. In addition, the twobase deletion inactivating TAS2R2 is polymorphic in modern human populations and archaic humans, and TAS2R64 also became a pseudogene in the orangutan because of a different nonsense mutation, showing convergent evolution [bib_ref] Lineage-specific loss of function of bitter taste receptor genes in humans and..., Go [/bib_ref] [bib_ref] Insights into hominin phenotypic and dietary evolution from ancient DNA sequence data, Perry [/bib_ref] [bib_ref] Probing the evolutionary history of human bitter taste receptor pseudogenes by restoring..., Risso [/bib_ref]. These lineage-specific gene losses likely reflect different responses to environmental changes, resulting from species-specific food preferences during primates' evolution [bib_ref] Lineage-specific loss of function of bitter taste receptor genes in humans and..., Go [/bib_ref] ; [bib_ref] Use it or lose it: molecular evolution of sensory signaling in primates, Liman [/bib_ref] [bib_ref] Eco-geographical diversification of bitter taste receptor genes (TAS2Rs) among subspecies of chimpanzees..., Hayakawa [/bib_ref] [bib_ref] Frequent expansions of the bitter taste receptor gene repertoire during evolution of..., Hayakawa [/bib_ref] [bib_ref] Probing the evolutionary history of human bitter taste receptor pseudogenes by restoring..., Risso [/bib_ref]. In particular, an in vitro study showed how the receptors encoded by the reconstructed human-specific pseudogenes and the respective chimpanzee orthologues recognized different ligands because of interspecific amino acid changes, likely allowing the adaptation to different environments and the identification of compounds of speciesspecific relevance [bib_ref] Probing the evolutionary history of human bitter taste receptor pseudogenes by restoring..., Risso [/bib_ref]. Similarly, a marked diversification of the TAS2R repertoire in terms of wholegene deletions, gene-conversion variations and copy number variations was identified among subspecies of chimpanzees, likely reflecting their subspecies-specific dietary habits [bib_ref] Eco-geographical diversification of bitter taste receptor genes (TAS2Rs) among subspecies of chimpanzees..., Hayakawa [/bib_ref] [bib_ref] Association of a bitter taste receptor mutation with Balkan Endemic Nephropathy (BEN), Wooding [/bib_ref]. # Conclusion Maynard Olson's "less-is-more" hypothesis sees gene loss as an engine of evolutionary change, representing a common response of different lineages undergoing similar environmental shifts [bib_ref] When less is more: gene loss as an engine of evolutionary change, Olson [/bib_ref] [bib_ref] Evolutionary biology: the lost appetites, Callaway [/bib_ref]. Some of the lineagespecific pseudogenization events discussed in this mini-review have been related to specific feeding behavior, dietary switches or environmental changes at both large and small scales, highlighting the plasticity and dynamism of the taste system [bib_ref] Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar, Li [/bib_ref] [bib_ref] Use it or lose it: molecular evolution of sensory signaling in primates, Liman [/bib_ref] [bib_ref] Pseudogenization of the umami taste receptor gene Tas1r1 in the giant panda..., Zhao [/bib_ref] [bib_ref] Genomic and genetic evidence for the loss of umami taste in bats, Zhao [/bib_ref] [bib_ref] Eco-geographical diversification of bitter taste receptor genes (TAS2Rs) among subspecies of chimpanzees..., Hayakawa [/bib_ref] [bib_ref] Major taste loss in carnivorous mammals, Jiang [/bib_ref] [bib_ref] Evolution of sweet taste perception in hummingbirds by transformation of the ancestral..., Baldwin [/bib_ref] [bib_ref] Massive losses of taste receptor genes in toothed and baleen whales, Feng [/bib_ref] [bib_ref] Birds generally carry a small repertoire of bitter taste receptor genes, Wang [/bib_ref] [bib_ref] Probing the evolutionary history of human bitter taste receptor pseudogenes by restoring..., Risso [/bib_ref]. In addition, the repertoire of bitter taste receptor genes has been associated with species' feeding behaviors, correlating with the fraction of plants in their diet as an evolutionary mechanisms protecting from ingesting potentially toxic compounds [bib_ref] Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire, Li [/bib_ref] [bib_ref] Birds generally carry a small repertoire of bitter taste receptor genes, Wang [/bib_ref] [bib_ref] Dietary specialization drives multiple independent losses and gains in the bitter taste..., Liu [/bib_ref] [bib_ref] The repertoire of bitter taste receptor genes in canids, Shang [/bib_ref]. However, birth-and-death events of taste receptor genes are not always correlated with species' feeding behaviors, where putative useless taste receptor genes are still present, making it possible to detect a taste quality that should not be present in their diet. These data suggest that there is still a gap in our understanding of the physiological function of these genes [bib_ref] Mismatches between feeding ecology and taste receptor evolution: an inconvenient truth, Zhao [/bib_ref] [bib_ref] Complex evolutionary history of the vertebrate sweet/umami taste receptor genes, Feng [/bib_ref] [bib_ref] Dietary specialization drives multiple independent losses and gains in the bitter taste..., Liu [/bib_ref]. This encourages further genetic and behavioral studies aimed at better identifying the role of taste receptor genes and pseudogenes in different species, in order to shed light on the evolutionary mechanisms that have inactivated genes exclusively in specific lineages. # Author contributions MA wrote the first draft of the manuscript. DR contributed to writing and editing the manuscript and created the figures/tables. [fig] FIGURE 1 |: Distribution of bitter taste receptor genes (blue) and pseudogenes (green) (i.e., TAS2Rs) among different lineages. Data were taken from Dong et al. (2009b), Shi and Zhang (2009); Jiang et al. (2012); Li and Zhang [/fig] [table] TABLE 1 |: Correlation of pseudogene and taste losses in different lineages. [/table]

Expense and benefit of neoadjuvant treatment in squamous cell carcinoma of the esophagus Background:The effectiveness of neoadjuvant treatment (NT) prior to resection of squamous cell carcinoma of the esophagus (SCCE) in terms of prolonged survival has not been proven by randomized trials. Facing considerable financial expenses and with concerns regarding the consumption of the patient's remaining survival time, this study aims to provide rationales for pretreating resection candidates.Methods: From March 1986 to March 1999, patients undergoing resection for SCCE were documented prospectively. Since 1989, NT was offered to patients with mainly upper and middle third T3 or T4 tumors or T2 N1 stage who were fit for esophagectomy. Until 1993, NT consisted of chemotherapy. Since that time chemoradiation has also been applied. The parameters for expense and benefit of NT are costs, pretreatment time required, postoperative morbidity and mortality, clinical and histopathological response, and actuarial survival.Results: Two hundred and three patients were treated, 170 by surgery alone and 33 by NT + surgery. Postoperative morbidity and mortality were 52% to 30% and 12% to 6%, respectively (p = n.s.). The response to NT was detected in 23 patients (70%). In 11 instances (33%), the primary tumor lesion was histopathologically eradicated. Survival following NT + surgery was significantly prolonged in node-positive patients with a median survival of 12 months to 19 months (p = 0.0193). The average pretreatment time was 113 ± 43 days, and reimbursement for NT to the hospital amounted to Euro 9.834.Conclusions: NT did not increase morbidity and mortality. Expenses for pretreatment, particularly time and costs, are considerable. However, taking into account that the results are derived from a non-randomized study, patients with regionally advanced tumor stages seem to benefit, as seen by their prolonged survival. # Background Neoadjuvant treatment (NT) of esophageal squamous cell cancer (SCCE) prior to surgery was thought to improve survival by reduction of the primary tumor lesion as well as of regional and systemic tumor spread [bib_ref] Randomized clinical trial of preoperative and postoperative adjuvant chemotherapy with cisplatin, vindesine,..., Roth [/bib_ref] [bib_ref] Preoperative therapy for esophageal cancer: a randomized comparison of chemotherapy versus radiation..., Kelsen [/bib_ref]. Meanwhile, to many gastroenterologists and surgeons, cytotoxic therapy prior to surgery appears to be a standard concept. However, until now, prospective randomized trials could not prove the effectiveness of chemoor chemoradiotherapy in terms of prolonged survival or a higher rate of cure, particularly in squamous cell carcinoma [bib_ref] A randomized study of chemotherapy, radiation therapy, and surgery versus surgery for..., Prise [/bib_ref] [bib_ref] Chemoradiotherapy followed by surgery compared with surgery alone in squamous-cell cancer of..., Bosset [/bib_ref] [bib_ref] Preoperative chemotherapy versus surgical therapy alone for squamous cell carcinoma of the..., Law [/bib_ref] [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref]. Additionally, there is an ongoing discussion on substantial risks of pretreatment to increase postoperative morbidity and mortality [bib_ref] Does neoadjuvant chemotherapy for carcinoma in the thoracic esophagus increase postoperative morbidity?, Tabira [/bib_ref] [bib_ref] Randomized trial of preoperative chemotherapy for squamous cell cancer of the esophagus, Schlag [/bib_ref]. Furthermore, considerable expenses in time and money have to be accepted when expecting neoadjuvant protocols to be beneficial. Facing considerable uncertainty upon effectiveness of NT we undertook a one-institution analysis to investigate whether application of NT nowadays is justified in terms of expenses and survival benefit. # Methods From May 1986 to March 1999, all patients carrying SCCE who were referred to our surgical department were documented prospectively. The patients underwent either transthoracic or transhiatal subtotal esophagectomy. Reconstruction of the intestinal route was achieved mostly using a gastric tube and in cases with previous gastric resection using colonic esophago-gastric interposition. Since 1989, NT was offered to patients with tumors mainly of the upper and middle third who all were staged by means of a computed tomography (CT) scan and endoscopic ultrasonography either T3/4 NX, or T2 N1, according to the 1992 UICC classification. Individuals obviously not suitable for esophagectomy were denied surgery and underwent endoscopic palliation. The majority of pretreatments were performed at our university hospital by the Departments of Gastroenterology (n = 20) and Hematology (n = 5), some in outside hospitals (n = 8). The neoadjuvant regimen during an initial period until 1993 consisted of chemotherapy with two cycles of cisplatin (100 mg/m 2 ) on day 1 along with 5-fluorouracil (400 mg/m 2 per day) on days 1 to 5. Participating in a multicenter trial comparing the effect of chemoradiotherapy + surgery to chemoradiation alone, we switched to three cycles of 5-fluorouracil, leucovorin, etoposid, and cisplatin, followed by an initial dose of etoposid and cisplatin, and radiation of 40 Gy with 5 × 2 Gy for 4 weeks [bib_ref] Combined preoperative chemotherapy and radiotherapy in patients with locally advanced esophageal cancer...., Stahl [/bib_ref] , so called FLEP + radiation . This resulted in 19 patients recieving chemotherapy alone, 14 cases underwent chemo-and radiotherapy. Esophagectomy was performed in all instances within 2-4 weeks after the end of pretreatment. Of particular interest was the length of the time period from histological confirmation of the diagnosis of esophageal malignancy to the day of surgery after NT. Additionally, the costs for the currently applied NT protocol FLEP + radiation were calculated. The response to pretreatment was classified clinically according to improvement of the ability to swallow and/or radiologically as, at least, a 50% reduction of the primary tumor size as "response", "no change", or "progression". Surgical morbidity was defined as "none", "minor" (e.g., insufficiency of the cervical anastomosis without systemic inflammatory response), or "major" (e.g., prolonged mechanical ventilation more than 7 days and any kind of abdominal or thoracic reoperation). Data on survival status were obtained by requests directed to the respective county administration. Results are expressed as means and standard deviation or percentages. To test for significant differences, the student's t-test (two-tailed) was applied for continuous data and Fisher's exact test was applied for categories. Overall survival including in-hospital deaths was estimated according to the Kaplan-Meier method [bib_ref] Nonparametric estimation from incomplete observations, Kaplan [/bib_ref] and comparison of significant differences in survival was calculated using the log-rank test. A p-value of < 0.05 was considered statistically significant. # Results Resection was performed in 203 patients; of these, 33 underwent pretreatment with the intention of performing subsequent resection. A comparison of both groups with regard to surgical procedures and patient characteristics is given in [fig_ref] Table 1: Baseline characteristics of 203 patients treated for squamous cell carcinoma of the... [/fig_ref]. Pretreated patients were, on average, somewhat younger and had a significantly shorter observation period. To compare tumor stages, the situation before NT was considered appropriate in the com- ## Figure 1 Schedule of cytotoxic interventions within the FLEP + radiation protocol. Medication is given for each cycle. bined treatment group. Because precise and prospective staging prior to resection was not sufficiently available in the surgery alone group, because it was not the prerequisite to decide on pretreatment or not, the final histopathological result was the source of TNM staging in those patients [fig_ref] Table 2: Tumor staging according to the UICC classification of 1992 [/fig_ref]. According to our inclusion criteria, there were significantly more T3 and T4 tumors and also a higher percentage of node positive patients in the combined modality group than in the group undergoing surgery alone. In contrast, the latter included considerably more early T1 and T2 stages. To the best of our knowledge, cytotoxic pretreatment, performed with no special prospective documentation in internal medicine departments, caused neither major morbidity nor mortality. With regard to the response to NT, there was relief of esophageal obstruction and or reduction of tumor size in 23 of 33 cases (70%), no change detected in 7 (21%), and progression in 3 (9%) patients. There was no difference in the response rate between preoperative chemotherapy with 13 of 19 (68%) and chemoradiotherapy with 10 of 14 (71%). Viable cells of the primary tumor lesion were no longer found in 11 of 33 (33%) resected specimens of patients after cytotoxic pretreatment, but two had either nodal or distant tumor spread. The percentage of potentially curatively resected cases was 70% in the surgery-alone group and 82% in the NT modality (p = n.s.). Hospital mortality following resection was 2 out of 33 (6%) in the NT group. It consisted of two septic multiorgan failures (MOF) post anastomotic leakage and postoperative pancreatitis, respectively. This was not elevated relative to surgery alone with 21 out of 170 (12%) (p = n.s.). Causes of death among patients with surgery alone were pneumonia and consecutive MOF (n = 8), anastomotic leakage (n = 4), pleura empyema (n = 3), bleeding and MOF (n = 2), hepatic failure (n = 2), and isolated heart disease (n = 2). Mortality numbers corresponded to fewer cases of postoperative morbidity after NT with 30% to 54% (p = 0.0215) and fewer days of postoperative hospital stay [23.4 ± 16.8 vs 30.1 ± 19.7 days (p = n.s.)]. Comparison of survival of all patients categorized in surgery alone and NT + surgery revealed a slight but not significantly better outcome after pretreatment [fig_ref] Figure 2: Comparison of survival of all patients undergoing surgery alone and NT +... [/fig_ref]. Two-year and five-year survival rates were 33% to 41% and 17% to 26%, respectively. Calculating survival only for T3-and T4-stages, the difference came close to but did not reach the level of statistical significance (p = 0.0538). Significantly longer survival following NT + surgery was found when comparing all node-positive patients. Two-year and five-year survival rates were 23% to 43% and 10% to 28%, respectively [fig_ref] Figure 3: Comparison of survival of all node positive patients undergoing surgery alone and... [/fig_ref]. Combining T3/4 stages with N1 resulted in even more pronounced prolongation of survival rates with 17% to 47% and 9% to 31% (p = 0.0096). Looking at the impact of the complete histopathological response of the primary tumor lesion on survival, we found a higher rate of survival at 2 years following complete response (61% vs 31%) but an even lower percentage at the 5-year margin (20% vs 31%). Pretreatment for all 33 cases required an average of 113 ± 43 days, counted from histologically confirmed diagnosis to surgery. Costs for NT were calculated for the most recently applied protocol concept of FLEP + 40 Gy of radiation. It consisted of 4 × 7 days of hospital stay at the Department of Gastroenterology (28 × Euro 233), 10 days at the Department of Radiation Oncology (10 × Euro 231), including Euro 7576 for cytotoxic drugs and another Euro 1022 for outpatient radiation. This adds up to Euro 9834, which will be the reimbursement for the hospital only for NT. This also has to cover expenses for the chemotherapy medication. # Discussion The prognosis of SCCE, at least in locally advanced stages, treated by surgery alone is disappointing. In particular, early local spread to adjacent structures and lymphatics and the natural limits for surgical radicality within the mediastinum render five-year survival rates in unselected patient cohorts to a level of about 15% [bib_ref] Surgical therapy of oesophageal carcinoma, Mÿller [/bib_ref] [bib_ref] Ratio of invaded to removed lymph nodes as a predictor of survival..., Roder [/bib_ref] [bib_ref] Transhiatal oesophagectomy compared with transthoracic resection and systematic lymphadenectomy for the treatment..., Horstmann [/bib_ref]. This was and is the rationale behind the attempt, using cytotoxic pretreatment, to reduce both the primary tumor size and the potentially regional and early single cell systemic spread. The phenomenon of response, in some instances disappearance of viable tumor cells histopathologically [bib_ref] Downstaging of esophageal cancer after preoperative radiation and chemotherapy, Vogel [/bib_ref] , has led to widespread application of neoadjuvant regimens without having proved by means of controlled randomized trials that this will improve long-term survival [bib_ref] Multimodal therapy for squamous carcinoma of the oesophagus, Lehnert [/bib_ref] [bib_ref] Preoperative (neoadjuvant) chemoradiotherapy in oesophageal cancer, Geh [/bib_ref]. One prerequisite to decide whether NT has the potential to prolong survival is to focus on a single tumor entity. Even recent randomized studies [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref] [bib_ref] The role of neoadjuvant therapy in surgically resectable esophageal cancer, Swisher [/bib_ref] have included adeno-and squamous cell carcinoma. Since the latter is known to be more susceptible to radiation, a potential benefit for this subgroup might be missed. The basis of comparing surgery alone with NT + surgery has to be the tumor status prior to pretreatment. Walsh et al. did find NT effective in adenocarcinoma of the esophagus but they gave no detailed information regarding the tumor stages prior to chemoradiation [bib_ref] A comparison of multimodal therapy and surgery for esophageal adenocarcinoma, Walsh [/bib_ref]. Response rates to neoadjuvant regimens range from 19% in previous studies [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref] to 70% in our setting. Complete disappearance of viable tumor cells after cytotoxic pretreatment was observed in 2.5% [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref] to 51% [bib_ref] Concurrent radiation therapy and chemotherapy followed by esophagectomy for localized esophageal carcinoma, Bates [/bib_ref]. These numbers probably reflect, to a certain degree, the quality of treatment or at least its consequent application, which might be a problem in large multicenter trials [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref]. Nowadays, response rates of more than 50% and pathologically complete eradication of the primary tumor in about 20% seem to be a standard to call pretreatment sufficiently effective [bib_ref] Chemoradiotherapy followed by surgery compared with surgery alone in squamous-cell cancer of..., Bosset [/bib_ref] [bib_ref] Downstaging of esophageal cancer after preoperative radiation and chemotherapy, Vogel [/bib_ref] [bib_ref] Role of preoperative chemoradiation in the management of upper third thoracic esophageal..., Tsujinaka [/bib_ref]. A certain hint that NT has its limitations in case of tumor spread to the lymphatic system or even distant organs is the fact that it caused 11 primary tumor lesions to be histopathologically eradicated but left two with regional or distant metastases. There is a continuing debate regarding whether cytotoxic regimens prior to resection carry the risk of increasing morbidity or mortality [bib_ref] Does neoadjuvant chemotherapy for carcinoma in the thoracic esophagus increase postoperative morbidity?, Tabira [/bib_ref]. During the eighties, mortality seemed to be negatively influenced by chemotherapy before surgery [bib_ref] Randomized trial of preoperative chemotherapy for squamous cell cancer of the esophagus, Schlag [/bib_ref]. In recent years, there have been reports of myelosuppression in those patients [bib_ref] Problems in neoadjuvant chemoradiotherapy preceding surgery for advanced squamous cell carcinoma of..., Ishida [/bib_ref] , but obviously this does not have a negative impact on the patient's final clinical outcome [bib_ref] A randomized study of chemotherapy, radiation therapy, and surgery versus surgery for..., Prise [/bib_ref] [bib_ref] The role of neoadjuvant therapy in surgically resectable esophageal cancer, Swisher [/bib_ref] [bib_ref] Role of preoperative chemoradiation in the management of upper third thoracic esophageal..., Tsujinaka [/bib_ref]. Also, in our experience, we found no significant difference between surgery alone and NT + surgery with regard to morbidity and mortality. Lower numbers after NT + surgery most likely are an expression of improved postoperative care in recent years. Looking at long-term survival of all patients [fig_ref] Figure 2: Comparison of survival of all patients undergoing surgery alone and NT +... [/fig_ref] , pretreated patients did better despite the fact that this group included more advanced tumor stages. However, this difference did not reach statistical significance. Reducing analysis to locally advanced stages T3 and T4 resulted in a difference being close to significant. This was clearly achieved by comparing only node-positive patients [fig_ref] Figure 3: Comparison of survival of all node positive patients undergoing surgery alone and... [/fig_ref] and was even more pronounced in those with advanced tumor stages combined with positive nodes. Despite small numbers and the non-randomized setting of this study, this might be interpreted as evidence that surgery alone was less able to control esophageal cancer with regional or even single cell systemic spread [bib_ref] Micrometastases in esophagogastric cancer: high detection rate in resected rib segments, O&apos;sullivan [/bib_ref]. Facing the recurrence pattern of esophageal carcinoma characterized by systemic organ metastases in more than 30%, the effectiveness of wide local excision with extensive lymph-node dissection is questioned [bib_ref] Recurrence pattern of oesophageal carcinoma after limited resection does not support wide..., Van Lanschot [/bib_ref] [bib_ref] Pattern of recurrence after oesophageal resection for cancer: clinical implications, Law [/bib_ref]. This might be one of the strongest arguments to fight regional and systemic disease using cytotoxic pre-treatment with final resection of the remaining disease. Some randomized studies comparing surgery alone and NT + surgery did not show significant differences in survival; these either included early stages, presumably mostly without systemic disease [bib_ref] Chemoradiotherapy followed by surgery compared with surgery alone in squamous-cell cancer of..., Bosset [/bib_ref] , or did not exclude them from entering the study [bib_ref] A randomized study of chemotherapy, radiation therapy, and surgery versus surgery for..., Prise [/bib_ref] [bib_ref] Preoperative chemotherapy versus surgical therapy alone for squamous cell carcinoma of the..., Law [/bib_ref] [bib_ref] Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer, Kelsen [/bib_ref] , which might be one reason for the failure in showing benefits of pretreatment. A survival advantage in our study is primarily shown for locally and regionally advanced stages. An increasingly important issue is the phenomenon of complete histopathological response of the primary tumor lesion to chemo-or chemoradiotherapy. It is discussed as an independent positive prognostic factor. Therefore, several authors call for means to identify responders before beginning the pretreatment [bib_ref] Induction chemo-radiotherapy for squamous cell carcinoma of the thoracic esophagus: long-term results..., Laterza [/bib_ref] [bib_ref] Only pathologic complete response to neoadjuvant chemotherapy improves significantly the long term..., Ancona [/bib_ref] , thus excluding non-responders to avoid unnecessary morbidity. To date, there are no means available to know beforehand whether a patient will respond or not [bib_ref] Van Dam J: Pretreatment staging by endoscopic ultrasonography does not predict complete..., Mallery [/bib_ref]. In our study, we compared patients with T0-stages after NT and resection with the other pretreated cases [fig_ref] Figure 4: Comparison of survival of patients with complete histopathological eradication of the primary... [/fig_ref]. Although there were higher survival rates at 2 years, both curves merge at 5 years, which means prolongation of median survival but not a higher chance of cure. Very well aware of the fact that these are small numbers and preliminary results, it might be hypothesized that the complete responders are a biologically privileged subgroup, identified by better reaction to cytotoxic pretreatment, which could have a better outcome also without NT. In our study the average time period from histologically proven diagnosis to resection was 113 ± 45 days in the NT + surgery group. This compared similarly to others [bib_ref] Neoadjuvant chemoradiotherapy for esophageal cancer: is it worthwhile?, Tamim [/bib_ref] with almost 20% of the median survival time of the respective patient. Since this loss of time is combined with treatment costs exclusively for the neoadjuvant modality of around Euro 10,000, several authors question the use of NT in resection of esophageal cancer [bib_ref] Neoadjuvant chemoradiotherapy for esophageal cancer: is it worthwhile?, Tamim [/bib_ref]. # Conclusion Because we found considerable evidence in this non-randomized historical case series that advanced tumor stages seem to benefit from pretreatment, we would advocate it for T3/4 and node-positive tumors in patients fit for resective surgery. These findings have to be confirmed in further well designed controlled randomized trials including particularly T3/4-stages. Facing the economic pressure to our health care system, it should only be executed in centers employing personnel well experienced in this technique, with close cooperation of medical and surgical faculties to minimize cytotoxic side effects, to reduce extra surgical morbidity, and to avoid pretreatment of the wrong patients. [fig] Figure 2: Comparison of survival of all patients undergoing surgery alone and NT + surgery. Median survival time was 13.9 and 20.3 months, respectively. [/fig] [fig] Figure 3: Comparison of survival of all node positive patients undergoing surgery alone and NT + surgery. Median survival time was 12.0 and 19.1 months, respectively. [/fig] [fig] Figure 4: Comparison of survival of patients with complete histopathological eradication of the primary tumor lesion post NT (T0) and those with residual viable tumor cells (T1-4). Median survival time was 27.8 and 13.6 months, respectively. [/fig] [table] Table 1: Baseline characteristics of 203 patients treated for squamous cell carcinoma of the esophagus. [/table] [table] Table 2: Tumor staging according to the UICC classification of 1992. [/table]

Short-Term Corticosteroid Therapy for Early Exacerbation of COVID-19 Pneumonia: A Case Report Objective:Management of emergency care Background:The effect of corticosteroids in the management of patients with coronavirus disease 2019 (COVID-19) is unclear.Case Report:A 67-year-old man who tested positive for COVID-19 by reverse-transcription PCR (RT-PCR) analysis was admitted to our hospital. On admission, he had no dyspnea and his oxygen saturation (SpO 2 ) level was normal. Chest imaging revealed ground-glass opacities (GGO) distributed in both lung fields. Four days after admission, bilateral lung shadows worsened, with a slight reduction in SpO 2 levels. Short-term corticosteroid therapy was initiated, and SpO 2 and radiographic findings promptly improved without use of antiviral agents.Conclusions:More data are required to ascertain the role of corticosteroids in the management of COVID-19 pneumonia. # Background Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was first reported at the end of 2019; it is now spreading worldwide. On, the WHO characterized COVID-19 as a pandemic. A Chinese study reported that the mortality rate of COVID-19 is 2.3% in China, which is not as high as that of SARS or Middle East respiratory syndrome (MERS); however, the elderly and patients with underlying comorbidities are at a higher risk of the disease and its related mortality. Although several antimicrobial agents, including arbidol [bib_ref] Therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics, Zhang [/bib_ref] , oseltamivir, ganciclovir [bib_ref] Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in..., Yang [/bib_ref] , lopinavir/ritonavir [bib_ref] The Author's Response: Case of the index patient who caused tertiary transmission..., Lim [/bib_ref] [bib_ref] Letter to the Editor: Case of the index patient who caused tertiary..., Kim [/bib_ref] , chloroquine [bib_ref] Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated..., Gao [/bib_ref] , and remdesivir [bib_ref] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The..., Lai [/bib_ref] , have been assessed for COVID-19 treatment, no effective treatment has been established thus far. Furthermore, corticosteroid treatment is not currently recommended for COVID-19 patients [bib_ref] Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury, Russell [/bib_ref]. Here, we present the case of a patient with COVID-19 whose condition improved with short-term systemic corticosteroid treatment early after exacerbation of pneumonia. ## Case report A 67-year-old man who had traveled on a cruise ship was hospitalized because of fever and headache 6 days before admission. Reverse-transcription PCR (RT-PCR)-based screening for SARS-CoV-2 from a throat swab performed on the ship revealed a positive result. In accordance with national standards at the time, hospitalization was required for all positive patients. Therefore, the patient was hospitalized and monitored, without oxygen therapy. His symptoms included only fever and headache. The patient had no remarkable medical history or comorbidities except for cholecystitis. He was not receiving any long-term medication. On admission, the patient's vital signs included a body temperature of 37.4°C; blood pressure, 134/77 mmHg; pulse, 78/min; respiratory rate, 16/min; and oxygen saturation (SpO 2 ), 96% on ambient air. Physical examinations revealed no abnormalities. Laboratory investigations revealed elevated C-reactive protein (CRP, 2.89 mg/L) and white blood cell count of 3.8×10 9 /L (neutrophils, 51.4%; lymphocytes, 30.1%) [fig_ref] Table 1: Laboratory data on admission [/fig_ref]. Chest X-rays revealed diffuse infiltrates [fig_ref] Figure 1: Clinical course of the patient [/fig_ref] and computed tomography (CT) imaging revealed bilateral ground-glass opacity (GGO) in subpleural areas [fig_ref] Figure 2: Chest computed tomography images on admission revealed bilateral ground-glass opacity [/fig_ref]. Based on imaging findings and RT-PCR testing, the patient was diagnosed with COVID-19 pneumonia. Initially, the patient's respiratory condition was stable, and he was examined almost daily by chest X-ray imaging, without medication and oxygen therapy. On hospitalization day 5, chest X-rays revealed worsening of diffuse infiltrates [fig_ref] Figure 1: Clinical course of the patient [/fig_ref] and laboratory investigations revealed CRP levels of 4.09 mg/L and a white blood cell count of 4.6×10 9 /L (neutrophils, 52.5%; lymphocytes, 27.2%), with a reduction in SpO 2 to 92% on ambient air. Short-term corticosteroid therapy (i.v. administration of 125 mg methylprednisolone every 12 hours) was administered on hospitalization days 5, 6, and 7; consequently, SpO 2 and radiographic findings improved from hospitalization day 6 [fig_ref] Figure 1: Clinical course of the patient [/fig_ref]. The symptoms did not exacerbate after discontinuation of corticosteroid treatment. However, despite improvement in SpO 2 , the patient could not be discharged owing to the positive result of an RT-PCR test performed on hospitalization day 14. Finally, RT-PCR tests of nasopharyngeal swabs and throat swabs yielded negative results on hospitalization days 16 and 17. The patient was then discharged on hospitalization day 18. # Discussion This study reports the case of a COVID-19 patient with improved overall conditions after short-term systemic corticosteroid treatment early after the exacerbation of pneumonia. Our findings suggest that systemic corticosteroid therapy may be a potential treatment alternative for exacerbated COVID-19 pneumonia. Moreover, the present case potentially provides novel insights into the application of corticosteroids for treating COVID-19. In some respiratory infections, a mortality benefit has been achieved through timely administration of corticosteroids [bib_ref] Adjunctive corticosteroids for Pneumocystis jiroveci pneumonia in patients with HIV infection, Ewald [/bib_ref]. A meta-analysis by Siemieniuk et al. reported that systemic corticosteroid therapy can reduce mortality in community-acquired pneumonia requiring hospitalization [bib_ref] Corticosteroid therapy for patients hospitalized with community-acquired pneumonia: A systematic review and..., Siemieniuk [/bib_ref]. Moreover, corticosteroids co-administered with anti-Pneumocystis therapy can decrease the mortality rate and respiratory failure associated with Pneumocystis pneumonia among patients with human immunodeficiency virus infections [bib_ref] Adjunctive corticosteroids for Pneumocystis jiroveci pneumonia in patients with HIV infection, Ewald [/bib_ref]. However, the benefits of corticosteroid treatment in influenza remain unknown [bib_ref] Corticosteroids as adjunctive therapy in the treatment of influenza, Lansbury [/bib_ref]. Corticosteroids were often prescribed empirically to SARS and MERS patients [bib_ref] Severe acute respiratory syndrome: Report of treatment and outcome after a major..., Sung [/bib_ref] [bib_ref] Corticosteroid therapy for critically ill patients with Middle East Respiratory Syndrome, Arabi [/bib_ref]. However, corticosteroid administration for COVID-19 patients is not recommended thus far; this includes patients with severe pulmonary injury or shock, based on evidence from other viral infectious diseases, including SARS or MERS [bib_ref] Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury, Russell [/bib_ref]. In the present case, SpO 2 and radiographic findings improved the day after systemic corticosteroid treatment was initiated, with no exacerbation of respiratory status or pneumonia, after discontinuation of corticosteroid treatment. Administration of lopinavir/ritonavir was considered if pneumonia had not improved the day after the initiation of corticosteroid therapy. However, the patient's condition improved without additional treatment, suggesting that some COVID-19 patients can benefit from systemic corticosteroid therapy. However, it remains unclear which COVID-19 patients benefit from corticosteroid treatment. Sung et al. reported that APTT -activated partial thromboplastin; PT -prothrombin time. the administration of corticosteroids to patients with SARS, relatively early after onset, yielded a favorable outcome [bib_ref] Severe acute respiratory syndrome: Report of treatment and outcome after a major..., Sung [/bib_ref]. Previous studies have reported that in acute viral respiratory infections, early-response cytokines, including interferon-tumor necrosis a, interleukin (IL)-1, and IL-6, are produced at high levels and mediate antiviral activity; however, they simultaneously potentially contribute to tissue injury [bib_ref] Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses:..., Cheung [/bib_ref] [bib_ref] In vivo studies on cytokine involvement during acute viral respiratory disease of..., Van Reeth [/bib_ref]. Early corticosteroid therapy among SARS patients may have prevented death by regulating cytokine responses [bib_ref] Severe acute respiratory syndrome: Report of treatment and outcome after a major..., Sung [/bib_ref]. Similarly, in the present case, early corticosteroid treatment may have suppressed such an inflammatory burst in a COVID-19 patient, thus preventing the need for ventilator management. However, corticosteroid treatment was reportedly ineffective among MERS patients in an intensive care unit; the general condition of the patients was not serious enough to warrant admission to the intensive care unit, whereas previously reported subjects had a severe pathological condition [bib_ref] Corticosteroid therapy for critically ill patients with Middle East Respiratory Syndrome, Arabi [/bib_ref]. The present case report included only 1 patient and the patient's condition might have improved spontaneously without corticosteroid treatment. Although the exact role of corticosteroid treatment cannot be determined owing to limited data, corticosteroid treatment in the early stage of COVID-19 pneumonia exacerbation may be considered a potential treatment alternative. # Conclusions Our findings indicate that corticosteroid treatment can benefit cases of mild and early-stage exacerbation of pneumonia among COVID-19 patients; however, this was an uncontrolled observation and warrants further controlled clinical trials. Additional cases need to be studied to determine the precise symptoms of COVID-19 patients who would benefit most from systemic corticosteroid therapy. [fig] Figure 1: Clinical course of the patient. Chest X-rays revealed worsening diffuse infiltrates (arrow) before systemic corticosteroid therapy. After administration of systemic corticosteroid therapy on days 5-7, chest X-ray images and SpO 2 levels displayed immediate and sustained improvement. SpO 2 -oxygen saturation; BT -body temperature. [/fig] [fig] Figure 2: Chest computed tomography images on admission revealed bilateral ground-glass opacity (GGO) in subpleural areas (arrow). [/fig] [table] Table 1: Laboratory data on admission. [/table]

Short- and midterm outcome of ruptured and unruptured intracerebral wide-necked aneurysms with microsurgical treatment To clip or coil has been matter of debates for several years and is the domain of interdisciplinary decision making. However, the microsurgical outcome has still been elusive concerning wide neck aneurysms (WNA). A retrospective single center study was performed with all patients with ruptured WNA (rWNA) and unruptured WNA (uWNA) admitted to author´s institute between 2007-2017. Microsurgical outcome was evaluated according to Raymond-Roy occlusion grade and follow-up angiography was performed to analyze the stability of neck/aneurysm remnants and retreatment poverty. Of 805 aneurysms, 139 were rWNA (17.3%) and 148 uWNA (18.4%). Complete occlusion was achieved in 102 of 139 rWNA (73.4%) and 112 of 148 uWNA (75.6%). Neck remnants were observed in 36 patients with rWNA (25.9%) and 30 patients with uWNA (20.3%), 1 (0.7%) and 6 (4.1%) patients had aneurysmal remnant, respectively. Overall complication rate was 11.5%. At follow-up (939/1504 months), all remnants were stable except for one, which was further conservatively treated with marginal retreatment rate under 1%. Even the risk of de-novo aneurysm was higher than the risk for remnant growth (2.6% vs 0% in rWNA; 8.7% vs 5.3% in uWNA) without significant difference. Microsurgical clipping is effective for complete occlusion of r/uWNA with low complication. Furthermore, the risk of remnant growth is marginal even lower than the risk of de-novo rate low retreatment rate. # Results Basic characteristics, clinical course and functional outcome. Of 805 patients in our database between 2007 and 2017, 287 patients had a WNA (35.7%). In this series, a total of 139 patients (48.4%) had a ruptured WNA and 148 patients (51.6%) had an unruptured WNA. Regarding patients with rWNA, the median age was 52 years of age (IQR 20-83) and 93 of 139 patients (66.9%) were female. About half of those patients were in good admission status (WFNS 1-3; 50%). Early hydrocephalus occurred in 95 patients (69.1%) and cerebral vasospasm (CVS) manifested in 106 of 139 patients (76.3%) with DCI in over 50% (73 of 139 patients). Favorable outcome (mRS 0-2) was achieved 37 patients at discharge (25.9%) and in 75 patients at FU (56%). Detailed information is listed in . Regarding patients with uWNA, basic characteristics (age, sex distribution) were comparable to rWNA-group. Eighteen of 148 patients (12.2%) had previous SAH. Favorable outcome (mRS 0-2) was achieved in 137 of 148 patients (92.6%) at discharge and 140 of 148 patients (94.6%) at 6 months' FU. Of note, 4 patients with uWNA, who were admitted in our clinic due to SAH from another aneurysm, were included in the analysis. All of them had initially worse admission status. Further details are reported in [fig_ref] Table 2: Patient and aneurysm characteristics in unruptured wide-neck aneurysm [/fig_ref]. Aneurysm characteristics, microsurgical treatment and short-term outcome. The most common rWNA was MCA-(50 of 139; 36%) followed by AcommA-(38 of 139; 28.1%) and ICA-aneurysm (28 of 139; 20.1%), particularly at the location of Pcomm (17 of 28; 60.7%). Mean neck size was 5.0 ± 2.0 mm and over 70% of aneurysm showed dome to neck ratio under 2. 94 of 139 rWNA (67.6%) had saccular perpendicular form of aneurysm and 102 of 139 rWNA (73.4%) were microsurgically completely occluded. In 36 of 139 patients (25.9%), there was a neck remnant (25.9%) and one patient (0.7%) had a residual aneurysm, which was just conservatively controlled at follow-up. For the aneurysm occlusion, mean number of clips was 1.8 ± 1.2. By doing so, volume reduction of rWNA was postoperatively achieved up to 85%. In total, the complication rate of operation was 15.2%. In 10 cases (7.2%), there were intraoperative rupture of those aneurysms, which were all surgically managed without relevant intraoperative complication except the necessity of blood product transfusion. Of note, relevant complication requiring revision due to secondary epidural hematoma or insufficient clipping was found in 4 of 139 patients (2.9%) and 4 of 139 patients (2.9%) had brain insult (4-13.5 cm 3 ) without relevant clinical impairment . The brain insult occurred due to Heubner artery occlusion (two patients), perforator vessel injury (one patient) and micro embolism after clip reposition (one patient). The location distribution of uWNA was similar to rWNA except there was more ophthalmic ACI-aneurysm than Pcomm ICA-aneurysm (10.1% vs 5.4%). Mean neck size was 5.3 ± 2.6 mm and 77.7% showed dome to neck ratio under 2. 71 of 148 uWNA (48%) had saccular perpendicular form of aneurysm and 112 of 148 uWNAs (75.6%) were microsurgically completely occluded. Neck remnant was observed in 30 of 149 uWNA (20.3%;and 6 of 148 patients (4.1%) had a residual aneurysm. One of the residual aneurysm (0.7%) was endovascularly treated at follow-up. For the aneurysm occlusion, mean number of clips was 2.1 ± 1.4. Overall 88% volume reduction of uWNA was postoperatively achieved. In total, complication rate was 8.1%, including intraoperative rupture (2%) and abruption of operation due to brain swelling under seizure (2%) . Among six patients (4.1%) with brain insult (3-10.5 cm 3 ), it occurred due to perforator vessel injury (four patients) and micro embolism after clip reposition (two patients). Notably, no revision operation was necessary in uWNA treatment. Microsurgical mid-term outcome. All patients with rWNA and uWNA were clinically followed up; however, radiological follow-up was performed in selected patient with either considerable aneurysm remnant or diagnosed additional aneurysms. 51 of 139 rWNA patients (36.7%) with 949 follow-up months (mean of 18.6 ± 19.4 months) and 68 of 148 uWNA patients (45.9%) with 1509 follow-up months (mean of 22.2 ± 19.9 months) were analyzed. Among 51 patients with rWNA, 38 (74.5%) were initially completely occluded and no recurrent aneurysm was detected at mean follow up of 14.9 ± 14.6 months. There was one de-novo aneurysm (2.6%) in other localization detected. 13 of 51 rWNA (25.5%) had aneurysm remnants, 12 neck-and 1 residual aneurysm. Even in 1 of 12 patients with neck-remnant (8.3%), the remnant disappeared and all other aneurysms were stable without any sign of growth at mean follow up of 29.9 ± 28.2 months. The residual aneurysm was stable up to 23 months' follow-up [fig_ref] Table 5: Follow-up analysis after microsurgical treatment [/fig_ref]. In the uWNA group, 46 of 68 uWNA (67.6%) were initially completely occluded and there was no recurrent aneurysm detected at mean follow up of 24.2 ± 20.9 months. Four de-novo aneurysm (8.7%) were detected in other localization. After initial microsurgical treatment, 19 patients had neck remnants (27.9%) and 3 patients had residual aneurysms (4.4%). 18 of 19 neck remnants were stable up to 19.6 ± 18.9 months and 1 of 19 showed aneurysm growth at follow up of 18 months, however, no additional treatment was required. Among three residual aneurysms, in one case the residual aneurysm disappeared (33%) and the remnant was stable at mean follow up of 7 ± 4.2 months [fig_ref] Table 5: Follow-up analysis after microsurgical treatment [/fig_ref]. To illustrate the case, one patient had a media bifurcation aneurysm of a size of 12 mm and was electively clipped Predictors for microsurgical WNA remnants. In the univariate analysis of rWNA cohorts, predictors for aneurysm remnants were worse admission status of patients (OR 2.7 Cl 95% 1.2-6.1), development of early hydrocephalus (OR 9.1 Cl 95% 3.5-23.8) and non-saccular non-perpendicular form of aneurysm (OR 2.5 Cl 95% 1.0-5.0). Aneurysm of vertebral artery showed a trend towards significance. Interestingly the neck sizes as well as the maximum aneurysm size were not significant predictors (Supplementary # Discussion Main findings. There are three main findings in this study. First, the complete occlusion rate of rWNA as well as uWNA is over 70% and stays stable at mid-term follow-up. Even the occurrence rate of de-novo aneurysm is higher compared to the risk of remnant growth indicating absolute stability of microsurgical outcome [fig_ref] Table 6: Summary of microsurgical treatment for wide neck aneurysm [/fig_ref]. Second, the procedural complication rate with 11.5% and retreatment rate of less than 1% at follow-up are reasonable values for the preference of surgical treatment of WNA [fig_ref] Table 6: Summary of microsurgical treatment for wide neck aneurysm [/fig_ref]. Third, independent predictors for remnant are neither the size of neck nor aneurysm but early hydrocephalus, complex configuration of aneurysm and number of clips, which were used. To clip or coil was and is still matter of debates, however, the direction of aneurysmal treatment dramatically changed since the publication of ISAT (International Subarachnoid Aneurysm Trial), which indicated better clinical outcome of endovascular arm at 1 year-up to 18 years of follow-up if treatment associated deaths within the first year were excluded from analysis 1,2 . On the other hand, ISAT itself (5-year data) and BRAT (Barrow Ruptured Aneurysm Trial) trial reported no significant difference of outcome at 3-, 6-and 10-years follow-up despite the higher likelihood of poor outcome in the clipping group compared to coiling group except for the aneurysms in the posterior circulation [bib_ref] The barrow ruptured aneurysm trial: 3-year results, Spetzler [/bib_ref] [bib_ref] The barrow ruptured aneurysm trial: 6-year results, Spetzler [/bib_ref] [bib_ref] The barrow ruptured aneurysm trial, Mcdougall [/bib_ref] [bib_ref] Ten-year analysis of saccular aneurysms in the barrow ruptured aneurysm trial, Spetzler [/bib_ref]. Even lower rate of complete aneurysm occlusion and higher retreatment rate was demonstrated in the coiling group supporting the beneficial factors of clipping. Of note, most of the published studies dealt with saccular aneurysms; however there is paucity of literatures regarding microsurgical treatment of WNA, in particular. One recent meta-analysis including 2794 ruptured and unruptured wide neck bifurcation aneurysms reported 43.8% adequate occlusion rate in endovascular arm compared to 69.7% in microsurgical arm, while the safety events were comparable, 21% and 24.3%, respectively 14 . Furthermore, Mascitelli et al. performed posthoc analysis of WNA based on BRAT data and demonstrated significant higher complete occlusion rate in the clipping group with 84.3% compared to coiling group with 51.2% 11 . Over the 3-to 10 year's follow-up, the occlusion grade dropped continuously in the coiling group whereas the outcome was stable with over 80% in the clipping group. Interestingly, the outcome did not differ at any time whereas the recanalization and retreatment rate was significantly higher in the coiling group [bib_ref] The barrow ruptured aneurysm trial: 3-year results, Spetzler [/bib_ref] [bib_ref] The barrow ruptured aneurysm trial: 6-year results, Spetzler [/bib_ref]. This particular analysis was limited first to ruptured aneurysm, second wider range of inclusion criteria, neck width ≥ 4 mm or neck-to-dome ratio < 2, and last, to different endovascular treatment options. Therefore we focused the WNA definition to neck width ≥ 4 mm to build more homogenous cohort and widened our analysis to unruptured WNA besides ruptured WNA. www.nature.com/scientificreports/ In general, WNA are difficult to treat due to wide-neck configuration, which is one of the prognostic factors for successful endovascular treatment [bib_ref] Endovascular treatment of ruptured intracranial aneurysms: factors affecting midterm quality anatomic results:..., Pierot [/bib_ref]. Indeed the endovascular field on WNA was limited in the past; however through the development of new techniques like flow diverter, stent-assisted or ballon-assisted coiling, and recently novel technique with intrasaccular occluding device (WEB device), endovascular treatment became more effective and successful with stable results in the mid-term follow-up; although the long term data (> 10 years) are still missing [bib_ref] Intrasaccular flow disruption in acutely ruptured aneurysms: A multicenter retrospective review of..., Liebig [/bib_ref] [bib_ref] WEB device for endovascular treatment of wide-neck bifurcation aneurysms, Lubicz [/bib_ref]. To date, there have been 2 prospective multicenter trials, WEBCAST and the French Observatory trial, which reported complete occlusions rate of 56-52% and adequate occlusion rate of 86 to 79%, respectively [bib_ref] Clinical and anatomical follow-up in patients with aneurysms treated with the WEB..., Pierot [/bib_ref]. Regarding complete occlusion rate, microsurgical treatment seems to be more effective compared to WEB-device, however, the question arises if it is necessary to achieve a complete occlusion of aneurysm [bib_ref] Efficacy and safety of the woven endobridge (WEB) device for the treatment..., Asnafi [/bib_ref]. In addition, a direct comparison is hard to perform since the main limitation would be the definition of an adequate occlusion and periprocedural complication. Besides recently, endovascular techniques like T-stenting assisted coiling has been introduced which show a comparable high complete occlusion grade of 73-83% in WNA with a stable result in www.nature.com/scientificreports/ the follow-up analysis and low complication rate [bib_ref] Long-term outcomes of wide-necked intracranial bifurcation aneurysms treated with T-stent-assisted coiling, Aydin [/bib_ref] [bib_ref] Safety and efficacy of stent-assisted coiling in the treatment of unruptured wide-necked..., Aguilar-Salinas [/bib_ref]. Still one should notice that in contrast to microsurgical treatment, the retreatment rate of all alternative endovascular treatment is still higher, which is associated with . Characteristic of ruptured aneurysms and microsurgical treatment with follow up. ACA anterior cerebral artery, AcomA anterior communicans antery, ICA internal carotid artery, MCA media cerebral artery, PCA posterior cerebral artery, PICA posterior inferior cerebellar artery, VA vertebral artery, CT computer tomography. www.nature.com/scientificreports/ a higher morbidity and mortality during long-term FU [bib_ref] Long-term follow-up of unruptured intracranial aneurysms repaired in California, Gonda [/bib_ref] [bib_ref] Small intracranial aneurysms in the barrow ruptured aneurysm trial (BRAT), Catapano [/bib_ref]. In our study, the complication rate of rWNA and uWNA was 15% and 8%, respectively. It seems a little high compared to general endovascular treatment (except WEB device), however, it is a matter of definition [bib_ref] Small intracranial aneurysms in the barrow ruptured aneurysm trial (BRAT), Catapano [/bib_ref]. We included radiological features like epidural hematoma or brain insult, which was not clinically apparent except in the routine postoperative scan. Moreover, intraoperative rupture of aneurysm is questionable to calculate as complication since this happening is a normal state in an open surgery. In summary, the open-end question: clip or "coil" in WNA has still not been answered and a meta-analysis comparing endovascular and surgical treatment on WNA is warranted. Therefore our study might be an important addition to the literature in future for further comparison studies regarding outcome of WNA in both, ruptured and unruptured situation. In our study, we tried to characterize the complexity of aneurysm configuration by comparing maximum-and perpendicular method. This method showed to be an independent predictor for microsurgical remnant besides early hydrocephalus and number of clips. Therefore this may serve as one of the preoperative measurement method to get a first insight of potential difficulty and alternative planning of operative approach. All in all, microsurgical treatment was effective and stable for ruptured as well as for unruptured WNA, thus, with respect . Aneurysm characteristic and surgical treatment with follow-up in unruptured wide-neck aneurysm. Limitations and generalizability. One limitation of this study is the limited follow-up of microsurgically treated patients. The reason for that was the absence of regular angiographic follow-up due to the known low number of recanalization after clipping [bib_ref] Endovascular and surgical treatment of internal carotid bifurcation aneurysms: Comparison of results,..., Konczalla [/bib_ref] [bib_ref] Natural history of postoperative aneurysm rests, Feuerberg [/bib_ref]. Therefore, this bias may underestimate the recanalization rate, however, the radiographic follow-up was selected for high-risked patients for recurrence, which may result in rather overestimation. Moreover, Tsutsumi et al. have previously shown even lower risk of aneurysm growth of clipped aneurysm compared to de-novo aneurysm in mean follow up of 9 years indicating the stability of clipping in long-term follow-up [bib_ref] Risk of aneurysm recurrence in patients with clipped cerebral aneurysms: Results of..., Tsutsumi [/bib_ref]. Secondly, due to monocentric and retrospective design of our study, a bias of retrospective nature could have occurred, although we think that this would be small in regard of prospectively collected database. At last, there was no direct comparison of microsurgical treatment to endovascular treatment in our study wherefore no conclusion regarding superiority of each modality can be made. On the other hand, we think that due to the diverse experience of endovascular treatment between clinicians, those two arms would be better comparable within the scope of meta analysis. # Conclusions This study is one of the few studies reporting microsurgical outcome in rWNA and uWNA. Microsurgical clipping was effective for treatment of WNA with low complication rate. Moreover, remnants were mostly stable after clipping; the risk of growth was marginal even lower than the risk of de-novo rate resulting in hardly retreatment necessity. Still, further comparing studies regarding clip or newly developed endovascular alternatives are needed for a general consensus. # Material and methods This study received the authorization of the local ethical standards committee (IRB 19-245). In accordance with institutional rules, informed consent of anonymized patient was not required for this study. From a prospective collected database, a retrospective analysis was conducted with patients microsurgically treated on a WNA, either ruptured (rWNA) or unruptured (uWNA), from 2007 to 2017 in author´s institute. Inclusion criteria was the diagnosis of WNA, which was defined as maximum neck width ≥ 4 mm measured by the preoperative 3-dimensional 4-vessel digital subtraction angiography (DSA) oblique "working view". Hendricks et al. summarized different definition of WNA in a systematic review reporting neck-to-dome ratio for further frequent used criteria. However, there were variable threshold defined for neck-to-dome ratio depending on the literatures, wherefore this parameter was not counted as definition criteria for WNA in our study. www.nature.com/scientificreports/ All patients underwent preoperative DSA. Neck, dome and height size were measured in order to analyze WNA configuration in detail using DSA. In so doing, two different methods were applied to differentiate saccular (simple) and non-saccular (complex) aneurysm: 1. Maximum-2. Perpendicular method. For the maximum-method, first the maximum neck diameter was measured and the maximal diameter regardless of perpendicularity of dome and height were calculated. For the perpendicular method, the neck size was the same as abovementioned; however, other parameters were calculated by drawing perpendicular line from neck to the height and additional dome line perpendicular to the height line. In case of less than 2 mm deviation of either neck or dome size, it was considered as saccular perpendicular (simple) aneurysm, otherwise the aneurysms were classified as non-saccular non-perpendicular (complex) aneurysm. Microsurgical treatments were typically performed with or without rapid ventricular pacing depending on the size of aneurysm 6 . Afterwards, patients with rWNA underwent standard treatment according to SAH-guideline and patients with uWNA were followed up for a week. Besides the intraoperative ICG-angiography, microsurgical outcome was determined by a postoperative DSA 7 days after microsurgical treatment. Clinical follow-up was set at 6 months after treatment and depending on the microsurgical outcome, MRA or DSA were additionally recommended. Those data were used to analyze the mid-term microsurgical outcome. The following information of each patient were completed: patient´s demographics including age, sex, smoker, family history of SAH, aneurysm information (location, size, neck/dome/height size), preoperative World Federation of Neurological Scale (WFNS), occurrence of hydrocephalus and cerebral vasospasm (CVS), delayed cerebral ischemia (DCI) and modified Ranking Scale (mRS) at discharge and 6 months' follow-up (FU) [bib_ref] Endovascular treatment of unruptured aneurysms, Roy [/bib_ref]. WFNS 1-3 was considered as favorable neurological status at admission and favorable outcome was defined as mRS 0-2, otherwise it was defined as an unfavorable outcome. To mention, ICA-pcomm aneurysm was counted as aneurysm of anterior circulation, if pcomm aneurysm was included in the subsection of ICA. For all volumetric measurement (volume of WA, aneurysm remnants, SAH associated intracerebral hemorrhage), the simplified ABC/2 formula was used. In general, this study was build up in 2 subsections: 1. Microsurgical outcome of rWNA at short-and midterm follow-up. 2. Microsurgical outcome of uWNA at short-and midterm follow-up. www.nature.com/scientificreports/ As primary aim of the study, microsurgical outcome was evaluated on Raymond Roy occluding grade: complete occlusion (class I), neck remnant (class II) and aneurysm remnant (class III). We particularly used this classification in order to facilitate comparative analysis concerning endovascular treatment on WNA in the future. The modified Raymond Roy occluding grade was not used, because it was not applicable for surgical analysis. Complete occlusion was defined either as no more detectable aneurysm after clipping or intended neck remnant for the preservation of one or more perforators. Neck or aneurysm remnants were analyzed without knowledge of clinical outcome. Outcome at follow-up was analyzed by performing CT-angiography or DSA with following parameters: no remnant, remnant stable, remnant growth or de novo aneurysm. The secondary aim of the study was to determine predictors indicating postoperative aneurysm remnant. Statistical analysis. IBM SPSS Statistics (Version 22, IBM Corp.) was used for data analysis. Continuous variables were described as mean and standard deviation. Categorical variables were shown as a numerical order and percentage of the analyzed population. Univariate analyses as well as multivariate logistic regression analysis were performed to determine predictors for aneurysm remnant. Only parameters with p-value ≤ 0.05 were included in the regression analysis. For parametric variables, t-test was used and for non-parametric variables, Fisher´s exact test or chi-squared test were used as appropriate. To assess the impact of those variables, odds ratio with 95% confidence intervals were calculated. Statistical significance was defined as p-value ≤ 0.05 and all tests were 2-tailed. Ethical approval and informed consent. This study was approved by the local ethics committee of the Goethe University Hospital Frankfurt am Main, the methods were carried out in accordance with the relevant guidelines and regulations. For this retrospective study, informed consent was waived by local ethic committee of the Goethe University Hospital Frankfurt. ## Data availability [fig] Figure 1: (A,B) A case with a saccular perpendicular ICA-Pcomm aneurysm and its complete occlusion by 4 clips. (C,D) A case with a nonsaccular non-perpendicular MCA aneurysm and its complete occlusion by 9 clips. (E,F) A case with AcommA aneurysm and its neck remnant. (G,H) A case with AcommA aneurysm and its aneurysmal remnant. Afterwards, postoperative endovascular treatment of the remnant was performed. ICA interal carotid artery, Pcomm posterior communicating artery, MCA media cerebral artery, AcommA anterior communicans artery. Scientific Reports | (2021) 11:4982 | https://doi.org/10.1038/s41598-021-84339-x [/fig] [fig] Figure 2: (A) Definition of wide neck aneurysm. (B) Differentiation between saccular perpendicular-(simple) and non-saccular non-perpendicular (complex) aneurysm. If width or height deviation was ≥ 2 mm, it was defined as a complex aneurysm. (C) Definition of Raymond-Roy occluding grade: complete, neck-and aneurysmal remnant. Scientific Reports | (2021) 11:4982 | https://doi.org/10.1038/s41598-021-84339-x [/fig] [table] Table 2: Patient and aneurysm characteristics in unruptured wide-neck aneurysm. [/table] [table] Table 5: Follow-up analysis after microsurgical treatment. [/table] [table] Table 6: Summary of microsurgical treatment for wide neck aneurysm. [/table]

A Review on the Use of the Educational Value Unit (EVU) among Teaching Hospitals 1) Background: In recent years, medical institutions across the U.S. have implemented a points system based on the Educational Value Unit (EVU) to assess and reward faculty for their educational efforts. The purpose of this narrative review is to summarize the current literature on EVU systems and to evaluate their utility in the U.S. healthcare system. (2) Methods: We searched the Ovid MEDLINE, Embase, Web of Science, and PubMed databases to identify literature describing the inception of EVU systems and current systems implemented by U.S. academic medical centers and medical schools. In total, a combined 48 studies and abstracts pertaining to EVU systems were reviewed, and a combined 26 published studies and abstracts from 1999 to 2022 pertaining to EVU systems were included. (3) Results: To our knowledge, at least 40 U.S. academic medical centers have used an educational metrics system, of which 21 institutions have published studies describing EVU systems in one or more of their medical departments. The outcomes associated with these selfdescribed EVU systems are the focus of this study. EVU systems increase the number of faculty who meet baseline educational requirements, promote educational productivity, redistribute educational burden and funding among faculty members, and shift physician priorities towards education. The monetary reward associated with EVU systems is unlikely to be a significant factor contributing to these changes; instead, intrinsic motivation and a sense of academic responsibility play a larger role. (4) Conclusions: EVU systems are an effective way to evaluate and reward individual and departmental educational efforts in U.S. academic medical centers and medical schools. The adoption of EVUs will likely become more commonplace as U.S. academic medical centers and medical schools place additional emphasis on medical education. # Introduction At the core of a successful medical institution is its faculty, who are responsible for providing high quality patient care, educating future medical professionals, and advancing an institution's academic mission through various educational pursuits. In the process of balancing both clinical and academic obligations, academic physicians face a unique set of challenges. Current payment models compensate physicians according to the number of work relative value units (wRVUs) assigned to various medical procedures [bib_ref] Estimating Physicians' Work for a Resource-Based Relative-Value Scale, Hsiao [/bib_ref] , which may or may not include research or teaching efforts. Additionally, reimbursement models for physicians have increasingly reduced payment rates, resulting in a need for greater clinical productivity among physicians. Academic physicians must also constantly adjust to evolving medical education pedagogy and compete against their colleagues for limited research funding, which adds additional burden to their high workloads. These factors, among others, contribute to increased burnout among academic physicians [bib_ref] Restoring Faculty Vitality in Academic Medicine When Burnout Threatens, Shah [/bib_ref] and has led to faculty proposing compensation and recognition for non-clinical work as one solution Healthcare 2023, [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] 2 of 10 to burnout [bib_ref] Professional Dissonance and Burnout in Primary Care, Agarwal [/bib_ref]. Furthermore, a survey of faculty in U.S. academic health centers has shown that faculty vitality, defined by the vibrancy, engagement, and motivation in work pursuits [bib_ref] Restoring Faculty Vitality in Academic Medicine When Burnout Threatens, Shah [/bib_ref] , is highest in young faculty and lowest in midcareer faculty members, in whom academic health centers have invested significant training and development. Faculty vitality [bib_ref] Faculty Vitality-Surviving the Challenges Facing Academic Health Centers: A National Survey of..., Pololi [/bib_ref] and satisfaction [bib_ref] Predictors of Workplace Satisfaction for U.S. Medical School Faculty in an Era..., Bunton [/bib_ref] are strongly predicted by the alignment of individual and institutional values, highlighting the need for institutions to recognize a faculty member's efforts towards achieving their institution's educational mission. To address a lack of supplemental compensation or recognition for physician teaching efforts and to better align institutional and individual values towards education, departments across various U.S. medical schools and teaching hospitals have implemented a metrics system based on the educational value unit (EVU). EVU systems assess a faculty member's performance using the sum of the activity or time weighted units for educational activities performed in the academic year [bib_ref] Measuring faculty effort and contributions in medical education, Nutter [/bib_ref]. These sums may be used to reallocate funds within [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] or between departments [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] to better support an institution's educational mission, to determine a year-end financial bonus [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] , or to reconsider an individual's faculty status [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] , among other things. To our knowledge, at least 40 U.S. academic medical centers have used an educational metrics system [bib_ref] How Do Medical Schools Use Measurement Systems to Track Faculty Activity and..., Mallon [/bib_ref] , and a total of 21 U.S. medical schools and teaching hospitals have published reports describing the EVU system implemented in one or more of their medical departments. Our summary of the literature provides a primer on EVU systems, describes current EVU systems in U.S. teaching hospitals and medical schools, and evaluates the utility of these systems in the U.S. healthcare system. # Materials and methods We conducted a narrative literature review to identify the inception of the EVU and to identify EVU systems implemented by U.S. academic medical centers, medical schools, and teaching hospitals. The searches were conducted using the Ovid MEDLINE, Embase, Web of Science, and PubMed databases. Search terms included "educational value unit(s)"; "educational relative value unit(s)"; "educational added value unit(s)"; "academic relative value unit(s)"; "teaching value unit(s)"; and "teaching relative value unit(s)". The bibliographies of articles produced in this search were examined to identify additional studies and search terms. Studies meeting the following criteria were excluded: described EVU systems implemented in locations outside the U.S., described EVU systems in settings other than U.S. academic medical centers, and described relative value unit systems whose purpose was to reward any activities, including administrative, clinical, and educational tasks not currently receiving compensation under a work relative value unit system. We additionally excluded studies describing theoretical frameworks for EVU systems when studies describing the implementation and outcomes of EVU systems containing similar elements were available. There was no language restriction for the searches. In total, a combined 48 studies and abstracts pertaining to EVU systems were reviewed, and a combined 26 published studies and abstracts from 1999 to 2022 pertaining to EVU systems were included [fig_ref] Figure 1: Method of article and abstract selection based on searches conducted in Ovid... [/fig_ref] was selected for review independently of the above search due to prior knowledge of article content. ## Inception of the evu In 2000, the Association of American Medical Colleges (AAMC)'s Mission-based Management Program [bib_ref] Measuring faculty effort and contributions in medical education, Nutter [/bib_ref] first proposed the use of an Educational Relative Value Unit (RVU), also known as the EVU [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] , Educational Added Value Unit (EAVU) [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] , Educational Relative Value Unit (eRVU) [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] , Teaching Value Unit (TVU) [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] , Teaching Relative Value Unit (TRVU) [bib_ref] Effects of a comprehensive faculty remuneration plan on educational productivity and innovation, Ghaemmaghami [/bib_ref] , or Academic Relative Value Unit (aRVU or ARVU) [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] , to provide medical institutions with a system by which they can measure faculty contributions to their educational mission. From our literature search, 21 U.S. teaching hospitalswas selected for review independently of the above search due to prior knowledge of article content. ## Inception of the evu In 2000, the Association of American Medical Colleges (AAMC)'s Mission-based Management Program [bib_ref] Measuring faculty effort and contributions in medical education, Nutter [/bib_ref] first proposed the use of an Educational Relative Value Unit (RVU), also known as the EVU [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] , Educational Added Value Unit (EAVU) [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] , Educational Relative Value Unit (eRVU) [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] , Teaching Value Unit (TVU) [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] , Teaching Relative Value Unit (TRVU) [bib_ref] Effects of a comprehensive faculty remuneration plan on educational productivity and innovation, Ghaemmaghami [/bib_ref] , or Academic Relative Value Unit (aRVU or ARVU) [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] , to provide medical institutions with a system by which they can measure faculty contributions to their educational mission. From our literature search, 21 U.S. teaching hospitals have published studies describing their EVU systems, which have come to serve several purposes. In specialties that do not offer reduced clinical hours or financial incentive for teaching activities, EVU systems can support physicians through financial compensation or through recognition of time spent on non-clinical activities [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref]. These systems also offer an alternative to clinical productivity-based payment systems, which may deemphasize teaching in favor of increased clinical time. Furthermore, EVU systems serve to more evenly distribute teaching obligations and educational efforts among faculty members [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] and may be used to reallocate funds within [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] or between departments [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] to better support educational activities. Aside from altering compensation, EVU systems allow faculty members to track their educational activities, which is particularly useful for junior faculty members whose educational endeavors strongly influence their chance of promotion [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref]. Though a variety of specialties may benefit from the use of an EVU system for the reasons mentioned above, self-described EVU systems have been implemented by Departments of Medicine [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] , Emergency Medicine [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Effects of a comprehensive faculty remuneration plan on educational productivity and innovation, Ghaemmaghami [/bib_ref] [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref] , Ophthalmology, Pediatric Surgery [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] , Surgery [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] The Impact of a Program for Systematically Recognizing and Rewarding Academic Performance, Williams [/bib_ref] , Pediatrics [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] , Internal Medicine [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Quantifying Physician Teaching Productivity Using Clinical Relative Value Units, Yeh [/bib_ref] [bib_ref] Educational value unit system at the university of Kansas 2005-2005: Updates and..., Babbott [/bib_ref] , Radiology [bib_ref] The academic RVU: A system for measuring academic productivity, Mezrich [/bib_ref] , Anesthesiology [bib_ref] Integration of academic and clinical performance-based faculty compensation plans: A system and..., Sakai [/bib_ref] , and Pathology, as well as Primary Care [bib_ref] Recruiting primary care physicians to teach medical students in the ambulatory setting:..., Denton [/bib_ref]. ## Calculating evus As described by the AAMC's Mission-based Management Program, an EVU is defined as the value, or weight, assigned to different educational activities in a manner specific to a given institution. Educational activities which accrue EVUs place an emphasis on medical student and resident education and include, but are not limited to, developing a course, clerkship, or laboratory program, mentoring students, teaching, publishing peer-reviewed articles, serving in education administration, or achieving scholarship in education. The weight placed on these activities is institution specific and may be influenced by factors such as time and effort spent on an activity, the level of expertise required to perform an activity, and the degree to which the activity contributes to the school's mission. The baseline calculation for the model EVU system created by the AAMC's Mission-based Management Program is EVU = activity weight × units of activity performed. This equation can be modified with several variables, including a solo/group adjustment for the number of participating faculty, the quality of the activity, the category weight, or the program weight [bib_ref] Measuring faculty effort and contributions in medical education, Nutter [/bib_ref]. A sample calculation using the EVU system established by the Cincinnati Children's Hospital Medical Center (CCHMC) is included below [fig_ref] Figure 2: Sample EVU Calculation based on the EAVU Tracking Tool used at the... [/fig_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref]. Medical institutions vary in how they determine which activities accrue EVUs and what factors contribute to the EVU calculation. Most departments that have implemented EVU-based systems created special committees, comprised of individuals like educational faculty, physicians, division directors, and vice chairs, to compile and approve a list of educational and teaching activities that earn EVUs [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. Committees may reference activities performed in the prior academic year [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] or use educational guidelines, like the American Academy of Medical Colleges Committee Educator Promotion categories [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] , to create their initial list of activities. equation can be modified with several variables, including a solo/group adjustment for the number of participating faculty, the quality of the activity, the category weight, or the program weight [bib_ref] Measuring faculty effort and contributions in medical education, Nutter [/bib_ref]. A sample calculation using the EVU system established by the Cincinnati Children's Hospital Medical Center (CCHMC) is included below [fig_ref] Figure 2: Sample EVU Calculation based on the EAVU Tracking Tool used at the... [/fig_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref]. Medical institutions vary in how they determine which activities accrue EVUs and what factors contribute to the EVU calculation. Most departments that have implemented EVU-based systems created special committees, comprised of individuals like educational faculty, physicians, division directors, and vice chairs, to compile and approve a list of educational and teaching activities that earn EVUs [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. Committees may reference activities performed in the prior academic year [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] or use educational guidelines, like the American Academy of Medical Colleges Committee Educator Promotion categories [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] , to create their initial list of activities. While similar activities accrue EVUs across institutions, departments primarily differ in factors contributing to their EVU calculations. Many factor in preparation for a given activity [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] [bib_ref] Quantifying Physician Teaching Productivity Using Clinical Relative Value Units, Yeh [/bib_ref] [bib_ref] Mission-based budgeting for education: Ready for prime time?, Sainté [/bib_ref] , the effort displayed by faculty members [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] The academic RVU: A system for measuring academic productivity, Mezrich [/bib_ref] , prestige garnered [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] , learner type (medical student/resident versus fellow) [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] , and the degree of seniority of faculty [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. Other institutions may weigh their EVU activities depending on the relative importance or quality of the activity to learners and teachers, which can be discerned through administering surveys to medical students, residents, and faculty [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. In these systems, learners may particularly benefit from the implementation of an EVU system because activities that are known to positively affect their future careers are assigned a greater weight. Departments may also add or subtract EVUs from a faculty member's annual EVU count depending on their performance in a given activity [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] and do not assign specific weights to activities, instead focusing on time required to complete each activity [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref]. Which factors and weights are incorporated into each institution's EVU calculation is subjective and ultimately reflects its culture and educational priorities. See [fig_ref] Table 1: Some Factors Contributing to EVU Calculations in Reporting U [/fig_ref]. While similar activities accrue EVUs across institutions, departments primarily differ in factors contributing to their EVU calculations. Many factor in preparation for a given activity [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] [bib_ref] Quantifying Physician Teaching Productivity Using Clinical Relative Value Units, Yeh [/bib_ref] [bib_ref] Mission-based budgeting for education: Ready for prime time?, Sainté [/bib_ref] , the effort displayed by faculty members [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] The academic RVU: A system for measuring academic productivity, Mezrich [/bib_ref] , prestige garnered [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] , learner type (medical student/resident versus fellow) [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] , and the degree of seniority of faculty [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. Other institutions may weigh their EVU activities depending on the relative importance or quality of the activity to learners and teachers, which can be discerned through administering surveys to medical students, residents, and faculty [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. In these systems, learners may particularly benefit from the implementation of an EVU system because activities that are known to positively affect their future careers are assigned a greater weight. Departments may also add or subtract EVUs from a faculty member's annual EVU count depending on their performance in a given activity [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] and do not assign specific weights to activities, instead focusing on time required to complete each activity [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref]. Which factors and weights are incorporated into each institution's EVU calculation is subjective and ultimately reflects its culture and educational priorities. See [fig_ref] Table 1: Some Factors Contributing to EVU Calculations in Reporting U [/fig_ref]. Institutions that have established EVU systems additionally differ in the EVU obligations assigned to their faculty. In many EVU systems, faculty members must spend a minimum number of hours performing educational activities. This minimum requirement may be influenced by how much annual teaching effort is available or how much teaching effort is necessary to achieve the department's educational mission and may range from as little as 30 h [10] to 400 h [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref]. Other systems simply assign a total number of EVUs to be accrued annually by each faculty member [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref]. Often, the EVU obligation assigned to faculty members is impacted by the faculty member's employment status, with full-time faculty members on educator career paths having larger EVU obligations than part-time faculty or fellows [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref]. For mandatory EVU systems, failure to achieve one's EVU obligations can result in funding cutbacks, increases in future teaching time, increases in clinical obligations, or reconsideration of one's faculty status [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref]. For voluntary EVU systems, failure to achieve one's EVU obligations may result in a lack of or reduced financial bonus at the academic year's end [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref]. ## Evu tracking systems To keep track of annual EVU totals for a given academic year, departments may use a combination of self-reporting, record-keeping systems, and administrative data. Physicians may self-report educational activities to academic leadership who provide final approval or log their activities in systems like RedCap or Microsoft Excel [bib_ref] Educational Added Value Unit: Development and Testing of a Measure for Educational..., Guiot [/bib_ref] [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref]. Administrative data, such as conference schedules, attendance records, or Medhub reports [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] , can also be used track educational activities. Some departments have also created web-based systems [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] The academic RVU: A system for measuring academic productivity, Mezrich [/bib_ref] [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref] or online dashboards to log educational activities and to calculate EVUs earned. Using one such system, faculty members received a monthly email detailing their EVU accruals [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref]. ## Evu systems and financial compensation Some U.S. teaching hospitals offer financial incentives for educational efforts performed under an EVU system [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref] [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] The Impact of a Program for Systematically Recognizing and Rewarding Academic Performance, Williams [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref] [bib_ref] Educational value unit system at the university of Kansas 2005-2005: Updates and..., Babbott [/bib_ref] [bib_ref] Recruiting primary care physicians to teach medical students in the ambulatory setting:..., Denton [/bib_ref] [bib_ref] Mission-based budgeting for education: Ready for prime time?, Sainté [/bib_ref]. Assuming a faculty member has met their baseline EVU requirements, this monetary benefit may be provided as a small percentage (≤5%) [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] of their annual or baseline salary, as a component of their annual financial bonus, or as a change in salary support [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref] [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref]. Alternatively, faculty may be awarded a dollar amount per EVU [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref] [bib_ref] Recruiting primary care physicians to teach medical students in the ambulatory setting:..., Denton [/bib_ref] [bib_ref] Mission-based budgeting for education: Ready for prime time?, Sainté [/bib_ref] , like at the Department of Medicine at the Medical University of South Carolina, where faculty were awarded USD 41.00 per EVU and 1 h spent on teaching = 2 EVU [bib_ref] Valuing the Education Mission: Implementing an Educational Value Units System, Clyburn [/bib_ref]. Departments may also compensate faculty based on their educational performance relative to their peers, where the financial bonus is either calculated using Individual EVUs earned/Group EVUs earned × teaching incentive dollars [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] or as lump sum amounts ranging from USD 0 to approximately USD 40,000 [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref]. Due to the relatively small compensation earned for educational efforts under EVU systems compared to the potential bonuses unlocked by meeting clinical productivity criteria, it is unlikely that financial compensation plays a significant factor in the success of these systems. Rather, other drivers, namely advancement in faculty position, are likely responsible for the increased educational efforts made by faculty participating in these systems. ## Outcomes of implementing an evu system Implementing an EVU system is an effective way to advance an institution's educational mission because these systems successfully redistribute educational burden among faculty members, increase the number of faculty members who meet baseline educational and teaching expectations, and promote educational productivity. In the Department of Emergency Medicine at Johns Hopkins Medicine and in the Department of Internal Medicine at St. Joseph Mercy Hospital in Ann Arbor, teaching responsibilities were more equally distributed among faculty after the implementation of their EVU systems [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref] [bib_ref] Quantifying Physician Teaching Productivity Using Clinical Relative Value Units, Yeh [/bib_ref]. Similarly, the Departments of Emergency Medicine at the University of Michigan Medical School and the New York University School of Medicine saw a significant increase in the number of faculty who attended conferences (p < 0.005, p < 0.001) and in the number of completed resident evaluations (p < 0.005, p < 0.001) after incorporating EVU systems into their departments [bib_ref] An Academic Relative Value Unit System: Do Transparency, Consensus, and Accountability Work?, Carmody [/bib_ref] [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref]. Educational productivity also increases under EVU systems, with some institutions reporting nearly a 30% increase in the mean individual productivity of faculty after implementation (p = 0.01) [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] , others reporting a 10% increase (84% to 94%) in the number of faculty meeting baseline educational requirements [bib_ref] Implementation of an Education Value Unit (EVU) System to Recognize Faculty Contributions, House [/bib_ref] and still others reporting a statistically significant increase in annual peer-reviewed publications [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref] [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref] [bib_ref] Integration of academic and clinical performance-based faculty compensation plans: A system and..., Sakai [/bib_ref]. EVU systems additionally motivate physicians with greater clinical obligations to engage in teaching, mentorship, and leadership activities. Physicians with the highest clinical load in pediatric emergency medicine departments affiliated with Emory University had a significant increase in teaching productivity post-implementation of an EVU system (p = 0.03) [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] , as did emergency medicine physicians at the University of Virginia School of Medicine (79% increase) [bib_ref] Effects of a comprehensive faculty remuneration plan on educational productivity and innovation, Ghaemmaghami [/bib_ref]. Similarly, ophthalmologists at Weill Cornell Medicine engaged in significantly more mentorship (p = 0.013) after the implementation of their points system. In the Department of Surgery at the Baylor College of Medicine, the number of faculty holding committee positions in academic organizations increased significantly after implementation of their EVU system (p < 0.001) [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref]. EVU systems may additionally contribute to changes in external funding received by physicians and medical departments. One Department of Pediatric Surgery saw an increase in annual external federal funding from USD 750,168 to USD 5,768,243 during the period in which their EVU system was implemented [bib_ref] Development of an academic RVU (aRVU) system to promote pediatric surgical academic..., Brigstock [/bib_ref]. In another surgical department, there was a statistically significant increase in total research funding, total NIH funding, total income from active grants, and income from industry sponsored trials after implementation of their EVU system (p < 0.001) [bib_ref] An Academic Relative Value Unit System for Incentivizing the Academic Productivity of..., Lemaire [/bib_ref]. This increase in extramural research funding also occurs in non-surgical specialties, such as emergency medicine. At Oregon Health & Science University, extramural research funding increased from USD 950,844 in the first year of their EVU system to USD 2,735,233 in the tenth year [bib_ref] The Academic RVU: Ten Years Developing a Metric for and Financially Incenting..., Ma [/bib_ref]. Though these departments saw an increase in external funding after implementation of their EVU systems, there are a variety of other factors that may contribute to this phenomenon, such as inflation or an increase in department size leading to a larger number of grant applications. While EVU systems can lead to increases in external funding for research, they can also lead to shifts in salary within departments. In the Department of Internal Medicine at the University of Kansas School of Medicine, 60% of faculty had a mean salary support reduction of USD 28,814 after implementation of their system, while the remaining 40% had a mean salary support increase of USD 29,453. In total, the department shifted USD 1.66 million in funding among faculty members [bib_ref] Aligning compensation with education: Design and implementation of the educational value unit..., Stites [/bib_ref] , with mixed responses from faculty: only 50% felt this shift in funds offset clinical salary losses incurred while engaging in educational activities [bib_ref] Educational value unit system at the university of Kansas 2005-2005: Updates and..., Babbott [/bib_ref]. In the Department of Medicine at the Medical University of South Carolina, over USD 500,000 was redistributed between departments over a four-year period based on the number of educational value units accumulated by each department. While physician salaries did not change under this system, the salary deficit between physicians in different ## Faculty response to evu systems Though there is limited data assessing the reception of EVU systems, current literature suggests the faculty response to these systems is mixed. Some faculty feel quantifying academic efforts could negatively impact their careers if their degree of activity is seen as lacking, while others feel academic activities are specialty specific; and thus, generalized categories in EVU systems do not accurately reflect their level of engagement [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. Faculty may additionally become upset when they do not meet baseline educational requirements, which in some cases, may lead to a departmental re-evaluation of the criteria used to determine EVUs [bib_ref] Establishing an educational value unit to promote teaching in an academic unit, Butani [/bib_ref]. Theoretical discussions of EVU systems have also proposed that these systems may introduce competition between members of a department rather than facilitate collaboration [bib_ref] Academic Relative Value Units: A Proposal for Faculty Development in Oral and..., Carlson [/bib_ref] due to the inherent nature of the system comparing one's performance to that of one's colleagues. In contrast, faculty at the University of Kansas Department of Medicine positively received their EVU system: the majority of faculty felt their system had a positive or neutral impact on their educational productivity, as well as on the quality and quantity of educational activities [bib_ref] Educational value unit system at the university of Kansas 2005-2005: Updates and..., Babbott [/bib_ref]. ## Limitations of an evu system Some limitations exist with the implementation of EVU systems in U.S. teaching hospitals and medical schools. EVU systems do not always assess the quality of edu-cational activities performed, so an increase in educational productivity may not reflect an equivalent increase in learning. However, the Department of Emergency Medicine at Johns Hopkins Medicine did not factor quality into their EVU calculations and still found equivalent learner satisfaction in educational programs before and after the implementation of their system [bib_ref] Educational Value Units: A Mission-Based Approach to Assigning and Monitoring Faculty Teaching..., Regan [/bib_ref]. If faculty in different departments do not equally participate in their institution's EVU system, the system may falsely reflect educational effort made by departments as a whole, which could lead to an inappropriate redistribution of funds across departments. With some EVU tracking systems, there may also be self-report bias from physicians and faculty; and therefore, their EVUs may not be truly reflective of their educational effort. By setting baseline expectations, some EVU systems may also disincentivize some faculty from engaging in educational activities beyond their minimum expectations [bib_ref] The Definition of Faculty Must Evolve: A Call to Action, Bellini [/bib_ref] , though there is limited evidence of this occurring. # Discussion Since its introduction in 2000 by the AAMC's Mission-based Management Program, the EVU has been used across U.S. teaching hospitals and medical schools to assess faculty contribution to an institution's educational mission. A limited number of studies describing current EVU systems have been published; however, the available literature supports the notion that these systems successfully advance an institution's educational mission in a myriad of ways. EVU systems redistribute educational burden and funding among faculty, increase the number of faculty meeting baseline educational requirements, promote educational productivity, support research endeavors, and encourage physicians with greater clinical obligations to engage in teaching, mentorship, and leadership activities. Department chairs can use EVUs to evaluate departmental contributions to educational achievement relative to other departments and use this knowledge to adjust incentives for clinical, research, administrative, or teaching activities. Additionally, by recognizing and rewarding educational efforts, EVU systems may help promote faculty vitality and reduce burnout-particularly in the midst of the COVID-19 pandemic which is actively reshaping employee motivation and workforce behavior [bib_ref] Frontline physician burnout during the COVID-19 pandemic: National survey findings, Melnikow [/bib_ref]. The implementation of EVU systems most greatly benefits academic physicians, junior faculty on an educational career path, and trainees at various stages. While EVU systems may encourage physicians with higher clinical loads to engage in more educational activity [bib_ref] Development and Implementation of a Relative Value Scale for Teaching in Emergency..., Khan [/bib_ref] , they are unlikely to motivate those physicians who are already uninterested in contributing to a school's academic mission. For faculty who dedicate a significant proportion of their careers to teaching and education; however, these systems provide recognition for their efforts and serve to reduce salary deficits incurred from lower clinical productivity. EVUs are of particular use to junior academic faculty, as they can be used to track the activities most likely to advance their career and may be used as a tangible, objective metric for promotion. Medical students, residents, and fellows also benefit from EVU systems since faculty are encouraged to engage in more teaching and research efforts, and many institutions have begun to factor learner satisfaction into their EVU weighting [bib_ref] Creating a Mission-based Reporting System at an Academic Health Center, Howell [/bib_ref]. By incentivizing physicians to mentor and educate trainees, EVU systems facilitate the process by which clinical knowledge and physicianship is passed from one generation of physicians to the next. Though this study does not address EVU systems implemented in settings other than U.S. academic medical centers, such as psychology or dentistry, these systems may additionally benefit from an EVU system for the reasons mentioned above. As mentioned previously, the monetary reward associated with EVUs is unlikely to be a significant factor contributing to the increased educational productivity seen under these systems. Faculty who already engaged in teaching, research, and administration will continue to perform these activities whether or not they accrue EVUs, especially under voluntary systems. Comparing a faculty member's EVU totals to their peers can motivate some to give a greater educational effort; this motivation may be driven by a sense of responsibility, a desire to meet the educational mission of their institution, or feelings of shame. # Limitations This narrative review has several limitations. We limited our search to the Ovid MEDLINE, Embase, Web of Science, and PubMed databases to identify relevant studies and abstracts. Due to the search terms used, it is possible that we did not include the entire body of literature self-reporting EVU systems; however, we attempted to minimize this chance by examining the bibliographies of selected studies to identify additional search terms. We did not include studies discussing the implementation of EVU systems in non-medical settings, such as psychology or dentistry, or in locations other than the U.S. In addition, when discussing the outcomes of EVU systems, we are only able to imply association, not causation, as several factors may have played a role in the changes seen with the implementation of EVU systems. # Conclusions In conclusion, EVU systems are an effective way to advance an institution's educational mission and reward individual and departmental educational efforts in U.S. academic medical centers and medical schools. As these institutions renew their commitment to their educational missions, EVU systems will likely become a permanent fixture in the evaluation and payment schemes of U.S. academic medical institutions. [fig] Figure 1: Method of article and abstract selection based on searches conducted in Ovid MEDLINE, EMBASE, Web of Science, and PubMed databases. Search results were examined to select articles and abstracts for review, and additional articles were identified from bibliographies of selected articles.Chen et al. (2021) [/fig] [fig] Figure 2: Sample EVU Calculation based on the EAVU Tracking Tool used at the Cincinnati Children's Hospital Medical Center from Guiot et al. (2017)[14]. [/fig] [fig] Author: Contributions: G.J.L., D.A.C. and A.H. were responsible for narrative review conception. A.H. was responsible for literature review and manuscript writing, and A.H., D.A.C. and G.J.L. were responsible for manuscript revisions. All authors have read and agreed to the published version of the manuscript. [/fig] [table] Table 1: Some Factors Contributing to EVU Calculations in Reporting U.S. Medical Institutions. [/table] [bib_ref] Restoring Faculty Vitality in Academic Medicine When Burnout Threatens, Shah [/bib_ref]

Purpura fulminans due to Vibrio vulnificus severe infection # Introduction Purpura fulminans is a life-threatening disease, characterized by disseminated intravascular coagulation and endovascular thrombosis, can often occur secondary to heterogeneous etiologies, such as sepsis, and to a lesser extent, secondary to sepsis due to halophilic bacteria, such as V. vulnificus, found in marine environments. Patients with specific comorbidities are at the highest risk of worst scenarios, without prompt treatment, infection can rapidly evolve to fatal, with a mortality rate close to 100 %. We present a case of Purpura fulminans due to V. vulnificus septicemia. ## Case report 52-year-old man, with type 2 diabetes and systemic arterial hypertension; bariatric surgery performed two years ago and history of raw seafoods consumption and skin trauma with an oyster while manipulating it ten days before to hospital admission. Upon admission, he referred 7 days with throbbing pain and burning legs, exacerbated by walking and improvement with rest and nonsteroidal anti-inflammatory analgesics ingestion; erythema and edema appeared later in both legs. During initial assessment, abnormal vital signs, such as, tachycardia, arterial hypotension, and undetectable oxygen saturation were found; physical examination highlighted a localized bilateral and symmetric dermatosis of the lower extremities constituted by edematous purpuric plaques with tense blisters of bloody content, excoriations, areas of necrosis and hyaline exudate [fig_ref] Figure 1: Clinical and histopathological findings [/fig_ref]. Laboratory findings suggested acute kidney injury, septic shock, and disseminated intravascular coagulation. Doppler ultrasound of pelvic limbs reported edema of superficial tissues from knee to foot, with no evidence of thrombi; abdominopelvic computed tomography reported compatible findings with cellulitis of the left pelvic limb. The patient rejected surgical treatment, and progressed to multiorgan failure, besides broad-spectrum antibiotics, and vasopressor therapy, and died 48 h after hospital admission. After-death, V. vulnificus was isolated in blood and wound cultures; changes in relation to Purpura fulminans were described in skin biopsies [fig_ref] Figure 1: Clinical and histopathological findings [/fig_ref]. # Discussion Purpura fulminans (PF) is a life-threatening disease characterized by disseminated intravascular coagulation and endovascular thrombosis resulting in a cutaneous purpura pattern [bib_ref] Purpura fulminans: mechanism and management of dysregulated hemostasis, Colling [/bib_ref]. Clinically, early stages present as well-demarcated erythematous and painful macule that progress rapidly to irregular central areas of blueblack hemorrhagic necrosis, typically surrounded by a thin border of erythema that fades into adjacent uninvolved skin. Early lesions may be reversible with prompt therapeutic intervention, but established lesions often progress within 24-48 h to full-thickness skin necrosis or more extensive soft tissue necrosis that may require surgical debridement, fasciotomies, or amputation [bib_ref] Purpura fulminans: mechanism and management of dysregulated hemostasis, Colling [/bib_ref]. Histopathological findings include occlusion of small dermal vessels with microthrombi, capillary dilation and congestion with red cells in early PF. In later stages, an irreversible endothelial ischemic injury with extravasation of blood cells into the dermis and gangrenous necrosis, sometimes with secondary infection, appears. Activation of procoagulant pathways, dysfunction of anticoagulant pathways and endothelial damage characterized this entity [bib_ref] Purpura fulminans: mechanism and management of dysregulated hemostasis, Colling [/bib_ref]. PF requires prompt recognition and treatment, targeting the coagulation system and management of the underlying cause [bib_ref] Purpura fulminans: mechanism and management of dysregulated hemostasis, Colling [/bib_ref] [bib_ref] Purpura fulminans induced by Vibrio vulnificus, Akoh [/bib_ref]. Although, PF may be triggered by several clinical scenarios, frequently occurs as consequence of infection, especially by gramnegative endotoxin-producing bacteria, but can also occur due to gram-positive, anaerobic, and viral infections. Less common cases of PF due to bacteria of genus vibrio have also been reported [bib_ref] Purpura fulminans: mechanism and management of dysregulated hemostasis, Colling [/bib_ref] [bib_ref] Purpura fulminans induced by Vibrio vulnificus, Akoh [/bib_ref] [bib_ref] A case of Vibrio vulnificus infection complicated with fulminant purpura: gene and..., Hori [/bib_ref]. Vibrio vulnificus is a gram-negative, opportunistic bacillus responsible of wound infections, gastroenteritis, and septicemia. Inhabits saltwater flora and fauna, multiple cases have been reported annually [bib_ref] A case of Vibrio vulnificus infection complicated with fulminant purpura: gene and..., Hori [/bib_ref] [bib_ref] Vibrio vulnificus: report of a potentially fatal skin infection, Dupont [/bib_ref] in regions with a subtropical climate [bib_ref] Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a..., Leng [/bib_ref] ; however, in recent years significant increases in expansion of the geographic area have been observed in infections caused by this pathogen, probably due to global warming increasing ocean temperature [bib_ref] Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a..., Leng [/bib_ref] [bib_ref] The role of Vibrio vulnificus virulence factors and regulators in its infection-induced..., Li [/bib_ref]. This bacillus is the deadliest foodborne pathogen with a fatality rate greater than 30 %, however, severity of this condition will depend too on host's comorbidities [bib_ref] Biochemical and virulence characterization of Vibrio vulnificus isolates from clinical and environmental..., Lydon [/bib_ref]. Two main routes of entry into the bloodstream have been identified in the development of this infection; ingestion of contaminated raw seafood, mainly oysters, and direct bacterial inoculation of skin wounds by direct contact between open wounds and contaminated seawater, or by skin trauma when fishing, handling, or preparation of seafoods [bib_ref] Vibrio vulnificus: report of a potentially fatal skin infection, Dupont [/bib_ref] [bib_ref] Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a..., Leng [/bib_ref] [bib_ref] Biochemical and virulence characterization of Vibrio vulnificus isolates from clinical and environmental..., Lydon [/bib_ref]. Three biotypes of V. vulnificus strains are known. Biotype 1 strains are responsible of most infections in humans, while biotype 2 strains (non-pathogenic for humans) are responsible for infections in marine eels. The strains of biotype 3, which cause fewer skin infections in humans, have been limited to people who handle fish [bib_ref] The role of Vibrio vulnificus virulence factors and regulators in its infection-induced..., Li [/bib_ref] [bib_ref] Biochemical and virulence characterization of Vibrio vulnificus isolates from clinical and environmental..., Lydon [/bib_ref]. V. vulnificus has virulence factors that cause direct cellular damage, including cytolysin, toxin A1, and extracellular proteases. In addition, V. vulnificus contains immunogenic lipoprotein A, which acts as an adhesin and immunogen to activate intracellular pathways, triggering an inflammatory cascade [bib_ref] The role of Vibrio vulnificus virulence factors and regulators in its infection-induced..., Li [/bib_ref] [bib_ref] Structure, function, and regulation of the essential virulence factor capsular polysaccharide of..., Pettis [/bib_ref]. V. vulnificus can cause mild infectious diseases in healthy hosts, which can resolve spontaneously without antibiotic treatment; however, in immunocompromised individuals, severity of the disease becomes much worse, with bacteremia being common, with a mortality rate greater than 50 % [bib_ref] A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play..., Duong-Nu [/bib_ref] ; fatal cases without prompt treatment who evolves to septic shock, mortality rate is close to 92 % [bib_ref] Vibrio vulnificus: review of mild to life-threatening skin infections, Coerdt [/bib_ref] [bib_ref] From sea to bloodstream: Vibrio vulnificus sepsis, Sheer [/bib_ref]. In case of oral ingestion, gastroenteritis may occur in the best-case scenario, meanwhile, wound infection can manifest heterogeneously, from mild symptoms with well-defined erythematous lesions, blisters, and cellulitis to deep infections such as myositis, necrotizing fasciitis, and purpura fulminans [bib_ref] Vibrio vulnificus: review of mild to life-threatening skin infections, Coerdt [/bib_ref]. Less common pneumonia, osteomyelitis, ocular infections, meningitis, peritonitis, and endocarditis may occur [bib_ref] From sea to bloodstream: Vibrio vulnificus sepsis, Sheer [/bib_ref] [bib_ref] Vibrio vulnificus infection: a persistent threat to public health, Yun [/bib_ref]. Septicemia may occur through both: ingestion of the microorganism and its passage into the bloodstream through the ileum or cecum, or as infected wounds and the ascent of the bacillus to the circulation [bib_ref] Vibrio vulnificus infection: a persistent threat to public health, Yun [/bib_ref] , however, of patients with chronic diseases, those with chronic liver disease, have an 80-fold higher risk of developing septicemia due to V. vulnificus compared to the rest of people with chronic affections, which place them at the top of the list [bib_ref] From sea to bloodstream: Vibrio vulnificus sepsis, Sheer [/bib_ref]. Cornerstone in the diagnostic approach, are both clinical presentation and epidemiological background. Growth of V. vulnificus in culture samples confirms the diagnosis. Other valuable diagnostic tests are conventional and real-time polymerase chain reactions (PCR), of which real-time PCR is characterized by its high sensitivity and specificity, as well as rapid execution and results, making it useful in patients with atypical manifestations or with recent antibiotic treatment [bib_ref] Vibrio vulnificus infection: a persistent threat to public health, Yun [/bib_ref]. Centers for Disease Control and Prevention (CDC) recommends the combine use of third generation cephalosporins and doxycycline for 7-14 days on the treatment of V. vulnificus infections. Patients with deep skin and soft tissue infections will often require urgent surgical debridement or amputation, in addition to promptly establishment of antibiotic therapy [bib_ref] Vibrio vulnificus infection: a persistent threat to public health, Yun [/bib_ref]. # Conclusion Sepsis and septic shock produced by V. vulnificus have a high fatality rate despite surgical and targeted antimicrobial treatment. In patients with immunosuppressive conditions, especially those with chronic liver disease, mild infections can easily evolve to catastrophic scenarios, since these people, constantly present high levels of transferrin saturation indexes, which favors the growth and reproduction of the microorganism and, with it, its blood dissemination. V. vulnificus infection should be suspected in immunocompromised patients with painful skin lesions and a history of raw shellfish ingestion, handling, or exposure of skin wounds to seawater. It is extremely important to inform vulnerable population about the importance of the use of protective gloves when handling shellfish for consumption or sale, as well as to avoid intake of raw shellfish, as well as exposition of open wounds to seawater. # Ethical approval This protocol was carried out in strict adherence to current regulations, considering the Declaration of Helsinki, Good Clinical Practices, International Ethical Guidelines for Biomedical Research and Experimentation on Human Beings and the Regulations of the General Health Law on Research for health. This protocol corresponds to an investigation without risk, since the information collected was carried out using retrospective documentary research techniques and methods (clinical file review) and no intentional intervention or modification was made in the physiological, psychological, and social variables. of the individual who participated in the study. ## Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. # Funding source In the making of this manuscript, there was none financial support provided by any private or public institution. [fig] Figure 1: Clinical and histopathological findings. a) Purpuric plaques characterized by hemorrhagic bullae, ulceration, necrosis, and diffuse edema on the left pelvic limb. b) and c) Biopsy sections of the patient's left thigh and left foot dorsum observed by electron microscopy. b) Dermoepidermal infarction with detachment of the superficial layer of the epidermis. c) Purpuric dermatitis associated with nascent thrombi in the deep vascular plexus. [/fig]

Possible Treatment Approaches of Sulfur Mustard-Induced Lung Disorders, Experimental and Clinical Evidence, an Updated Review Sulfur mustard (SM) is one of the major potent chemical warfare that caused the death of victims in World War I and the Iraq-Iran conflict (1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988). The respiratory system is the main target of SM exposure and there are no definitive therapeutic modalities for SM-induced lung injury. The effects of the new pharmaceutical drugs on lung injury induced by SM exposure were summarized in this review. Literature review on PubMed, ScienceDirect, and Google Scholar databases was performed to find papers that reported new treatment approach on SM-exposure-induced injury in the respiratory system until October 2019. The search was restricted to sulfur mustard AND induced injury (in vitro studies, animal experiments, and clinical trials) AND respiratory system OR lung, AND treatment in all fields. Two hundred and eighty-three relevant articles were identified that 97 retrieved articles were eligible and were included in the review. Some new pharmaceutical drugs have shown therapeutic potential in controlling various characteristics of lung injury due to SM exposure. Recent studies showed therapeutic effects of mucolytic drugs, non-steroidal drugs, and antibiotics on reducing lung inflammation, oxidative stress responses, and modulating of the immune system as well as improving of respiratory symptoms and pulmonary function tests. Studies on the therapeutic effects of new agents with amelioration or treatment of SM-induced lung injury were reviewed and discussed. # Introduction Sulfur mustard (SM) is a toxic vesicant that was first used in 1917 as a chemical weapons (CW) agent and was repeatedly used during Iraq-Iran conflict . For a period (5 years) from August 1983 to July 1988, Iran was attacked several times with CW by the Iraqi army. Iraq used large amounts of CW against Iranian military and civilian people. Sardasht (the Kurdish cities, North West of Iran) was attacked several times in July 1987 and June 1988. The last Iraq chemical attack was in Feb 1988 in the town of Oshnaviyeh (North West of Iran) which injured thousands of civilians. The use of CW against civilian population in eight locations in Oshnaviyeh (Sheikh Othman District) has been confirmed by United Nation's experts [bib_ref] Iraq-Lran chemical war: calendar, mortality and morbidity, Seyed [/bib_ref]. Sulfur mustard is a cell poison agent which can alter DNA and other nuclear components [bib_ref] Nuclear dependence of sulfur mustard-mediated cell death, Lodhi [/bib_ref] , and cause short and long term injury to heart, lung, nervous, and digestive systems [bib_ref] Regular review: chemical weapons, Evison [/bib_ref] depending on the dose and duration of the exposure [bib_ref] Medical aspects of sulphur mustard poisoning, Kehe [/bib_ref]. The lungs are major targets of SM which led to destruction of bronchial tissues (7), obstruction of airways [bib_ref] Chronic pulmonary sequelae of sulfur mustard gas exposure in man: a report..., Emad [/bib_ref] , and oxidative stress [bib_ref] Oxidants and antioxidants in sulfur mustard-induced injury, Laskin [/bib_ref]. Edema and erythema of the pharynx and bronchial tree have occurred minutes to several hours after exposure to SM [bib_ref] Bronchiolitis obliterans: an update, Chan [/bib_ref] , hemorrhagic pulmonary edema, secondary pneumonia, and respiratory failure 24-48 h after severe exposure [bib_ref] Diagnostic and therapeutic value of short-term corticosteroid therapy in exacerbation of mustard..., Ghanei [/bib_ref] and bronchopneumonia 36-48 h after SM exposure [bib_ref] Pulmonary manifestations of mustard gas injury: a review of 61 cases, Hosseini [/bib_ref] in the veterans. Various pathological changes of the lungs (13) in SM exposed individuals can lead to asthma and/or chronic obstructive pulmonary disease (COPD) like symptoms [bib_ref] Respiratory effects of sulfur mustard exposure, similarities and differences with asthma and..., Khazdair [/bib_ref]. Releasing of several inflammatory mediators by phagocytic leukocytes [bib_ref] Protection from halfmustard-gas-induced acute lung injury in the rat, Mcclintock [/bib_ref] [bib_ref] Macrolide antibiotics improve chemotactic and phagocytic capacity as well as reduce inflammation..., Gao [/bib_ref] , increased bronchoalveolar lavage fluid (BALF) cells [bib_ref] Pathologic changes in rat lungs following acute sulfur mustard inhalation, Anderson [/bib_ref] [bib_ref] Inflammatory effects of inhaled sulfur mustard in rat lung, Malaviya [/bib_ref] which were shown in animals exposed to SM that indicates the role of phagocytic leukocytes and their inflammatory mediators in the pathogenic response to inhaled SM [bib_ref] Sulfur mustard toxicity in macrophages: effect of dexamethasone, Amir [/bib_ref]. Pulmonary and systemic inflammation such as changes in serum levels of cytokines [bib_ref] Serum levels of IL-8 and IL-6 in the long term pulmonary complications..., Pourfarzam [/bib_ref] , increased C-reactive protein, [bib_ref] Highly sensitive C-reactive protein levels in Iranian patients with pulmonary complication of..., Attaran [/bib_ref] and the serum levels of the pro-apoptotic protein, soluble Fas l [bib_ref] Serum soluble fas ligand and nitric oxide in long-term pulmonary complications induced..., Ghazanfari [/bib_ref] were observed in individuals long time after exposing to SM. Increased capillary leakageand permeability in the cultured vascular endothelial cell monolayers [bib_ref] Activation of poly [ADP-ribose] polymerase in endothelial cells and keratinocytes: role in..., Hinshaw [/bib_ref] were also reported due to SM exposure. Hematological changes such as increased WBC count, increased respiratory symptoms, and reduced pulmonary function test (PFT) were reported in veterans long term after SM exposure [bib_ref] Long term effect of sulfur mustard exposure on hematologic and respiratory status,..., Khazdair [/bib_ref]. The possible mechanisms of SM-induced lung disorders were shown in . Presently, there is no approved medication for the treatment of SM exposure-induced lung injury [bib_ref] Preventive measures against the mustard gas: a review, Razavi [/bib_ref] , but a combination of vitamin E and corticosteroids was used for protection against acute phases of SM-induced lung injury [bib_ref] Treatment with dexamethasone or liposome-encapsuled vitamin E provides beneficial effects after chemical-induced..., Wigenstam [/bib_ref]. Moreover, Lnitroarginine methyl ester (L-NAME) and L-thiocitrullline (L-TC) are two protective drugs against the acute toxicity of SM in the in vitro and in vivo studies [bib_ref] Characterization of the protective effects of L-nitroarginine methyl ester (L-NAME) against the..., Sawyer [/bib_ref] [bib_ref] Protection against systemic poisoning by mustard gas, di (2-chloroethyl) sulphide, by sodium..., Callaway [/bib_ref]. The results of these studies showed that different treatments still have no optimal effectiveness and also have known adverse effects in this stage of disease [bib_ref] Treatment for sulfur mustard lung injuries; new therapeutic approaches from acute to..., Poursaleh [/bib_ref]. After the acute phase, respiratory problems such as chronic bronchitis (59%), tracheobronchial stenosis (24%), asthma (11%), or bronchiectasis (9%) are the leading causes of long-term disability among patients with exposure to SM [bib_ref] Fibrogenic cytokine levels in bronchoalveolar lavage aspirates 15 years after exposure to..., Aghanouri [/bib_ref] [bib_ref] Mustard gas exposure and carcinogenesis of lung, Hosseini-Khalili [/bib_ref]. Inhaled corticosteroids are extensively used to resolve exacerbation of respiratory symptoms for the treatment of longterm toxic effects of SM [bib_ref] Effect of high dose inhaled steroid on cells, cytokines, and proteases in..., Culpitt [/bib_ref]. In addition, bronchodilators can be applied for treatment of increased airway hyper-reactivity in SM exposed patients. The combination of β-agonist and anticholinergic with corticosteroids has been found to be more effective than any of the other bronchodilators used alone in SM exposed patients [bib_ref] Inhaled corticosteroids and long-acting β2-agonists in treatment of patients with chronic bronchiolitis..., Ghanei [/bib_ref]. The possible therapeutic effects of natural products on SM induced complication were also reported [bib_ref] Possible treatment with medicinal herbs and their ingredients of lung disorders induced..., Khazdair [/bib_ref]. Exposure to SM could induced lung injury including longterm effects like COPD and fibrosis, even decades after exposure. It is essential to identify efficacious treatments for chronic diseases induced by SM. Therefore, this review aimed to present available experimental and clinical publications on the efficacy of the synthesized drugs in the prevention and/or treatment of lung disorders due to SM exposure. ## Possible treatment approaches for sm induced lung disorders ## Anti-inflammatory agents ## Interferon-gamma Interferon-gamma (IFN-γ) and transforming growth factor-beta1 (TGF-β1) showed inhibitory effects on several aspects of the inflammatory process. TGF-β1 induced stimulation of collagen transcription in fibroblasts independent of Stat1promoter interactions but IFN-γ inhibited these effects [bib_ref] Inhibition of type I collagen mRNA expression independent of tryptophan depletion in..., Yufit [/bib_ref]. IFN-γ and TGFβ may also provide opposing signals to macrophages [bib_ref] Transforming growth factor beta 1 and gamma interferon provide opposing signals to..., Hausmann [/bib_ref]. ## Experimental studies Interferon-gamma modified mesenchymal stem cells (MSCs) induced apoptosis in lung tumor cells through caspase-3 activation. The percentage of activated-caspase-3-positive tumor cells in IFN-γ modified cultured MSCs was significantly higher than in control cultured MSCs. The results of this study provide a new strategy for tumor therapy that utilizes IFN-γ modified MSCs [bib_ref] IFN-γ-secreting-mesenchymal stem cells exert an antitumor effect in vivo via the TRAIL..., Yang [/bib_ref]. Anti-inflammatory effect of IFN-γ via down-regulation of TGF-β1 and pro-collagen I and III gene expression in a mouse model of lung fibrosis was demonstrated [bib_ref] Molecular mechanisms of antifibrotic effect of interferon gamma in bleomycin-mouse model of..., Gurujeyalakshmi [/bib_ref]. Interactions of IFN-γ and IL-13, on the mouse model of airway inflammation, showed a negative correlation between increased IFN-γ (number of Th1 cells) with airway hyperreactivity. Also, IFN-γ is able to modulate the effects of IL-13-induced airway hyperreactivity and goblet cell hyperplasia. Intranasal administration of IFNγ inhibits IL-13-induced goblet cell hyperplasia and airway eosinophilia. Co-administration of IFN-γ and IL-13 showed synergic effects on increased IL-6 level as well as numbers of natural killer (NK) cells and CD11c-positive cells in the airways of mice [bib_ref] IL-13 and IFN-γ: interactions in lung inflammation, Ford [/bib_ref]. ## Clinical studies Interferon-gamma has been shown to be effective in the treatment of patients with idiopathic pulmonary fibrosis (IPF) [bib_ref] A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone..., Ziesche [/bib_ref]. Decreased IFN-γ levels in leukocyte cultures from SM FIGURE 1 | Possible mechanisms of lung injury induced by SM exposure. MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; TGF-β1, transforming growth factor-beta1; IL, interleukin; IFNγ, interferon gamma; TNFα, tumor necrosis factor-α; MMPs, matrix metalloproteinase; FVC, forced volume capacity; FEV1, volume in one second; PEF, peak expiratory flow; MEF, maximal expiratory flow. exposed patients were also reported [bib_ref] Immunological consequence of sulfur mustard exposure, Hassan [/bib_ref]. In a clinical study, 36 SM exposed patients with SM bronchiolitis were divided into case and control groups. The case group was treated with a combination of 200 mg IFN-γ and 7.5 mg prednisolone for 6 months. Pulmonary function tests (PFT) including FEV 1 and forced vital capacity (FVC) were not significantly different at the baseline between the two groups. However, FEV 1 and FVC were significantly increased in the case group during the subsequent months [bib_ref] Effect of gamma interferon on lung function of mustard gas exposed patients,..., Ghanei [/bib_ref]. In another similar study, the effect of interferon IFN-γ on respiratory symptoms, quality-of-life, and oxidative parameters in SM exposed patients was investigated. IFN-γ (100 µg) was administered every other day for 6 months. Severity and frequency of respiratory symptoms, quality-of-life, serum levels of different cytokines, and oxidative stress parameters were assessed at the baseline and at the end of the study. The results showed that IFN-γ therapy not only elevated FEV 1 , but also reduced the severity of cough, dyspnea, and frequency of sputum occurrence. IFN-γ therapy also is associated with improvements in quality-of-life. Serum levels of IL-4, IL-6, IL-10, calcitonin gene-related peptide (CGRP), MMP-9, tumor necrosis factoralpha (TNFα), TGFβ, and malondialdehyde (MDA) as oxidative stress marker were also decreased while the level of IL-2, IFN-γ, and glutathione (GSH) were increased at the end of study [bib_ref] Effect of recombinant human IFNγ in the treatment of chronic pulmonary complications..., Panahi [/bib_ref]. The above studies suggest the therapeutic potential of IFN-γ in SM-induced lung injury by reduction of inflammatory mediators and oxidative stress as well as respiratory symptoms but increased PFT values in SM exposed patients with lung disorders. ## Theophylline Theophylline in high-dose has bronchodilatory effects and in low concentration may have immunomodulatory and anti-inflammatory properties [bib_ref] Immunomodulation by theophylline in asthma. Demonstration by withdrawal of therapy, Kidney [/bib_ref]. Theophylline suppressed TGF-β-induced type I collagen mRNA expression in lung fibroblasts and also inhibited fetal bovine serum (FBS)-stimulated fibroblast proliferation and TGF-β-induced α-smooth muscle actin protein [bib_ref] Antifibrotic effects of theophylline on lung fibroblasts, Yano [/bib_ref]. ## Experimental studies The administration of theophylline (10 or 20 µg/ml) 2 h before human TGF-β1 stimulation suppressed TGF-β-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation [bib_ref] Antifibrotic effects of theophylline on lung fibroblasts, Yano [/bib_ref]. It was reported that theophylline (1-50 mg/kg-1, i.p.) significantly inhibited the inflammation at the early (4 h) and late (48 h) phases of inflammatory reaction induced by carrageenin (1%) in a murine model of pleurisy. Theophylline reduced airway inflammation and pathologic changes of lung tissue induced by cigarette smoke in a COPD model of rats [bib_ref] Low-dose theophylline restores corticosteroid responsiveness in rats with smoke-induced airway inflammation, Sun [/bib_ref]. Theophylline (3 and 30 mg/kg) reduced the influx of inflammatory cells into the BALF of Guinea pigs exposed to LPS (30 µg/mL). Furthermore, theophylline improved histological changes induced by LPS, including accumulation of inflammatory cells in the lung parenchyma, swelling of the alveolar walls, and goblet cell hyperplasia in the airways [bib_ref] Effects of theophylline on chronic inflammatory lung injury induced by LPS exposure..., Kaneko [/bib_ref]. ## Clinical studies Treatment of children with moderate to severe cystic fibrosis (CF) by theophylline for 10 days showed protective effects on arterial oxygen desaturation during sleep, increased wakefulness and decreased sleep efficiency [bib_ref] Effect of theophylline on lung function tests, sleep quality, and nighttime 8a02..., Avital [/bib_ref]. Histone deacetylase complex (HDAC) is the main enzyme responsible for regulating inflammatory gene expression activity induced by oxidative stress. Theophylline can restore reduction of this enzyme. Moreover, theophylline may be able to reverse steroid resistance in COPD and other inflammatory lung diseases [bib_ref] Histone deacetylation: an important mechanism in inflammatory lung diseases, Adcock [/bib_ref]. Treatment of asthmatic smokers with theophylline (400 mg) in combination with inhaled beclometasone (200 mg) per day after four weeks significantly improved peak expiratory flow (PEF) and FVC but a borderline increase in pre-bronchodilator FEV 1 [bib_ref] Effect of theophylline plus beclometasone on lung function in smokers with asthmaa..., Spears [/bib_ref]. In a clinical study, SM-exposed patients were treated with oral slow releasing (SR) theophylline (250 mg), omeprazole (20mg), NAC (600 mg), salmetrol, and fluxitide (2 puffs) twice a day, during 8 weeks of therapy. The results showed that the low dose of theophylline and other mentioned drugs was partially able to decelerate the reductions in PFT values of these patients [bib_ref] Study on effectiveness of low dose theophylline as add-on to inhaled corticosteroid..., Panahi [/bib_ref]. The decline of the reductions in PFT values in SM-exposed patients and its effect on CF and improvement of PFT values in asthmatic smokers by theophylline suggest its therapeutic effect on SM-induced lung disorders. ## Protease inhibitor agents Doxycycline Doxycycline (DOX) is an antibiotic used to treat bacterial infections, pneumonia, and other respiratory tract infections that has been reported to exhibit non-specific matrix metalloproteinase (MMPs) inhibitory activity [bib_ref] Doxycycline inhibition of aneurysmal degeneration in an elastase-induced rat model of abdominal..., Petrinec [/bib_ref]. The important role of MMPs in the toxicity of SM in different tissues including, lungs, skin, and eyes were indicated (57). ## Experimental studies It was reported that doxycycline (2 mg/kg, p.o.) reduced lung pathology (neutrophil migration, septal thickening, and alveolar destruction), intra-alveolar inflammatory cells (mainly neutrophils), bacterial number, and MMP-2 and MMP-9 lipopolysaccharide-induced lung injury in C57Bl/6 mice (58). The effects of doxycycline (20 mg/kg or 60 mg/kg, orally), on virulent influenza A/Aichi/2/68 (H3N2) virus-induced lung injury in mice showed a significant decrease in inflammation and protein leakage in the lungs. Treatment with doxycycline also reduced the levels of MMP-2 and MMP-9 activity, T1-α (type I alveolar epithelium protein), and thrombomodulin (endothelial protein) compared to the non-treated group. These results demonstrated that doxycycline treatment is able to reduce lung damage but it did not affect virus titers and body weights [bib_ref] Doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza..., Ng [/bib_ref]. Administration of doxycycline (2 mg/kg) in the drinking water in mice challenged with intratracheal LPS, significantly decreased the number of neutrophils and shed syndecan-1 in BALF. In addition, doxycycline had no significant effect on total BALF protein and the whole lung caspase-3 activity (60). The protective effect of doxycycline on acute lung injury induced by cardiopulmonary bypass (CPB) in 30 healthy mongrel dogs showed that administration of doxycycline (30 mg/kg) in feeding food significantly decreased WBC count, alveolar-arterial oxygen difference, respiratory index, total protein, and myeloperoxidase (MPO) activity in the BALF compared to the control group. Also, doxycycline (60 mg/kg) significantly decreased the concentration of MMP-9 compared to the control group [bib_ref] Inhibition of matrix metalloproteinase-9 with low-dose doxycycline reduces acute lung injury induced..., Zhang [/bib_ref]. Administration of doxycycline (2 mg/kg) in drinking water prior to intratracheally administration of bleomycin which induced pulmonary fibrosis, significantly decreased the number of neutrophils, although the total number of cells in the BALF remained unchanged. Dxycycline also attenuated gelatinase activities and reduced production of gelatinase B in BALF [bib_ref] Doxycycline attenuated pulmonary fibrosis induced by bleomycin in mice, Fujita [/bib_ref]. Pretreatment of SM exposed guinea pigs with doxycycline resulted in the reduction of gelatinases activity (MMP-2 and MMP-9), decreased lung inflammation (cellularity and protein levels in BAL), and decreased lung pathological changes (epithelial lesions) [bib_ref] Effect of doxycycline on sulfur mustard-induced respiratory lesions in guinea pigs, Guignabert [/bib_ref]. These results indicated that doxycycline has potent therapeutic effects on LPS and bleomycin-induced pulmonary fibrosis and inflammation in animal models with a similar action to SM-induced lung injury. ## Clinical studies In a clinical study, pharyngeal Chlamydia trachomatis patients were treated with azithromycin (n = 78) and doxycycline (n = 64). Treatment failure in patients treated with azithromycin and doxycycline was 8.78 (10%) and 1.64 (2%), respectively. Treatment with doxycycline (100 mg) twice a day for 7 days was associated with less treatment failure of oropharyngeal chlamydia compared with azithromycin (1 g), [bib_ref] Comparison of doxycycline with azithromycin in treatment of pharyngeal chlamydia infection, Manavi [/bib_ref]. The effect of doxycycline on cystic fibrosis exacerbation in a randomized, double-blind, placebo-controlled study was studied and bio-specimens were collected at the start and the end of the study. Treatment with doxycycline (100 mg) orally twice daily in participants (n = 20) was given over an 8-day period during hospitalization, significantly reduced the levels of MMP-9 in sputum, which was also associated with the reduction in active MMP-9 levels (56.5%), and increased TIMP-1 in sputum. Doxycycline also improved forced expiratory volume in the first second (FEV1) and increased the time to next exacerbation compared to the placebo participants (n = 19). Furthermore, treatment with doxycycline reduced total hospital days compared with the placebo-treated group [bib_ref] Doxycycline improves clinical outcomes during cystic fibrosis exacerbations, Xu [/bib_ref]. Using nuclear magnetic resonance (NMR)-based metabolomics to obtain serum metabolic profiles of doxycyclinetreated (n = 60) and standard therapy of COPD patients (n = 40), doxycycline (100 mg) significantly increased the values of FEV1/forced vital capacity (FVC) and improved the COPD assessment test (CAT) scores after 3 months' treatment. In addition, doxycycline significantly down-regulated serum levels of lactate and fatty acid, while, up-regulated the levels of formate, citrate, imidazole, and L-arginine compared to the pre-treatment level. The post doxycycline treatment significantly reduced serum level of the folate compared to COPD patients [bib_ref] Metabolomic profiling of doxycycline treatment in chronic obstructive pulmonary disease, Singh [/bib_ref]. The results of experimental and clinical studies indicated that doxycycline could attenuate lung injury and pulmonary edema through anti-inflammatory effects such as degradation of the cell membrane, pulmonary neutrophil infiltration, and PFT test. These results suggest its therapeutic value in COPD and lung injury induced by SM. ## Tissue plasminogen activator Tissue plasminogen activator (tPA) is a potent fibrinolytic agent that currently used as first-line therapy in clot-associated diseases, such as stroke (67). ## Experimental studies Sulfur mustard analog exposure led to the formation of fibrinrich in the airways which caused airway obstruction [bib_ref] Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation, Veress [/bib_ref]. The intra-tracheal administration of tPA (0.15-0.7 mg/kg, 5.5 and 6.5 h) in adult rats exposed to SM analog completely eliminated mortality at 48 h (0%), and improved morbidity (90-100%). Treatment with tPA also normalized plastic bronchitis and hypercarbia. It also improved respiratory distress, pulmonary gas exchange, and oxygen utilization while reduced airway fibrin casts [bib_ref] Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction, Veress [/bib_ref]. Intra-tracheal tPA treatment reduced mortality at 48 h (0%) and significantly improved lung injury after lethal SM inhalation (100% death in controls). In addition, tPA improved respiratory distress and normalized hypoxemia, hypercarbia, and lactic acidosis induced by SM. Moreover, tPA was given via airway 6h after SM exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects [bib_ref] Airway tissue plasminogen activator prevents acute mortality due to lethal sulfur mustard..., Veress [/bib_ref]. ## Clinical studies The effect of intravenous recombinant human tPA (rt-PA) was compared to urokinase in 45 patients with pulmonary embolism (PE). The results showed improvement in lung scan reperfusion in the two treatment groups after 24 h. The reduction in fibrinogen did not differ significantly between the two treated groups. These results also indicated that rt-PA acts more rapidly and is safer than urokinase in the treatment of acute PE [bib_ref] Randomised controlled trial of recombinant tissue plasminogen activator versus urokinase in the..., Goldhaber [/bib_ref]. Therefore, tPA reduced mortality and respiratory distress as well as normalized hypoxemia and hypercarbia induced by lethal SM inhalation which suggest the therapeutic effect of tPA on SMinduced lung disorders. ## Tissue factor pathway inhibitor Tissue factor pathway inhibitor (TFPI) is a 276 amino acid glycoprotein with three distinct structural domains and an acidic N terminus which is an important physiologic inhibitor of the extrinsic pathway of the coagulation system [bib_ref] Tissue factor pathway inhibitor: structure, biology and involvement in disease, Lwaleed [/bib_ref]. It was reported that TFPI-2 inhibits tumor invasion and angiogenesis in vitro and in vivo, and also suggested a potentially important therapeutic role for recombinant TFPI-2 in the treatment of malignant esophageal carcinomas [bib_ref] A novel role for tissue factor pathway inhibitor-2 in the therapy of..., Ran [/bib_ref]. ## Experimental studies Pre and post-exposure treatment of rats with recombinant TFPI (rTFPI) significantly inhibited LPS-induced pulmonary vascular injury and coagulation abnormalities. Treatment with rTFPI also significantly inhibited increases of TNF-α, cytokine-induced neutrophil chemo-attractant, and myeloperoxidase in lung tissue. The administration of rTFPI significantly reduced the expression of TNF-α messenger RNA (mRNA) and inhibited TNF-α production in the lungs after stimulation by LPS. The results of this study suggested the effect of rTFPI on pulmonary vascular injury by inhibiting leukocyte activation in LPS-administered rats [bib_ref] Recombinant tissue factor pathway inhibitor reduces lipopolysaccharideinduced pulmonary vascular injury by inhibiting..., Enkhbaatar [/bib_ref]. Intravenous injection of rTFPI immediately before the introduction of tumor cells also reduced metastasis by 83% in mice. Intra-tracheal administration of TFPI decreased fibrincontaining formation and limited severe hypoxemia in SM analog-induced lung injury in animals. TFPI limited thrombin activation in airways by reduction of prothrombin consumption and decreased thrombin-antithrombin complex (TAT) in BALF which led to a reduced airway obstruction and an improved gas exchange in rats [bib_ref] Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due..., Rancourt [/bib_ref]. Therefore, the therapeutic effect of TFPI on LPS and SM analog-induced lung injury was documented. ## Antioxidant agents ## N-acetyl cysteine N-acetyl cysteine (NAC) is a thiol compound and categorized as a mucolytic drug-containing sulfhydryl groups. It is a reactive oxygen species (ROS) scavenger and also reduces GSH and therefore can regulate the oxidation status in cells. In addition, it interferes with several signaling pathways including regulating apoptosis, cell growth and arrest, angiogenesis, redox-regulated gene expression, and inflammatory response [bib_ref] The use of N-acetylcysteine in intensive care, Atkinson [/bib_ref]. ## Experimental studies The involvement of IL-8 in the pathogenesis of Bleomycin (BLM)-induced lung injury has been suggested and its level was elevated in a bronchial epithelial cell line (BEAS-2B cells). However, BLM-induced expression of IL-8 protein and mRNA in BEAS-2B cells was inhibited by NAC [bib_ref] N-acetyl-L-cysteine inhibits bleomycin-induced interleukin-8 secretion by bronchial epithelial cells, Gon [/bib_ref]. Pre-incubation with NAC (5 × 10 −5 M, 24 h) also significantly reduced peroxynitrite (ONOO-) and O 2− production in lung macrophages obtained from systemic sclerosis (SSc) patients [bib_ref] Effect of N-acetyl-L-cysteine on peroxynitrite and superoxide anion production of lung alveolar..., Failli [/bib_ref]. Administration of NAC in lead-exposed animals, reduced or reversed lead-induced oxidative stress. Lead-exposed rats resulted in signs of anemia such as anisocytosis, poikilocytosis, and alterations in hemoglobin and hematocrit. It also causes an alteration in lipid peroxidation such as, increased MDA content and a decrease in GSH which are reversed by NAC [bib_ref] Antioxidant effects of N-acetylcysteine and succimer in red blood cells from lead-exposed..., Gürer [/bib_ref]. It has been reported that pretreated animals with NAC or liposomally-entrapped N-acetylcysteine (L-NAC) (25 mg/kg, iv), and challenged with lipopolysaccharide (LPS) (Escherichia coli, LPS 0111:B4) prevents increased lung weights, decreased lung angiotensin-converting enzyme (ACE) (the injury marker for pulmonary endothelial cells), and significantly reduced the LPS-induced increase in plasma TNF-α levels. In addition, NAC significantly decreased myeloperoxidase (MPO) and chloramine concentrations in the lungs and the extent of lipid peroxidation in liver tissues [bib_ref] Effectiveness of liposomal-N-acetylcysteine against LPS-induced lung injuries in rodents, Mitsopoulos [/bib_ref]. The enhancement of endothelial survival via GSH against SM injury of the endothelium was also reported. In this study, exposure to 500 µM SM resulted in increased activation of the nuclear transcription factor (NF kB ) binding to its consensus sequence 5 h after exposure. Pretreatment with NAC suppressed SM-induced NF kB activation which is an important transcription factor for a number of cytokine genes (e.g., TNF) and is activated following stress in endothelial cells [bib_ref] N-acetylcysteine and endothelial cell injury by sulfur mustard, Atkins [/bib_ref]. GSH-induced enhancement of cell viability at 2.5 and 5 µM was shown after SM exposure. Pretreatment with hioninesulfoximine (BSO) an inhibitor of GSH synthesis, alone did not show toxicity effect but potentiated the toxicity of SM [bib_ref] N-acetylcysteine and endothelial cell injury by sulfur mustard, Atkins [/bib_ref]. Similarly, SM exposure led to a dose and time-dependent decrease in GSH content in human skin fibroblast cell line (HF2FF). NAC increased intracellular GSH level and protected the cells against SM-induced reactive oxygen species formation and lactate dehydrogenase leakage. In contrast, BSO pretreatment reduced cellular GSH and enhanced the cytotoxic effects of SM on HF2FF cells [bib_ref] Critical role of GSH in sulfur mustard-induced oxidative stress and cytotoxicity in..., Mahmoudabad [/bib_ref]. Treatment with intra-tracheal administration of NAC on pulmonary edema formation induced by phosgene exposure in rabbit lungs lowered pulmonary artery pressure, lung weight gain, the concentration of peptide leukotrienes LTC 4 , D [bib_ref] Toxicodynamics of sulfur mustard, Somani [/bib_ref] , and E 4 , and lipid peroxidation which were increased following exposure to phosgene [bib_ref] Protective effects of N-acetylcysteine treatment after phosgene exposure in rabbits, Sciuto [/bib_ref]. Animal exposure to SM leads to inflammatory cell accumulation in the airways and lung as well as structural and functional alterations in the respiratory tract [bib_ref] Inflammatory effects of inhaled sulfur mustard in rat lung, Malaviya [/bib_ref] [bib_ref] Acute and chronic respiratory effects of sulfur mustard intoxication in guinea pig, Calvet [/bib_ref]. The role of oxygen species and free radicals in the pathophysiology of inflammation-induced pulmonary lesions was described previously [bib_ref] Anti-inflammatory effects of macrolide antibiotics, Čulić [/bib_ref]. However, treatment with NAC reduced the number of neutrophils in mice that were exposed to SM and developed lung injuries [bib_ref] Treatment of sulfur mustard (HD)-induced lung injury †, Anderson [/bib_ref]. Following animal exposure to SM vapor (100 µg/kg) for 10 min, arterial blood oxygen saturation levels (SaO 2 ), arterial blood pH and bicarbonate (HCO − 3 ) were significantly decreased. Also, arterial blood carbon dioxide (PaCO 2 ) and shunt fraction were significantly increased. After treatment with inhaled doses of NAC (1 ml of 200 mg) arterial blood oxygen saturation, HCO − 3 level, and shunt fraction were significantly improved compared to those of the SM exposure. In addition, infiltration of neutrophils and concentrations of protein in BALF were significantly decreased compared to the SM group [bib_ref] N-acetyl-Lcysteine protects against inhaled sulfur mustard poisoning in the large swine, Jugg [/bib_ref]. ## Clinical studies In clinical studies, treatment with NAC in patients with COPD, asthma, and acute bronchitis improved respiratory symptom, lung function, and quality of life. Treatment with NAC also scavenged reactive oxygen species and inhibited mediator release [bib_ref] Mucolytics in acute and chronic respiratory tract disorders. I. Pathophysiology and mechanisms..., Kupczyk [/bib_ref]. It was reported that the administration of NAC, 600 mg daily for 12 months in patients with COPD, reduced oxidative stress [bib_ref] A controlled trial of intermittent oral acetylcysteine in the long-term treatment of..., Grassi [/bib_ref] and also the oxidative burst of poly morphonuclear (PMN) cells and showed protective effects on peripheral granulocytes in COPD patients [bib_ref] Human neutrophil oxidative bursts and their in vitro modulation by different N-acetylcysteine..., Allegra [/bib_ref]. Intravenous administration of NAC (190 mg/kg/day) in adult patients (n = 42) with respiratory distress syndrome (Pao2/Fio2 ≤ 200 mm Hg) significantly decreased the lung injury score between days 1 and 3 compared to the placebo group [bib_ref] Treatment with N-acetylcysteine during acute respiratory distress syndrome: a randomized, double-blind, placebo-controlled..., Domenighetti [/bib_ref]. Treatment with NAC (40 mg/kg/day) intravenously for 3 days in 61 adult patients with mild-to-moderate acute lung injury (NAC, n = 32 and the placebo groups, n = 29 patients) improved ventilation compared to the placebo group. The oxygenation index (PaO 2 /FIo 2 ) significantly was improved from day 0 to day 3 only in the NAC treated group and the lung injury score was improved in the NAC treated group during the first 10 days of treatment but, no change was observed in the placebo group [bib_ref] N-acetylcysteine enhances recovery from acute lung injury in man: a randomized, double-blind,..., Suter [/bib_ref]. Treatment of SM-induced bronchiolitis obliterans patients (n = 144) with NAC (1,200 mg daily), improved dyspnea, wakeup dyspnea, and cough after 4 months of treatment compared to the control group. Additionally, NAC reduced sputum from (76.9%) of cases before the study to (9.6%) of cases at the end of trial. The values of FEV1, FVC, and FEV1/FVC were also significantly improved in NAC treated patients compared to the placebo group [bib_ref] N-acetylcysteine improves the clinical conditions of mustard gasexposed patients with normal pulmonary..., Ghanei [/bib_ref]. In another clinical study, treatment with NAC (1,800 mg daily) in 144 bronchiolitis obliterans patients 18 years after SM exposure improved clinical signs and symptoms of patients including; dyspnea, cough, sputum, wake-up dyspnea, and also increased PFT values [bib_ref] Therapeutics effect of N-acetyl cysteine on mustard gas exposed patients: evaluating clinical..., Shohrati [/bib_ref]. It has been reported that administration of NAC (oral, IV, IP or IT) is effective in the management of SM induced acute lung injury due to inhibition of oxidative stress, inflammatory responses and apoptosis in a time-dependent manner. Furthermore, oral NAC alone at high doses for 4 months or in combination with clarithromycin (500 mg/day) at a dose 600 mg/day for 6 months improved clinical and paraclinical pulmonary parameters of patients with bronchiolitis obliterans induced by SM exposure [bib_ref] The role of N-acetylcysteine in the management of acute and chronic pulmonary..., Shohrati [/bib_ref]. The efficacy of NAC in reducing SM toxicity in different studies (in vitro and in vivo) model of lung injury as well as the safe and efficacy of NAC in treating patients suffering the long-term and chronic pulmonary effects of SM exposure in veterans was reported (100). Both experimental and clinical studies suggest the possible therapeutic effect of NAC on SM-induced lung disorders and other respiratory diseases via possible anti-inflammatory and anti-oxidant mechanisms. ## Vitamin e experimental studies The effects of vitamin C, vitamin E, and rINN, alone or in combination in placental villi culture after exposure to nicotine-induced endothelial dysfunction reduced placental cell proliferation until cell death [bib_ref] Potential therapeutic effects of vitamin E and C on placental oxidative stress..., Gallo [/bib_ref]. Vitamin E reduced phospholipidosis in cultured human skin fibroblasts chronically exposed to amiodarone and desethylamiodarone (DEA) and inhibited cumulative uptake of the drugs in a dose-dependent manner [bib_ref] Vitamin E reduces accumulation of amiodarone and desethylamiodarone and inhibits phospholipidosis in..., Honegger [/bib_ref]. Vitamin E pretreatment on human fibroblast cells significantly protected against ciprofloxacin (CPFX)-induced cytotoxicity. It also significantly increased the total GSH content and reduced the level of lipid peroxidation [bib_ref] Cytotoxicity in ciprofloxacin-treated human fibroblast cells and protection by vitamin E, Gürbay [/bib_ref]. The effects of liposome-encapsuled vitamin E on a mouse model for airway inflammation induced by inhalation exposure to the alkylating nitrogen mustard melphalan showed that vitamin E (50 mg/kg) reduced inflammatory cell influx, and inhibited collagen formation in lung tissue [bib_ref] Treatment with dexamethasone or liposome-encapsuled vitamin E provides beneficial effects after chemical-induced..., Wigenstam [/bib_ref]. The administration of vitamin E (600 mg/kg), dexamethasone (5 mg/kg) or their combination in guinea pigs exposed to SM caused improvement in the pathological changes in the livers and kidneys [bib_ref] The effect of vitamin E on pathological changes in kidney and liver..., Boskabady [/bib_ref]. The effect of vitamin E on tracheal responsiveness (TR) and lung inflammation in SM exposed animals also showed that TR to methacholine, total and differential WBC count in lung lavage, and serum levels of IL-4 were significantly decreased in treated animals with vitamin E compared to the untreated SM-exposed group [bib_ref] The effect of vitamin E on tracheal responsiveness and lung inflammation in..., Boskabady [/bib_ref]. In addition, vitamin E treatment reduced pathological changes in guinea pigs exposed to SM. The antioxidant effect of tocopherol acetate (vitamin E) was also reported (106, 107) which supports the above results. Tocopherol acetate (200 mg/kg, i.p.) increased GSH levels and decreased MDA level in systemic toxicity due to percutaneous administration of SM in animals. Furthermore, total antioxidant status was increased but red blood corpuscles and hemoglobin content were significantly decreased in the tocopherol acetate treated group [bib_ref] Comparative evaluation of some flavonoids and tocopherol acetate against the systemic toxicity..., Vijayaraghavan [/bib_ref]. Liposomes containing tocopherol (α, γ, δ) showed protective effects on the accumulation of RBC in the bronchi, alveolar space, arterioles and veins, and fibrin and collagen deposition in the alveolar space on 2-chloroethyl ethyl sulfide (CEES) (a monofunctional analog of sulfur mustard)-induced lung injury in a guinea pig. Moreover, tocopherol decreased lipid peroxidation level and hydroxyproline in the lungs [bib_ref] Protection of half sulfur mustard gas-induced lung injury in guinea pigs by..., Mukherjee [/bib_ref]. The treatment with liposome of tocopherol (α, γ, δ) significantly blocked the CEES-induced increase in the protein levels of the cell cycle protein (cyclin D1), and a cell differentiation marker (PCNA). Additionally, tocopherol protected the lungs against CEESinduced inflammation and alveolar infiltration of neutrophils and eosinophils [bib_ref] Role of MAPK/AP-1 signaling pathway in the protection of CEES-induced lung injury..., Mukhopadhyay [/bib_ref]. The effect of vitamin E on CEES and alkylating nitrogen mustard-induced lung inflammation, as well as its effect on lung oxidative stress, lung inflammation, lung pathology, and tracheal responsiveness due to similar mechanisms in SMexposed subjects indicated its therapeutic value in SM and other chemical-induced lung injuries. ## Clinical studies Dietary intakes of vitamin E in 178 men and women aged indicated that intake of vitamin E may influence lung function by increasing FVC, FEV1 and showed protective effects on wheeze in elderly in three European countries including Finland (n = 1,248), Italy (n = 1,386), and the Netherlands [bib_ref] Does dietary intake of vitamins C and E influence lung function in..., Dow [/bib_ref] [bib_ref] Dietary factors and pulmonary function: a cross sectional study in middle aged..., Tabak [/bib_ref]. Treatment of 33 mild atopic asthmatics subjects for 16 weeks, with high doses of -α-tocopheryl acetate significantly decreased F2-isoprostanes, and reduced allergenprovoked concentrations of IL-3 and IL-4, and increased levels of IL-12 in BALF. In addition, natural source of vitamin E improved airway responsiveness to methacholine [bib_ref] Natural-source D-αtocopheryl acetate inhibits oxidant stress and modulates atopic asthma in humans..., Hoskins [/bib_ref]. In a placebo-controlled randomized clinical trial the effects of natural vitamin E (500 mg) or matched placebo in 72 participants aged (18-60 years) for 6 weeks, had no significant impact on FEV1, asthma symptom scores, or serum immunoglobulin levels [bib_ref] Vitamin E supplements in asthma: a parallel group randomised placebo controlled trial, Pearson [/bib_ref]. Investigation of the influence of maternal antioxidant intake in pregnancy on the development of asthma and eczema in children showed that maternal vitamin E intake during pregnancy is negatively associated with wheeze in the absence of "cold" and childhood eczema in the children's with 24 months of age [bib_ref] Antioxidant intake in pregnancy in relation to wheeze and eczema in the..., Martindale [/bib_ref]. The therapeutic effects of pharmaceutical drugs on SMinduced lung disorders in experimental and clinical studies were summarized in Tables 1, 2, respectively. The possible mechanisms of the therapeutic effects of pharmaceutical drugs on SM-induced lung disorders were also summarized in [fig_ref] FIGURE 2 |: Possible mechanisms of lung injury induced by SM exposure and the effect... [/fig_ref]. # Conclusion Sulfur mustard, CEES, and nitrogen mustard exposure due to similar actions induces various acute and chronic diseases such as lung disorders. Unfortunately, there is still no definitive medication for SM-induced diseases in clinics. However, several studies suggested the therapeutic effects of individual or a combination of pharmaceutical drug products on SM and its analogs-induced lung disorders in animals as well as in some clinical studies. In this review, published papers regarding the new approach for the treatment of SM-induced lung disorders by pharmaceutical drugs were discussed. The results of the reviewed papers showed that pharmaceutical drugs improved inflammatory cells, inflammatory mediators, oxidative stress biomarkers, and lung pathological changes in lung injury induced by various agents including SM exposure. The pharmaceutical agents also showed a reduction of clinical symptoms, improvement of PFT values, and health-related quality of life as well as systemic and lung inflammation and oxidative stress in patients suffering from various chronic lung diseases including SM-induced pulmonary complications. Therefore the present review present various potential treatment strategy for preventing and treatment of lung injury patients who are exposed to SM in mean of inflammation and oxidative stress. However, more studies especially clinical trials are needed to examine the exact mechanisms of action of pharmaceutical drugs and their long-term outcome as well as exploring new drugs for the treatment of the serious condition of lung disorders induced by SM exposure. # Author contributions MRK searched the literature and wrote the first draft of the manuscript. MHB designed the study and critically edited and revised the manuscript. Both authors contributed to the article and approved the submitted version. [fig] FIGURE 2 |: Possible mechanisms of lung injury induced by SM exposure and the effect of pharmaceutical drugs in these changes. T1-α, type I alveolar epithelium protein; MMPs, matrix metalloproteinase; BALF, bronchoalveolar lavage fluid; TGF-β1, transforming growth factor-beta1; IL, interleukin; IFNγ, interferon gamma; TNFα, tumor necrosis factor-α; NO, nitric oxide; MDA, malondialdehyde; FVC, forced volume capacity; FEV1, volume in one second. [/fig] [table] TABLE 1 |: Therapeutic effects of pharmaceutical drugs on SM-induced lung injuries, experimental evidence. [/table] [table] TABLE 2 |: Therapeutic effects of pharmaceutical drugs on SM-induced lung injuries, clinical studies. [/table]

Delay of transfer from the intensive care unit: a prospective observational study of incidence, causes, and financial impact Introduction: A paucity of literature exists regarding delays in transfer out of the intensive care unit. We sought to analyze the incidence, causes, and costs of delayed transfer from a surgical intensive care unit (SICU). of all 731 patients transferred from a 20-bed SICU at a large tertiary-care academic medical center. Data were collected on patients who were medically ready for transfer to the floor who remained in the SICU for at least 1 extra day. Reasons for delay were examined, and extra costs associated were estimated. Results: Transfer to the floor was delayed in 22% (n = 160) of the 731 patients transferred from the SICU. Delays ranged from 1 to 6 days (mean, 1.5 days; median, 2 days). The extra costs associated with delays were estimated to be $581,790 during the study period, or $21,547 per week. The most common reasons for delay in transfer were lack of available surgical-floor bed (71% (114 of 160)), lack of room appropriate for infectious contact precautions (18% (28 of 160)), change of primary service (Surgery to Medicine) (7% (11 of 160)), and lack of available patient attendant ("sitter" for mildly delirious patients) (3% (five of 160)). A positive association was found between the daily hospital census and the daily number of SICU beds occupied by patients delayed in transfer (Spearman rho = 0.27; P < 0.0001). Conclusions: Delay in transfer from the SICU is common and costly. The most common reason for delay is insufficient availability of surgical-floor beds. Delay in transfer is associated with high hospital census. Further study of this problem is necessary. # Introduction In recent years, increasing attention has been paid to the sizable and growing costs of critical care services. The percentage of the United States' gross domestic product used for critical care services increased from the year 2000 to 2005 by 13.7%, from 0.58% to 0.66% [bib_ref] Critical care medicine in the United States 2000-2005: an analysis of bed..., Halpern [/bib_ref]. Optimal use of intensive care unit (ICU) resources is an important goal for individual hospitals and healthcare systems and is an essential component of the effort to contain healthcare expenditures. At our institution, we found that many patients who met criteria for transfer out of the ICU remained in the ICU for a longer time than necessary. We investigated the incidence of delayed transfer of surgical ICU patients and sought to determine the causes for delays and to estimate the costs associated with them. We hypothesized that transfers out of our ICU are delayed by 1 or more days at least 20% of the time, and that the majority of delays are due to a lack of available floor beds. # Materials and methods ## Setting After approval by the hospital's Institutional Review Board, the study was conducted in the Surgical Intensive Care Unit (SICU) of the Massachusetts General Hospital from January 24, 2010, to July 31, 2010. Informed consent was not necessary because of the observational nature of the study. Patient care was not affected by the study, and individual patient information was not used during the analysis of data. MGH is a 900-bed university-affiliated tertiary care center with 1.5 million outpatient visits and 47,000 inpatient admissions annually. The hospital is a level 1 trauma center. The SICU is a 20-bed unit with >1,400 annual admissions, primarily from the complex trauma, vascular, thoracic, and general surgical populations. It is staffed 24 hours per day by attending intensivists in addition to critical care fellows and residents. ## Transfer process A patient was classified as ready for transfer to the floor by consensus among the patient's intensivist, surgeon, and SICU nurse, based on guidelines for discharge criteria, with emphasis on hemodynamic and respiratory stability. After a patient was deemed ready for transfer, a request to the admissions office was placed to locate an appropriate floor bed. Hospital-based nursing triage supervisors were aware of the bed availability throughout the hospital and supported the process. When a bed became available, the patient was transferred out of the SICU. ## Reasons for delay Before the study, we surveyed intensivists, surgeons, SICU nurses, nursing triage supervisors, and administrators to determine the perceived reasons for transfer delay. Based on these responses, we predefined the following four reasons: lack of available surgical-floor bed, lack of an appropriate bed for infectious contact precautions, change of primary service (Surgery to Medicine), and lack of available patient attendant ("sitter" for mildly delirious patients). ## Demographic and clinical factors All patients who were admitted to the SICU during the study period were included in the study, without regard for demographic or clinical factors. ## Data collection and definition of delay During the study period, a SICU intensivist (DWJ) contacted the SICU charge nurse each night to identify those patients who were delayed in transfer. Patients were classified as delayed in transfer when the intensivist, surgeon, and SICU nurse were in agreement that the patient could be transferred to the floor, yet the patient remained in the SICU past 00:01 that night. The intensivist and charge nurse discussed each case to determine which of the four reasons (or "other") best characterized the patient's reason for delay. Duration of ICU stay, duration of hospital stay, admitting surgical service, destination (type of surgical floor), date deemed ready for transfer, date/time of actual transfer, and daily hospital census were recorded throughout the study period. # Cost analysis We used the difference between the 2005 national average cost of a day in the ICU ($3,518) and a day in a non-ICU hospital bed ($1,153) to estimate the extra costs associated with patients remaining in the SICU unnecessarily [bib_ref] Critical care medicine in the United States 2000-2005: an analysis of bed..., Halpern [/bib_ref]. This difference ($2,365) was multiplied by the total number of delay days (246) to generate the estimated total costs of the delays. ## Statistics All data analysis was performed by using Stata 10 (Stata-Corp LP, College Station, TX, USA). Continuous variables with a normal distribution are expressed as mean and standard deviation (SD). Ordinal variables are expressed as median and range. The χ 2 test was used to compare absolute numbers and proportions. The Spearman rho was used to evaluate the association between the daily hospital census and the daily number of SICU beds occupied by patients delayed in transfer. # Results During the study period, 731 patients were transferred from the SICU to the floor. Of these patients, 160 (22%) experienced a delay in transfer of at least 1 day. The delays in transfer ranged from 1 to 6 days (mean, 1.5 days; median, 2 days; [fig_ref] Figure 1: Distribution of length of delay in intensive care unit [/fig_ref]. The most common reason for delay was lack of availability of a surgical-floor bed (71% (114 of 160)). The lack of an appropriate room for infectious contact precautions accounted for 18% (28 of 160) of delays. The remaining causes were change of primary service (Surgery to Medicine) at 7% (11 of 160), and lack of available patient attendant ("sitter" for mildly delirious patients) at 3% (five of 160). The cost associated with delays in transfer was estimated to be $581,790 for the entire study period, or $21,547 per week. The daily hospital census was positively correlated with the number of SICU beds occupied by delayed patients (Spearman rho = 0.27; P < 0.0001). Delayed patients were significantly more likely than nondelayed patients to be transferred during night shifts, between 19:00 to 06:59, (21% (33 of 160) versus 12% (67 of 571); χ 2 = 10.6; P < 0.005). # Discussion In this study, we found that delay in transfer from the SICU occurred in 22% of cases, with lack of availability of surgical-floor beds as the most common reason, accounting for 71% of delayed transfers. The ICU beds in our hospital constitute approximately 15% of the total number of beds. Although the absolute numbers and ratios of ICU beds-to-floor beds vary from institution to institution, our hospital's ratio of 15% ICU-to-total hospital beds is equal to the national average of 15% [bib_ref] Critical care medicine in the United States 2000-2005: an analysis of bed..., Halpern [/bib_ref]. Recent studies describe similar incidence and financial impacts [bib_ref] Optimizing patient flow out of the ICU: impact of "no-bed days" on..., Vandy [/bib_ref] [bib_ref] The "green light" program: improving patient throughput in a rapid turnover surgical..., Young [/bib_ref]. Our results are similar to those from a 2004 study in an Australian combined medical-surgical ICU that found that 27% of ICU transfers were delayed by at least 8 hours, and that 81% of these delays were due to lack of available floor beds [bib_ref] Delayed discharges from an adult intensive care unit, Williams [/bib_ref]. Whereas it is currently impossible to estimate the overall incidence of ICU-transfer delays in the United States and abroad, our study and these reports from other institutions suggest that the problem is widespread and contributes significantly to inefficiencies in healthcare systems. ICU transfer delays have a large financial impact. Time spent in the ICU costs more than time spent on a regular floor, so delays in transfer naturally increase the overall cost of care. The cost difference between ICU days and floor days is driven mostly by the difference in staff-to-patient ratios in the ICU versus on the floor, including higher ratios of nurses, physicians, and therapists to patients. The cost analysis in this study was performed by using data from the report of the United States critical care bed numbers, occupancy rates, payer mix, and costs published by Halpern and Pastores in 2010 [bib_ref] Critical care medicine in the United States 2000-2005: an analysis of bed..., Halpern [/bib_ref]. By using national average costs, the results of this study are more generally applicable to the healthcare community at large. A limitation of this method of cost analysis is that it assumes that ICU costs remain constant throughout the ICU stay. A study by Dasta and colleagues in 2005 [bib_ref] Daily cost of an intensive care unit day: the contribution of mechanical..., Dasta [/bib_ref] showed that costs are highest during the first 2 days of an ICU stay, and that higher costs are associated with mechanical ventilation. Patients who are stable and ready for discharge to the floor incur fewer costs than do critically ill patients. At our institution, the majority of daily ICU cost per patient is attributable to room and board, which includes nursing care. The daily cost of room and board in our ICU does not change after a patient is deemed ready for transfer to the floor, and the amount is not dramatically different from the estimated cost of an ICU day in the Halpern report; therefore, the cost-analysis model is valid for estimation of increased costs. We analyzed the actual costs for delayed versus nondelayed patients (from the time each patient was deemed ready for transfer out of the ICU). Although actual patient-charge information is not publishable per our hospital's policies, the total additional costs of delays in transfer were greater than what would be predicted by the cost data in the Halpern report. The increased costs associated with delayed transfer in our single-ICU study should prompt clinicians and administrators to examine closely ways to reduce or eliminate such delays. Preliminary results from a recent retrospective study showed similar increases in hospital costs associated with delayed transfer from the ICU [bib_ref] Optimizing patient flow out of the ICU: impact of "no-bed days" on..., Vandy [/bib_ref]. Although the methods used in that study were different from ours, both studies came to the same conclusion: delays in transfer out of the ICU constitute a significant and costly problem. A large number of delays were related to infectious contact precautions (28%). In accordance with Centers for Disease Control (CDC) guidelines, patients in our institution known to be infected or colonized with multidrugresistant organisms or Clostridium difficile are required either to have single rooms or to share a room with patients with the same organism. Providers caring for these patients are required to use contact precautions: gowns and gloves in addition to hand cleaning. A possible contribution to this element of the delay problem is that a majority of our hospital's non-ICU rooms are not private. Recently published studies appear to offer conflicting results regarding the efficacy of contact precautions; still, the recommendation to keep affected patients separated from unaffected patients is unlikely to change in the near future [bib_ref] Veterans Affairs Initiative to prevent methicillin-resistant Staphylococcus aureus infections, Jain [/bib_ref] [bib_ref] for the STAR*ICU Investigators: Intervention to reduce transmission of resistant bacteria in..., Huskins [/bib_ref]. Delays in transfer were associated with high hospital census. At times during the study period, our hospital's census was >95% occupancy, and these times correlated with an increased number of SICU beds occupied by delayed patients. It has been shown that efficiency of acute care units is impaired when hospital occupancy rates exceed 85% [bib_ref] Dynamics of bed use in accommodating emergency admissions: stochastic simulation model, Bagust [/bib_ref]. Excessive hospital occupancy can lead to a bottleneck effect in which completely occupied floor beds prohibit transfers out of the ICU and thus prohibit the admission of critically ill surgical patients into the ICU. Our hospital is a level-1 trauma center and referral center for complex surgical operations. Prevention of SICU admissions because of excessively occupied floor beds results in trauma victims and critically ill surgical patients being admitted to nonsurgical ICUs. Patients who were delayed in transfer were more likely than nondelayed patients to be transferred during night shifts. This is notable because of the previously published data that show that patients who are transferred at night have an increased likelihood of readmission to the ICU [bib_ref] The association between nighttime transfer from the intensive care unit and patient..., Hanane [/bib_ref] [bib_ref] Impact of intensive care unit discharge time on patient outcome, Priestap [/bib_ref] and that patients who are readmitted to the ICU have an increased risk of hospital mortality [bib_ref] Patients readmitted to the intensive care unit during the same hospitalization: clinical..., Chen [/bib_ref]. The reason for the observed association between ICU transfer delay and nighttime transfer is not clear and requires further study. Investigators recently reported a marked reduction in the incidence of delayed OR-to-SICU transfer after the implementation of measures aimed at facilitating early transfer of medically suitable patients out of the SICU [bib_ref] The "green light" program: improving patient throughput in a rapid turnover surgical..., Young [/bib_ref]. In this study, the authors noted delays in OR-to-ICU transfer related to impeded SICU throughput and demonstrated a reduction in such delays after interventions reduced time to transfer out of the SICU. Similar delays occur in our institution, and they place a tremendous burden on OR personnel, surgeons, anesthesiologists, and OR administrators. The successful interventions by Young etal. [bib_ref] The "green light" program: improving patient throughput in a rapid turnover surgical..., Young [/bib_ref] demonstrate that reduction in delayed transfer from the SICU can have a measurably positive impact on the hospital as a whole. Possible contributions to the problem include the occupation of floor beds by patients ready to be discharged from the hospital, inefficiency in discharging patients from the ICU to home, and the subspecialization of surgical-floor beds. When a surgical floor is at capacity and discharge-ready patients remain in the floor's beds, transfers to the floor from the SICU are potentially delayed. Interventions to reduce the number of floor beds occupied by discharge-ready patients would likely reduce SICU-transfer delays. Physicians and nurses in our ICU have relatively little experience in discharging patients home. Some of the patients who experienced long delays in transfer from the SICU to the floor were likely ready for discharge home. Efforts to improve recognition of discharge-ready patients and to educate staff members in the process of discharging home might be beneficial in reducing the unnecessary occupation of ICU beds. Patients in our hospital are transferred from the SICU to service-specific floors (for example, aortic surgery patients are transferred to a floor dedicated solely to vascular surgery). Only when clinically essential (for example, to create an available SICU bed for a patient with severe traumatic injuries) are patients transferred to floors other than their service-specific floor. Despecialization of surgical floors might reduce the incidence of delayed transfer from the SICU. The benefit of specialty surgical floors to patients, presumed by intensivists and surgeons at many large tertiary care centers, requires study for confirmation. The discussion regarding the United States' need to curb its ever-increasing healthcare expenditures must include consideration of the costs associated with provision of critical care. Multiple reports have projected a shortfall of critical care services in the years to come [bib_ref] for the Committee on Manpower for Pulmonary and Critical Care Societies (COMPACCS):..., Angus [/bib_ref]. Considering these projections and the budgetary constraints that the healthcare system faces, intensivists and hospital administrators must optimize the efficiency of each dollar spent in the ICU. In a system with too few ICU beds and inadequate financial resources, floor-ready patients occupying ICU beds represent a double-edged sword. Although planning for the projected increased need for ICU beds seems prudent, improved utilization of existing ICU resources is an essential component of the strategic planning needed to address the problem. Reduction or elimination of delays in transfer from the ICU have the potential to increase ICU-bed capacity effectively without the physical creation of new ICU beds. Previous publications have discussed the importance of ICU outflow in overall ICU resource utilization, yet few studies of this problem have been conducted [bib_ref] The process of intensive care triage, Levin [/bib_ref]. The paucity of literature addressing the problem in our study suggests that the issue has not been adequately quantified and analyzed. Further study of ICU-transfer delays, including studies of interventions to improve the problem, will likely improve patient care and resource utilization. This study has several limitations. The study was performed in a single surgical ICU of a major academic medical center. Similar academic centers have reported delays in SICU transfer, but it is unknown whether such delays occur commonly in nonacademic settings. We used days as the time variable. It might be beneficial to use hours in future studies, more precisely to quantify the problem. Our cost analysis was based on national average costs of ICU and floor days. Future studies could collect and analyze cost data of the hospital or hospitals where the study is being conducted and account for the reduction in actual costs that occurs as patients' care needs lessen over time. Hospital census was used as a proxy for surgical-floor census. Future studies could analyze the actual census of destination floors for delayed ICU patients. # Conclusions Delay in transfer from the SICU is common and is associated with increased cost of hospitalization. Because the most common reason for this delay was insufficient availability of surgical-floor beds, future efforts should include emphasis on increasing the availability of floor beds. ## Key messages - Delay in transfer from the SICU is common and costly. - The most common reason for delay is insufficient availability of surgical-floor beds. - Delay in transfer is associated with high hospital census. Abbreviations CDC: Centers for Disease Control; ICU: intensive care unit; IRB: institutional review board; OR: operating room; SICU: surgical intensive care unit. ## Competing interests The authors declare that they have no competing interests, financial or nonfinancial. Authors' contributions DWJ designed the study, engaged in daily communication with ICU charge nurses, collected and recorded all data, analyzed all data, and drafted the preliminary, revised, and final versions of the manuscript. US provided input on the design of the study, assisted in designing the data-collection instrument, and edited the manuscript extensively. EAB provided input on the design of the study, performed all statistical analysis, created [fig_ref] Figure 1: Distribution of length of delay in intensive care unit [/fig_ref] , and edited the manuscript. BC and RL provided expertise on the financial elements of the study, conducted analysis of cost differences between delayed and nondelayed patients, and edited the manuscript. RMP conceived of the study, managed the IRB application, obtained IRB approval, provided input on the design of the study, and edited the manuscript. All authors read and approved the manuscript before submission. [fig] Figure 1: Distribution of length of delay in intensive care unit (ICU) transfer (in days). [/fig]

Screening and identification of differentially expressed transcripts in circulating cells of prostate cancer patients using suppression subtractive hybridization Background: Tumor metastasis and changes in host immunosurveillance are important components in cancer development. Tumor cell invasion into the bloodstream is an essential step for systemic metastasis. Currently, the detection of tumor cells in the circulation is mainly dependent upon the utilization of known epithelial cell markers. However, expression of these molecules is not limited to cancer patients; healthy people also have a small number of epithelial cells in their circulation. Utilizing these markers to detect circulating tumor cells (CTCs) cannot adequately explain the mechanisms of tumor cell survival or their development of metastatic potential in peripheral blood. The immune system can also evolve along with the cancer, actually promoting or selecting the outgrowth of tumor variants. Unfortunately, both metastasis and immunosurveillance remain mysterious and are debatable because we have yet to define the molecules that participate in these processes. We are interested in identifying the existence of expressed genes, or mRNA species, that are specifically associated with circulating cells of cancerbearing patients using prostate cancer (PCa) as a model.Results:We established two comprehensive subtracted cDNA libraries using a molecular technique called suppression subtractive hybridization. This technique selectively amplifies transcripts that are specifically expressed in circulating cells of either PCa patients or healthy men. Following sequencing reaction, we showed that 17 out of 23 (73.9%) sequenced clones did not match any mRNAs in the GenBank database. This result suggests that genes associated with alterations in circulating cells of cancer-bearing patients are largely unknown. Semi-quantitative RT-PCR confirmed that two genes are up-regulated in circulating cells of PCa patients, whereas another two genes are down-regulated in the same patients.Conclusion:The comprehensive gene expression analysis is capable of identifying differentially expressed genes in circulating cells of healthy men and PCa patients. We did not attempt to enrich specific cell types in this study because phenotypes of CTCs and subsets of leukocytes participating in immunosurveillance remain largely unknown. Continuous studies of these differentially expressed genes will eventually lead us to understand the mechanisms involved in tumor metastasis and immune modulation during cancer development. # Background Metastasis is a sequential, multi-step process in which tumor cells detach from the primary tumor, migrate through the basement membrane and extracellular matrix, and invade the lymphatic and/or blood systems [bib_ref] The detection of circulating breast cancer cells in blood, Gilbey [/bib_ref]. This is followed by the establishment of secondary tumors at distant sites. It has been suggested that tumor cell invasion into the bloodstream can occur earlier than the time of primary diagnosis [bib_ref] Micrometastasis detection and treatment with monoclonal antibodies, Pantel [/bib_ref]. The ability to detect occult tumor cells with metastatic potential could have a substantial clinical impact on the management of cancer patients. Most, if not all, markers developed to detect occult tumor cells of epithelium origin in peripheral blood have been based on the concept that circulating tumor cells (CTCs) continue to express epithelial cell markers [bib_ref] Increased detection of mammary carcinoma cells in marrow smears using antisera to..., Dearnaley [/bib_ref]. Based on this concept, several epithelial cell markers have been evaluated for detecting disseminated tumor cells in the blood circulation. Frequently used molecules include cytokeratins (CKs) 7, 19, and 20 [bib_ref] Identification of carcinoma cells in peripheral blood samples of patients with advanced..., Felton [/bib_ref] [bib_ref] Detection of disseminated urothelial cancer cells in peripheral venous blood by a..., Fujii [/bib_ref] [bib_ref] Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer..., Soeth [/bib_ref] , carcinoembryonic antigen (CEA) [bib_ref] Persistent evidence of circulating tumor cells detected by means of RT-PCR for..., Jotsuka [/bib_ref] [bib_ref] Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by..., Gerhard [/bib_ref] , epidermal growth factor receptor [bib_ref] Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using..., Hildebrandt [/bib_ref] including HER-2/neu [bib_ref] Detection of HER-2/neu-positive circulating epithelial cells in prostate cancer patients, Ady [/bib_ref] , mucin-1 [bib_ref] Detection of breast cancer micrometastases in axillary lymph nodes by means of..., Noguchi [/bib_ref] , β-subunit of human chorionic gonadotropin (β-hCG) [bib_ref] Detection of metastatic breast cancer by b-hCG polymerase chain reaction, Hoon [/bib_ref] , and α-fetoprotein [bib_ref] Detection of a-fetoprotein mRNA, an indicator of hematogenous spreading hepatocellular carcinoma, in..., Matsumura [/bib_ref]. In prostate cancer (PCa) patients, the expression of prostate specific antigen (PSA) [bib_ref] Early postoperative peripheral blood reverse transcription PCR assay for prostate-specific antigen is..., Shariat [/bib_ref] [bib_ref] Detection of circulating tumor cells in patients with localized and metastatic prostatic..., Ghossein [/bib_ref] [bib_ref] Detection of circulating tumor cells in men with localized prostate cancer, Seiden [/bib_ref] , prostate-specific membrane antigen (PSMA) [bib_ref] Detection of circulating prostate cells during radical prostatectomy by standardized PSMA RT-PCR:..., Schmidt [/bib_ref] [bib_ref] Ts'o PO: Identification and characterization of circulating prostate carcinoma cells, Wang [/bib_ref] , and human glandular kallikrein 2 (hK2)along with other epithelial cell markers has been investigated individually or in combination [bib_ref] Terstappen LW: Multigene reverse transcription-PCR profiling of circulating tumor cells in hormone-refractory..., O&apos;hara [/bib_ref] for their ability to detect CTCs in patients with localized and metastatic PCa. This detection strategy involves the amplification of target mRNAs species by reverse transcriptasepolymerase chain reaction (RT-PCR) [bib_ref] Utilization of polymerase chain reaction technology in the detection of solid tumors, Raj [/bib_ref] [bib_ref] Molecular detection of micrometastases and circulating tumor cells in solid tumors, Ghossein [/bib_ref] [bib_ref] The molecular detection of circulating tumour cells, Johnson [/bib_ref]. However, the use of these markers to detect CTCs fails to explain mechanisms that regulate tumor cell survival in the circulation and the development of their metastatic capability. Recent reports have also emphasized that the immune system actively participates in cancer formation and development. Although this concept of immune response was formulated more than half a century ago [bib_ref] Cancer immunoediting: from immunosurveillance to tumor escape, Dunn [/bib_ref] , the existence of cancer "immunosurveillance" is still largely unknown and debatable because we know very little about the molecules participating in this event. If cancer "immunoediting" is present under the concept of cancer immunosurveillance, we hypothesized that genes expressed in immune cells participating in this event are significantly different from their counterparts in healthy persons. We also hypothesized that both CTCs and immune cells need to evolve through their gene expression at stages of cancer formation and progression. The identified mRNA species associated with circulating cells of cancer-bearing patients will serve as independent markers for future tumor staging and help us understand metastasis and immunosurveillance. In this study, using PCa as a model, we applied the suppression suppressive hybridization (SSH) technique [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref] to establish two libraries consisting of mRNA species that are either present or absent in circulating cells of PCa patients. We sequenced a small number of clones present in these libraries, and identified that the majority, 17 out of 23 (73.9%), of the sequenced clones did not match previously identified mRNA species. From the sequenced clones, we confirmed that four genes are differentially expressed in circulating cells of healthy men and PCa patients using semi-quantitative RT-PCR. Two mRNA species were identified to be significantly elevated in PCa patients, and two mRNA species were identified to be significantly suppressed in PCa patients. # Results and discussion Current protocols for detecting CTCs mainly utilize known epithelial cell markers or other tissue-specific molecules [bib_ref] Fodstad : Detection and clinical importance of micrometastatic disease, Pantel [/bib_ref]. However, the presence of these markers in CTCs does not correlate with their survival in the circulation and their metastatic capability. Furthermore, molecules that participate in the process of "immunosurveillance" remain poorly understood. In this report, we used a PCR-based, genome-wide gene expression analysis named SSH to establish comprehensive, subtracted cDNA libraries to catalogue mRNA species either present or absent in circulating cells of PCa patients. The SSH libraries were constructed from two age-and race-matched, pooled sample populations, healthy men and PCa patients. Each pooled sample consisted of 25 individual double-stranded cDNA libraries derived from circulating cell poly(A) + RNA of 25 men. We used a PCRbased method to evaluate the hybridization efficiency. After two rounds of hybridization, β-actin was amplified from a subtracted population using a pair of gene-specific primers located within the very 3' end of Rsa I digested βactin. No DNA product was detectable after 40 cycles of amplification , whereas the corresponding unsubtracted library showed the presence of abundant βactin. This result demonstrated that β-actin, and possibly the majority of commonly expressed genes between the two sample populations, formed heterohybrids, and could not be amplified using the suppression PCR technique [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref]. To demonstrate that the subtracted cDNA libraries contain potential differently expressed mRNA species, the PCR products were electrophoresed on an agarose gel following the second round of PCR. A series of DNA fragments, ranging from 300 to 1,000 bp in size, representing mRNA species specifically expressed in PCa patients were detected. The various PCR products represent the Rsa I digested cDNA fragments are shown in To reveal the identities of the cDNA clones, the second round PCR products from both subtracted libraries were subcloned into the pCRII TA cloning vector. A total of 23 clones from both subtracted libraries were randomly selected for sequencing using M13 reverse primer. Identi-ties of these clones are listed in [fig_ref] Table 1: Identities of selected cDNA clones present in subtracted libraries [/fig_ref]. A majority of the sequenced clones, 17 out of 23 (73.9%), matched to genomic DNA fragments in the GenBank database, but not previously identified mRNA species. These results might reflect that some of these clones were amplified from rare CTCs in PCa patients and mRNA species reflecting these cells' biology or pathology have not been identified using traditional cDNA construction and Evaluation of subtraction efficiency and the presence of potential differentially expressed genes in the subtracted libraries Evaluation of subtraction efficiency and the presence of potential differentially expressed genes in the subtracted libraries. To determine the subtraction efficiency, β-actin was PCR amplified using a primer set located within the very 3'-end of Rsa I digested β-actin fragment following the second round of hybridization. PCR products were electrophoresed on an agarose gel. No β-actin product was detected after 40 cycles of PCR amplification in a subtracted library, whereas β-actin was detected after 25 cycles of amplification in corresponding un-subtracted library (A). To amplify differentially expressed genes in circulating cells of healthy men and PCa patients, two rounds of PCR amplification was performed following hybridization steps described in Materials and Methods. To demonstrate the presence of potential differentially expressed genes in the subtracted libraries, the final PCR products were analyzed on a 1.5% agarose gel followed by ethidium bromide staining. We detected a series of distinct bands ranging from 300 to 1,000 bp. These DNA fragments represented genes that are either present (B, lane 1) or absent (B, lane 2) in circulating cells of PCa patients. sequencing. In addition, it is possible that genes identified from both subtracted libraries may represent previously un-identified molecules participating in tumor-immune system interactions [bib_ref] Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human..., Nakano [/bib_ref] [bib_ref] Regulation of cutaneous malignancy by gd T cells, Girardi [/bib_ref] [bib_ref] Induction of tumor-specific T cell memory by NK cellmediated tumor rejection, Kelly [/bib_ref] [bib_ref] A novel ligand for the NKG2D receptor activates NK cells and macrophages..., Diefenbach [/bib_ref]. Since a portion of the subtracted cDNAs may be false positive clones [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref] , we needed to confirm the identified clones as truly differentially expressed genes in our two sample populations. We used semi-quantitative RT-PCR to confirm that the cloned cDNAs are associated with peripheral blood circulating cells of cancer-bearing patients. Samples were collected from 12 PCa patients and 8 age-and race-matched healthy men for this analysis. RT-PCR was performed on individual samples. A total of 20 samples, 8 healthy men and 12 PCa patients, total RNA was analyzed for selected genes. Four target genes were selected to be confirmed by RT-PCR. Two genes, PCa-001 and PCa-002, were selected from the library that consists of mRNA species only present in circulating cells of PCa patients. PCR primers were designed according to the Homo sapiens chromosome 5 clone RP11-52M14, complete sequence * PCa clones were selected from the subtracted cDNA library that represents mRNA species only present in circulating cells of PCa patients. ** Nrml clones were selected from the subtracted cDNA library that represents mRNA species absent in circulating cells of PCa patients but present in their counterparts in cancer-free healthy men. . β-actin was also amplified from the same samples using a β-actin primer set (BD Bioscinces Clontech) to serve as an internal control for standardizing the quantity of the RNA applied in each reaction. After PCR amplification, aliquots (10 µl) of these PCR products were electrophoresed into 2% agarose gels followed by ethidium bromide staining. ## Nrml-001 Nrml-002 PCa-001 PCa-002 β-actin Healthy Men PCa patients sequencing results and the designed primers were subjected to a BLAST search to ensure that these sequences do not match any identified mRNA with high homology. As expected, PCa-001 and PCa-002 were expressed at significantly higher levels in circulating cells obtained from PCa patients than in healthy men [fig_ref] 2: PCR primers and conditions for detecting levels of mRNA expression in circulating... [/fig_ref]. The detection of low levels of PCa-001 and PCa-002 may due to high sensitivity of RT-PCR-based detection. Martin et al. also reported low levels of Mdm-2 and Groalpha expression in peripheral blood mononuclear cells of healthy samples using RT-PCR, whereas array-based analysis did not show a detectible signal for these two genes [bib_ref] High-sensitivity array analysis of gene expression for the early detection of disseminated..., Martin [/bib_ref]. It is also possible that a very small number of epithelial cells can be present in the peripheral blood of healthy men [bib_ref] Trial results boost circulating tumor cell field, Garber [/bib_ref] and PCa-001 and PCa-002 are epithelial cells markers. Another two genes, Nrml-001 and Nrml-002, were selected as absent in PCa patients' circulating cells. These two genes were expressed, as expected, at higher levels in healthy men than in PCa patients [fig_ref] 2: PCR primers and conditions for detecting levels of mRNA expression in circulating... [/fig_ref]. β-actin has been shown to be constantly expressed in [bib_ref] Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple..., Vandesompele [/bib_ref]. We also demonstrated that level β-actin expression is similar in all our samples [fig_ref] 2: PCR primers and conditions for detecting levels of mRNA expression in circulating... [/fig_ref]. The relative levels of PCa-001, PCa-002, Nrml-001, and Nrml-002 expression were then normalized to β-actin and illustrated in . Levels of these genes' expression were statistically different between healthy men and PCa patients. ## Relative levels of target genes expression in peripheral blood circulating cells of healthy men and pca patients In order to ensure that the PCR products were not resulted from genomic DNA contamination, we performed two pre-cautionary procedures. First, all total RNA samples were subjected to RNase-free, DNase I digestion to remove residual genomic DNA. Second, we noticed that, in reactions with reverse transcription, β-actin signal was detected after 26 cycles of PCR amplification [fig_ref] 2: PCR primers and conditions for detecting levels of mRNA expression in circulating... [/fig_ref] , whereas β-actin signal was absent in reactions without reverse transcription even after 35 cycles of amplification using amplimers within the same exon (data not shown). However, before we can confirm our statement, fulllength cDNAs corresponding to our identified sequences need to be cloned. The genome-wide screening and identification of mRNA species associated with circulating cells of tumor-bearing patients has been described for various cancers. Twine et al. reported the use of the microarray approach to differentiate gene expression patterns of mononuclear cells in patients with advanced renal cell carcinoma [bib_ref] Disease-associated expression profiles in peripheral blood mononuclear cells from patients with advanced..., Twine [/bib_ref]. However, due to the low number of tumor cells present in the circulation compared to the large number of normal leukocytes, hybridization array (cDNA array and oligonucleotide array) may not be an ideal tool for identifying rare molecular events occurring in small number of CTCs. To overcome this problem, Smirnov et al. used magnetic separation of epithelial cell adhesion molecule (EpCAM) expressing cells from peripheral blood circulating cells and compared gene expression patterns between EpCAMenriched and EpCAM-depleted cells using microarray analysis in cancer-bearing patients [bib_ref] Global gene expression profiling of circulating tumor cells, Smirnov [/bib_ref]. Another comprehensive gene expression method, named mRNA differential display, has been conducted to compare genes that are expressed in peripheral blood mononuclear cells of tumor-free individuals with those from lung, breast, and colon cancer patients [bib_ref] A strategy to identify genes associated with circulating solid tumor cell survival..., Fournier [/bib_ref]. This study found a total of 21 mRNA species expressed in tumor patients' blood samples but not in samples from tumor free volunteers. In addition, Martin et al. reported the use of the differential display to first identify transcripts differentially expressed in breast cancer cells and normal breast epithelial cells followed by an array analysis of these transcripts in the circulating cells of breast cancer patients [bib_ref] High-sensitivity array analysis of gene expression for the early detection of disseminated..., Martin [/bib_ref]. Results from these experiments demonstrate that the detection of disseminated cancer cells in peripheral blood is attainable. It has been suggested that selective enrichment of the tumor cell population from both bone marrow and blood before analysis can increase the sensitivity for detecting CTCs [bib_ref] Detection and characterization of carcinoma cells in the blood, Racila [/bib_ref] [bib_ref] Detection of circulating tumor cells in blood using an optimized density gradient..., Gertler [/bib_ref] [bib_ref] Comparison of two density gradient centrifugation systems for the enrichment of disseminated..., Rosenberg [/bib_ref] [bib_ref] Increased sensitivity for detection of micrometastases in bone-marrow/peripheralblood stem-cell products from breast-cancer..., Naume [/bib_ref]. Various cell separation techniques have been devised to enrich the CTC population from whole blood. However, these methods may also introduce artifacts into the sample preparation steps. For example, the addition of anticoagulants to blood samples affect leukocyte gene expression ex vivo [bib_ref] Influence of collection and separation of blood samples on plasma IL-1, IL-6..., Riches [/bib_ref] [bib_ref] In-vitro production of TNF-a in blood samples, Freeman [/bib_ref] [bib_ref] Stabilization of mRNA expression in whole blood samples, Rainen [/bib_ref]. Efforts were made to avoid RNA degradation and alteration in gene expression during in vitro processing of blood cells and to avoid under-and over-estimation of in vivo mRNA expression. We used direct isolation of poly(A) + RNA and total RNA from whole blood circumventing a prior cell separation step. Our intention was also to prevent the loss of rare, unidentified target cells from blood samples during enrichment procedures since we do not know much about tumor cells' genotypes/phenotypes or any type(s) of immune cells' participation in cancer development. Although it has been suggested that cancers are composed of a heterogeneous collection of cells with different degrees of tumor marker expressions [bib_ref] The molecular detection of micrometastatic breast cancer, Baker [/bib_ref] [bib_ref] Tumor-antigen heterogeneity of disseminated breast cancer cells: implications for immunotherapy of minimal..., Braun [/bib_ref] , CTCs of all types might need to develop a "common" mechanism(s) to survive in the circulation and acquire metastatic capability. We speculate that universal "tumor-specific" markers can be identified in occult tumor cells from different cancers. Moreover, we expect to identify "tissue-specific" molecules in CTCs if tumor cells continue to express their tissue-specific markers. The identification of tissue-specific markers will help to identify the origins of CTCs. Emerging evidence also demonstrates that detection of tumor cells disseminated in peripheral blood can provide clinically important data for tumor staging, prognostication, and identification of surrogate markers for early assessment of the effectiveness of adjuvant therapy. Furthermore, by comparing gene expression profiling of all circulating cells, we expect to identify genes that might play a role in "immunosurveillance". Our future objectives include the identification of cell types that expressed differentially regulated mRNA species. We also intend to study the functional activities of these molecules in circulating cells during cancer development and establish an association between these genes' expression and cancer stages. # Conclusion Using the PCR-based SSH technique, we established two comprehensive subtracted cDNA libraries consisting of potentially differentially regulated genes in circulating cells of PCa patients. We further confirmed that both elevated and suppressed transcripts can be detected in circulating cells of PCa patients. This is an initial attempt to perform genome-wide gene expression analysis in peripheral blood circulating cells and demonstrate the presence of previously un-identified mRNA species in circulating cells of cancer-bearing patients. This is the first step toward understanding tumor metastasis and tumorinduced immune reactions in the development of cancer. We will continue to investigate these molecules' physiological/pathological function and their use in cancer detection. # Methods ## Patient selection ## Blood collection and rna isolation For SSH, whole blood (5 ml) drawn from each individual was immediately mixed with 10x volume of RNA stabilization reagents for blood/bone marrow (Roche). The cells were then lysed. Poly(A) + RNA was immediately isolated by a two-step procedure through magnetic separation using the mRNA isolation kit for blood/bone marrow (Roche). The poly(A) + enriched samples were finally eluted from magnetic beads with H 2 O. Purified poly(A) + RNA was quantitated spectrophotometrically and stored in liquid nitrogen until use. For RT-PCR, blood (2.5 ml) from each individual was colleted into a PAXgene™ Blood RNA tube (QIAGEN) following the manufacturer's protocol. Whole blood was thoroughly mixed with PAXgene stabilization reagent and stored at room temperature for 6 hours prior to RNA extraction. Total RNA was then extracted using the PAX-gene™ Blood RNA kit according to the manufacturer's directions (QIAGEN). As the resulting RNA was usually contaminated with genomic DNA [bib_ref] Impact of pre-analytical handling on bone marrow mRNA gene expression, Breit [/bib_ref] , total RNA samples absorbed to the PAXgene™ Blood RNA System spin column were incubated with DNase I (QIAGEN) at 25°C for 20 min to remove genomic DNA. Total RNA was eluted, quantitated, and stored in liquid nitrogen. ## Suppression subtractive hybridization (ssh) procedures SSH was performed according to procedures described by Diatchenko et al. [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref]. All reagents are now commercially available from BD Biosciences Clontech. Briefly, reverse transcription was performed with 2 µg poly(A) + RNA from an individual patient sample in the presence of a mixture of three 3' anchored primers (5'-TTTGCATGCTCGAG-(T) [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref] [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref]. To form heterohybrids between two sample populations, the adapter A and adaptor B ligated cDNAs (20 ng) were combined with excess Rsa I digested cDNAs (400 ng) from healthy men in two separate reactions, heat-denatured, and hybridized at 68°C for 10 hours. In a second hybridization step, the two separate samples from adapters A and B containing reactions were combined. A fresh aliquot of 150 ng heat-denatured Rsa I digested cDNAs derived from healthy men was added to the combined reaction. Hybridization was continued for another 10 hours at 68°C. Commonly expressed sequences between controls and PCa patients formed hybrids in these two sequential hybridization steps. The heterohybrids are less likely to be amplified in the following PCR step due to the design of SSH adaptors [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref]. Genes specifically expressed in PCa patients' circulating cells were amplified by two consecutive rounds of PCR according to the procedures reported by Diatchenko et al. [bib_ref] Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA..., Diatchenko [/bib_ref]. The PCR-amplified products were then ligated to the pCRII vector (Invitrogen) followed by transformation. The bacteria were plated on agar plates containing ampicillin and overlaid with X-gal and IPTG. After overnight incubation, white colonies were picked and used for subsequent sequencing reaction. Sequencing results were used to design PCR primer sets to determine the genes' expression levels in healthy controls and PCa patients. To detect sequences present in circulating cells of healthy men but absent in circulating cells of PCa patients, the initial adaptors ligation reaction was reversed. Aliquots of Rsa I digested pooled cDNAs derived from healthy men were ligated to adapters A or B followed by hybridization and PCR amplification as described above. ## Rt-pcr confirmation First strand cDNAs were reverse transcribed from 2.5 µg of the total RNA in the presence of oligo d(T) primer (Invit-rogen), 20 µM each of dNTPs, and 200 units of M-MLV reverse transcriptase (Invitrogen). This was done in a total of 50 µl at 42°C for 2 hours. PCR reactions were performed by mixing 1 µl of first-strand cDNAs, 0.2 µM genespecific 5' and 3' primers , and 5 units Taq DNA polymerase (Invitrogen) in a total of 50 µl. Reactions were performed by heat activation at 94°C for 2 min, followed by cycling through 94°C for 30 sec, 50-55°C for 1 min, and 72°C for 1 min. The minimal numbers of PCR cycles required for detecting these gene products were first determined and is indicated in . β-actin (NM_001101) was also amplified and used as an internal control for comparing relative levels of target gene expression. We also included RNA samples without reverse transcription for β-actin amplification to determine levels of genomic DNA contamination. Following gel electrophoresis, images were captured using a Bio-Rad Gel Doc system; and band intensities were analyzed by the Quantity One ® software (Bio-Rad). # Statistical analysis Levels of target gene expression were expressed as mean ± standard deviation (SD) following normalization to βactin. A Student's t test was used to compare means of these genes expressions between the healthy controls and PCa patients. A probability value of p < 0.05 was considered significant. ## List of abbreviations # Authors' contributions XL and HKL conducted sample preparations, subtracted library construction, transcript abundance analysis, and data analysis. CW, RM, GS, AM, JK, and CSM participated in patient selection, patient enrollment, and sample collection. DJC, BPK, and HKL participated in study design, data interpretation, and manuscript preparation. [fig] 2: PCR primers and conditions for detecting levels of mRNA expression in circulating cells of healthy men and patients with PCa Clone I.D. GenBank Accession No. PCR primers cycles PCa-001 AC132068 5'-AGG AAT AAG TCA CAC CGT GGA-3' 5'-ACC TGT TGG GAC TAG ACG CAT-3' 20 nested 5'-TGG TCT GTA ACC CTT AGG AGA-3' 5'-TCT GCC CTT TGA GTC CAA GT-3' 25 Pca-002 AC019106 5'-AGG TCA GCA GAG ATG TCT GT-3' 5'-TAG TCC CCG AGA AAG AAT TA-3' 32 Nrml-001 AC004690 5'-TGA GCA GTT TCT TCA GCC TCA-3' 5'-TGA TAA GTC CAA CCC AAA GGC T-3' 20 nested 5'-TAT CTG GGT GAC ACT GGG AAA-3' 5'-AGA GAC CAG CGT AAT ATC CCT-3' 30 Nrml-002 AK095899 5'-AGG TAA AGG AAA CCC CAA CAT GCA-3' 5'-AAC CAA CGA GGT GGC CGA ATC TT-3' 35 Confirmation of differential gene expression in circulating cells of healthy men and PCa patients using semi-quantitative RT-PCR Figure 2 Confirmation of differential gene expression in circulating cells of healthy men and PCa patients using semiquantitative RT-PCR. RT-PCR was performed on individual samples from 8 healthy controls and 12 PCa patients to confirm the SSH results. After sequencing reaction to reveal the identities of a total of 23 clones present in the subtracted libraries, PCR primers were designed [/fig] [fig] Figure 3 < 0: Relative levels of target genes expression in peripheral blood circulating cells of healthy men and PCa patients.Images obtained fromFigure 2were captured and analyzed using the Quantity One ® software. For each target gene, levels of gene expression were normalized to the level of β-actin expression for each individual sample. * indicates statistical significance between healthy men and PCa patients at p Gene Expression (Relative to β-actin) [/fig] [table] Table 1: Identities of selected cDNA clones present in subtracted libraries [/table]

Socioeconomic status, financial hardship and measured obesity in older adults: a cross-sectional study of the EPIC-Norfolk cohort Background: Socioeconomic status is strongly associated with obesity. Current economic circumstances are also independently associated with self-reported weight status in Finnish civil servants. We aimed to examine three types of financial hardship in relation to measured general and central obesity in a general population of older adults, while considering conventional socioeconomic indicators.Methods: Data from 10,137 participants (≥50 years) in the EPIC-Norfolk cohort who responded to a postal Health and Life Experiences Questionnaire (1996)(1997)(1998)(1999)(2000) and attended a clinical assessment (1998)(1999)(2000)(2001)(2002). Multivariable logistic regression models assessed likelihood of general obesity (BMI ≥30 kg/m 2 ) and central obesity (women: ≥88 cm; men: ≥102 cm) calculated from measured anthropometrics. Results: Obesity prevalence was consistently patterned by standard socioeconomic indicators, with over-50s in the lowest social class being twice as likely to be obese than those in the highest class (women OR 2.10 [CI95: 1.41-3.13]; men OR 2.36 [1.44-3.87]). After adjustment for socioeconomic status, reporting having less than enough money for one' s needs (compared to more than enough) was associated with obesity in women (OR 2.04 [1.54-2.69]) and men ). Similar associations were demonstrated between obesity and always or often not having enough money for food/clothing (women OR 1.40 [1.03-1.90]; men OR 1.81 [1.28-2.56]), compared to reporting this never occurred. The strongest independent associations were seen for obesity and reported greatest level of difficulty paying bills (women OR 2.20 [1.37-3.55]; men 2. 40 [1.38-4.17]), compared to having no difficulties. Findings for central obesity were slightly higher in women and lower in men. Conclusions: Obesity in British over-50s was more likely in study participants who reported greater financial hardship, even after education, social class and home ownership were taken into account. Public health policies need to consider the hitherto neglected role of financial hardship in older people, especially difficulty paying bills, as part of strategies to prevent or reduce obesity. # Background Obesity is one of the biggest public health challenges faced by high income countries, accounting for a large and growing disease burden (cardiovascular disease, type 2 diabetes, some cancers) and imposing substantial cost burden on both the healthcare system and society at large [bib_ref] The lifetime medical cost burden of overweight and obesity: implications for obesity..., Finkelstein [/bib_ref]. Both general (weight status) and central (excess abdominal fat) obesity significantly increase a person's risk of, for example, cardiometabolic conditions [bib_ref] Necessity of both waist circumference and waist-to-height ratio for better evaluaion of..., Wakabayashi [/bib_ref]. Obesity is strongly and inversely related to socioeconomic status (SES) as measured by conventional indicators of education or social class, particularly in the case of women [bib_ref] Consortium TE-P: Tackling the social and economic determinants of nutrition and physical..., Pomerlau [/bib_ref] [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref]. But, conventional SES has been acknowledged as insufficient to understand how a person's economic situation is associated with their body mass index (BMI) or waist circumference (WC) [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] , and differences in obesity by education or income are not consistently observed in UK adults. A limitation with conventional SES indicators is that they do not fully capture people's material circumstances and spending power, whereas everyday financial troubles may be a stronger antecedent to obesity than income, occupational status, or education [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Multiple socioeconomic determinants of weight gain: the Helsinki Health Study, Loman [/bib_ref]. Wider research on poverty has highlighted the added value of material hardship measures to concepts of inequality. Evidence from two occupational cohorts supports the notion that financial hardship reflects a distinct set of economic factors that independently impacts health beyond the reported influence exerted by conventional SES [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Multiple socioeconomic determinants of weight gain: the Helsinki Health Study, Loman [/bib_ref] [bib_ref] Economic difficulties and physical functioning in Finnish and British employees: contribution of..., Laaksonen [/bib_ref] [bib_ref] Economic difficulties and common mental disorders among Finnish and British white-collar employees:..., Laaksonen [/bib_ref] [bib_ref] Self-reported economic difficulties and coronary events in men: evidence from the Whitehall..., Ferrie [/bib_ref] [bib_ref] Change in economic difficulties and physical and mental functioning: evidence from British..., Lallukka [/bib_ref]. Another study found an association between financial hardship and smoking even within high-income groups [bib_ref] The contribution of lone parenthood and economic difficulties to smoking, Rahkonen [/bib_ref]. To date, one longitudinal study of US adolescents explored separate types of financial hardship and found suggestive evidence that having trouble paying bills may be related to obesity in women, but not men. Financial hardship is closely correlated, but not interchangeable, with conventional SES and therefore deserves specific attention [bib_ref] Socioeconomic status in health research: one size does not fit all, Braveman [/bib_ref]. Since individuals at all income levels can experience financial hardship with consequent health effects, the implications for public health and policy are that income (or other standard SES indicators) should not be seen as the sole criteria for targeting interventions [bib_ref] Economic difficulties and physical functioning in Finnish and British employees: contribution of..., Laaksonen [/bib_ref] [bib_ref] Socioeconomic status in health research: one size does not fit all, Braveman [/bib_ref] [bib_ref] Financial strain over the life course and health among older adults, Kahn [/bib_ref] [bib_ref] Economic difficulties and subsequent sleep problems: evidence from British and Finnish occupational..., Lallukka [/bib_ref]. One of the largest drains on disposable income, especially in older people, is paying bills and affording adequate food and clothing, and yet, health research and policy has tended to neglect current financial difficulties as a unique economic domain determining health. Consequently, evidence remains limited on the role of financial hardship in obesity and whether separate types of hardship differ in their associations with obesity, particularly among older adults who comprise a growing population [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Financial strain over the life course and health among older adults, Kahn [/bib_ref] [bib_ref] The sense of mastery as a mediator and moderator in the association..., Pudrovska [/bib_ref]. Older adults with greater hardships may purchase less food and have lower weights due to fewer calories; however, they may also purchase cheaper food high in energy density which could contribute to excess weight. We therefore investigated the sex-specific associations between three types of self-reported financial hardships and obesity measured objectively in adults aged 50 and older. We hypothesised that greater levels of financial hardship may be associated with greater odds of obesity, overall and centrally, with sex differences in magnitude of associations. We further hypothesised that associations would remain significant after adjusting for conventional socioeconomic status. # Methods ## Study population We used data collected as part of the EPIC-Norfolk prospective cohort study-a component of the European Prospective Investigation of Cancer (EPIC) study in 10 European countries [bib_ref] EPIC-Norfolk: study design and characteristics of the cohort. European Prospective Investigation of..., Day [/bib_ref]. As we were interested in adults near the end of working life and beyond to place findings in a healthy ageing context, we included over-50s (n = 20,274) from the population-based cohort who were recruited from age-sex registers of general practices and who attended a first health check at entry (1993-97). At entry, over-50s were similar to the total cohort (n = 25,639) in terms of health behaviours and other socio-demographic factors. Ethnicity of 99.7% of EPIC-Norfolk participants was of white origin. Financial hardship was assessed using the postal "Health and Life Experiences Questionnaire" (HLEQ) designed to assess social and psychological circumstances [bib_ref] The shackles of misfortune: social adversity assessment and representation in a chronic-disease..., Surtees [/bib_ref] [bib_ref] Psychosocial aetiology of chronic disease: a pragmatic approach to the assessment of..., Surtees [/bib_ref]. Completed responses from over-50s ranged between 17,953 and 17,998. Outcome data on BMI (n = 11,982) and WC (n = 12,000) were measured objectively during a second clinical assessment . Our available sample therefore included over-50s who responded to financial hardship questions, had covariates and follow-up anthropometry (range: 10,113-10,137) with 99% complete data (see Additional file 1: . The sample was similar in characteristics and lifestyle to responders in the full cohort, and over-50s not responding to hardship questions were similar to cohort non-responders. All volunteers gave written informed consent and the study was approved by the Norwich district ethics committee. ## Measures ## Financial hardship and conventional socioeconomic indicators Financial hardship was measured by three self-reported questions in line with Pearlin's list of chronic strains as used in similar studies [bib_ref] Economic difficulties and physical functioning in Finnish and British employees: contribution of..., Laaksonen [/bib_ref] [bib_ref] Economic difficulties and subsequent sleep problems: evidence from British and Finnish occupational..., Lallukka [/bib_ref]. Questions covered sufficiency of money to meet needs (more than enough, just enough, less than enough), frequency of not having enough money to afford adequate food or clothing (5 responses, range 'always' to 'never'), and difficulty paying bills (6 responses, range 'very great' to 'none'). Responses 'always' and 'often', or 'great' and 'very great', were combined for analysis due to low numbers in the bottom two categories. We analysed three conventional indicators of SES. Education level (no qualification, O-level (16 years), Alevel (18 years), degree (>18 years)) and occupation were self-reported at cohort entry, with occupation used to classify participants into six hierarchical categories of the Registrar General's classification scheme of social class (professional, managerial and technical, skilled non-manual, skilled manual, partly skilled, and unskilled). Social class in women was based on her partner's occupation (68%) unless it was unclassified, missing, or they were single and then her own occupation was used [bib_ref] Occupational social class, risk factors and cardiovascular disease incidence in men and..., Mcfadden [/bib_ref]. We also employed a measure of housing tenure (home-owner, public renting, private renting) as a conventional SES proxy, given that previous research has documented the utility of home ownership as a measure of wealth in older populations [bib_ref] Should health studies measure wealth?: a systematic review, Pollack [/bib_ref]. ## Obesity Trained nurses used standardised protocols to measure weight, height, and WC of all participants attending the second clinic assessment, as reported elsewhere [bib_ref] Occupational social class, risk factors and cardiovascular disease incidence in men and..., Mcfadden [/bib_ref] [bib_ref] Obesity, confidant support and functional health: cross-sectional evidence from the EPIC-Norfolk cohort, Surtees [/bib_ref]. BMI was calculated as weight in kilograms divided by the square of height in metres. Participants who had a BMI ≥ 30 kg/m 2 were classified as obese overall. Central obesity was calculated using sex-specific threshold criteria for WC: women with WC ≥ 88 cm and men with WC ≥ 102 cm were classified as centrally obese. ## Socio-demographic variables Participants self-reported smoking status (current, former, never), marital status (married/living as married, single, widowed, separate, divorced), and general health status (excellent, good, moderate, poor), at second health check . Regular car use (yes/no) was self-reported in the Environment and Physical Activity questionnaire (EPAQ2) . Socio-demographic variables for date of birth (continuous age) and sex were measured at cohort entry. # Data analysis Descriptive statistics summarised socio-demographic characteristics (sex, education, social class, home-ownership, car use, health status, smoking status, marital status), and crude prevalence of obesity across financial hardship levels. We used a correlation matrix to examine interrelationships among the financial hardship indicators after recoding two indicators into three levels: frequency of not having enough money for food or clothing (never; sometimes/seldom; often/always), and difficulty paying bills (none; very little/slight; some/great/very great). Odds ratios of prevalent obesity for the six socioeconomic indicators were examined by fitting logistic regression models a priori sex-stratified and adjusted for age, smoking status and marital status. As known confounders, each is associated with socioeconomic factors and independently with obesity [bib_ref] Association of workplace chronic and acute stressors with employee weight status: data..., Diana [/bib_ref] [bib_ref] Negative aspects of close relationships as a predictor of increased body mass..., Kouvonen [/bib_ref] [bib_ref] Prospective effect of job strain on general and central obesity in the..., Brunner [/bib_ref] [bib_ref] Race and weight change in US women: the roles of socioeconomic and..., Kahn [/bib_ref]. For each categorical measure of financial hardship, we fitted sequential logistic regression models to base models (sex-stratified and adjusted for age, smoking and marital status); first by education, followed by occupational social class, and then housing tenure. The final model for each hardship indicator mutually adjusted for all conventional SES and covariates and therefore the remaining sexspecific odds ratios for general and central obesity were interpreted as independent associations of the financial hardship variable in question. In secondary analyses, we further adjusted for concurrent lifestyle variables: total energy intake (Kcal), total alcohol consumption (units/week), and physical activity and energy expenditure (PAEE) score. Self-reported total energy and alcohol intake were assessed by food frequency questionnaire [bib_ref] Comparison of dietary assessment methods in nutritional epidemiology: weighed records v. 24..., Bingham [/bib_ref] ; and PAEE by the EPAQ2 questionnaire, previously validated against individually calibrated heart rate against energy expenditure [bib_ref] Validity and repeatability of the EPIC-Norfolk physical activity questionnaire, Wareham [/bib_ref]. For women, we also adjusted for menopause and HRT status in secondary models. Statistical analyses were performed separately for women and men using Stata 12.1. Results are presented as odds ratios (ORs) and 95 percent confidence intervals (95% CIs). # Results The mean age of participants was 62.5 years (SD 7.5) with 54% of the sample made up of women. A majority (81%) reported being in good or excellent general health, and 51% were ever smokers. For the whole sample, 11% were educated to degree-level; 14% of men and 9% of women were educated to this level. Professional (class I), and managerial and technical (class II), occupations comprised 42% of the sample; few women (4%) and men (3%) had unskilled occupations. Mean BMI was 27 kg/ m 2 (SD 3.3) in men, and 26.8 kg/m 2 (SD 4.4) in women; 16% of men and 20% of women were classified as obese overall. Women's average WC was 82.9 cm (SD 10.6) and men's was 96.7 cm (SD 9.6); 29% of women and 27% of men were classified as being centrally obese. There was a close inter-relationship between the three measures of self-reported financial hardship and other socio-demographic measures (see Additional file 1: [fig_ref] Table 2: Odds ratios of general obesity across levels of financial hardship in women... [/fig_ref]. We found that the three financial hardship measures were moderately related to each other. The indicator having enough money for needs shared 23% and 26% of its variability with the indicators frequency of not having enough money for food or clothing (r = 0.48) and difficulty paying bills (r = 0.51), respectively. Frequency of not having enough money for food or clothing shared 38% of its variability with difficulty paying bills (r = 0.62). ## Conventional ses indicators and odds of obesity There was a clear pattern of association between lower levels of social class, education, and housing tenure and general and central obesity in sex-specific models adjusted for covariates [fig_ref] Table 1: Odds ratios of general and central obesity in women and men [/fig_ref]. The lowest social class (V) was significantly associated with greater odds of general obesity in women (OR 2.10; 1.41-3.13) and men (OR 2.36; 1.44-3.87) aged 50 and over. Similar sex-specific associations were observed between social class and central obesity, but reached significance only in women. Women and men who reported having no educational qualification were more likely to be obese centrally and overall, although odds ratios were larger in men for both outcomes. Similarly, obesity was more likely in women and men who reported renting public or private accommodation (compared with owning); magnitudes were largest for general obesity in men who rented accommodation. Further adjustment for total energy intake, physical activity and alcohol intake attenuated or made little difference to most of the associations between conventional SES indicators and obesity (see Additional file 1: [fig_ref] Table 3: Odds ratios of central obesity across levels of financial hardship in women... [/fig_ref]. Addition of menopause and HRT status made no difference to overall magnitude or direction of findings for women (data not shown). ## Financial hardship and odds of obesity In an analysis adjusting for age, smoking status, and marital status, all three measures of financial hardship were strongly associated with obesity in both sexes (Model A, [fig_ref] Table 2: Odds ratios of general obesity across levels of financial hardship in women... [/fig_ref]. The magnitude of association was greater than that seen for the more traditional measures of SES. In general the measures of association were similar in men and women. A somewhat different pattern was observed for associations with central obesity which tended to be stronger in women than men (Model A, [fig_ref] Table 3: Odds ratios of central obesity across levels of financial hardship in women... [/fig_ref]. Adjusting for education, social class, and housing tenure attenuated the associations between financial hardship and odds of general and central obesity (Model D, [fig_ref] Table 2: Odds ratios of general obesity across levels of financial hardship in women... [/fig_ref]. Additional adjustment for other lifestyle variables had little effect on the measures of association either for general or central obesity (see Additional file 1: . # Discussion # Synopsis of results This cross-sectional, population-based study of UK over-50s showed social class, education, and housing tenure gradients in obesity. It further demonstrated strong associations with obesity for three types of financial hardship that were independent of social class, education and housing tenure. Independent associations were particularly strong for central obesity in women compared to men. # Methodological considerations The financial hardship variables were self-reported and like all such variables may be subject to recall or social desirability bias. Interpretation of the meaning of financial hardship can also vary widely across the population; equivalent levels of financial strain can be perceived and experienced as a normative status of daily living for some groups but as deprivation for others [bib_ref] Financial strain over the life course and health among older adults, Kahn [/bib_ref]. Precedent exists, however, for the measures used here as findings of independent associations are consistent with other studies of self-reported and objective health outcomes in similarly-aged groups [bib_ref] Self-reported economic difficulties and coronary events in men: evidence from the Whitehall..., Ferrie [/bib_ref] [bib_ref] Financial strain over the life course and health among older adults, Kahn [/bib_ref] [bib_ref] Economic difficulties and subsequent sleep problems: evidence from British and Finnish occupational..., Lallukka [/bib_ref]. Although financial Sex-specific odds ratios (95% confidence intervals) for general obesity (BMI ≥ 30 kg/m 2 ) obtained by multivariable logistic regression analysis adjusting for age, marital status and smoking status. Final numbers (Model D) of women and men, respectively, for money (n = 5 526; n = 4 588); frequency of not enough money (n = 5 536; n = 4 591); difficulty paying bills (n = 5 542; n = 4 596). hardship was assessed before anthropometry measurement, we could not ascertain the duration of, or transition in, hardship in relation to obesity as the survey was administered once. Thus, there may have been misclassification of exposures stemming from changes to participants' hardship levels in the interval between assessment of financial circumstances and anthropometric measurement. Such misclassification would be non-differential since it was unlikely to have been related to our outcomes and hence would have biased results towards the null. Our findings may also be subject to residual confounding in two ways. First, income was not collected in EPIC-Norfolk and thus we could not account for income-based differences. However, income is not consistently shown to have an impact on weight in older adults or for both sexes [bib_ref] The impact of income on the weight of elderly Americans, Cawley [/bib_ref] [bib_ref] Socioeconomic status in relation to obesity and abdominal obesity in korean adults:..., Yoon [/bib_ref] , and may not sufficiently reflect structural resources in our sample of older adults since they likely Sex-specific odds ratios (95% confidence intervals) obtained by multivariable logistic regression analysis adjusting for age, marital status and smoking status. Central obesity for women (waist circumference, WC ≥ 88 cm) and for men (WC ≥ 102 cm). Final numbers (Model D) of women and men, respectively, for money (n = 5 533; n = 4 594); frequency of not enough money (n = 5 543; n = 4 597); difficulty paying bills (n = 5 549; n = 4 602). also use savings to fund their expenses [bib_ref] Sociodemographic determinants of diet quality of the EU elderly: a comparative analysis..., Irz [/bib_ref]. Our study nevertheless examined six socioeconomic indicators separately and also included education, social class and housing tenure in analyses of financial hardship. Second, parity was not analysed but may have confounded or mediated our associations between socioeconomic factors and obesity [bib_ref] Childbearing and obesity in women: weight before, during, and after pregnancy, Gunderson [/bib_ref] [bib_ref] Number of children associated with obesity in middle-aged women and men: results..., Weng [/bib_ref]. Notwithstanding some limitations, the study's strengths include a large sample size, sex-specific analyses, adjustment for multiple known confounders and lifestyle variables, and two objective obesity measures. Finally, this cohort had similar characteristics to the general UK population apart from fewer smokers and lack of ethnic diversity [bib_ref] EPIC-Norfolk: study design and characteristics of the cohort. European Prospective Investigation of..., Day [/bib_ref] [bib_ref] Obesity, confidant support and functional health: cross-sectional evidence from the EPIC-Norfolk cohort, Surtees [/bib_ref] , and so findings could be generalised to other white European-origin older adults. ## Relationship to previous work Our work is novel in at least three ways. First, we examined three separate financial hardship exposure measures and thus provide unique information on how different types of the financial hardship domain might be associated with higher prevalence of obesity. We therefore add depth to previous studies which combined hardship questions into one summary indicator [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Multiple socioeconomic determinants of weight gain: the Helsinki Health Study, Loman [/bib_ref] [bib_ref] Self-reported economic difficulties and coronary events in men: evidence from the Whitehall..., Ferrie [/bib_ref] [bib_ref] Change in economic difficulties and physical and mental functioning: evidence from British..., Lallukka [/bib_ref] [bib_ref] The sociological study of stress, Pearlin [/bib_ref] [bib_ref] Reduced food access due to a lack of money, inability to lift..., Burns [/bib_ref] [bib_ref] Multiple socioeconomic circumstances and healthy food habits, Lallukka [/bib_ref] [bib_ref] Changes in economic difficulties and subsequent sickness absence: a prospective register-linkage study, Lallukka [/bib_ref]. Second, our focus on over-50s contributes new knowledge in support of a healthy ageing agenda as no studies of economic strain and health in older populations have assessed obesity, to our knowledge [bib_ref] Financial strain over the life course and health among older adults, Kahn [/bib_ref] [bib_ref] The sense of mastery as a mediator and moderator in the association..., Pudrovska [/bib_ref]. Third and most notably, our study is clinically relevant. We used two objective measures of obesity recommended as separate predictors of health risk, particularly central adiposity [bib_ref] Beyond BMI: The value of more accurate measures of fatness and obesity..., Burkhauser [/bib_ref] , rather than self-reported weight which is prone to systematic bias from inaccuracy of height and underreporting and misclassification for obese categories [bib_ref] Waist circumference and cardiometabolic risk: a consensus statement from shaping America's health:..., Klein [/bib_ref] [bib_ref] Waist circumference and not body mass index explains obesity-related health risk, Janssen [/bib_ref]. Our finding that financial hardship showed independent associations with BMI is consistent with current evidence which notably comes from occupational cohorts [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Self-reported economic difficulties and coronary events in men: evidence from the Whitehall..., Ferrie [/bib_ref] [bib_ref] Multiple socioeconomic circumstances and healthy food habits, Lallukka [/bib_ref]. The Helsinki Health Study of middle-aged employees, mostly women, reported increased odds of self-reported BMI for frequent financial hardship independent of conventional SES and early life factors [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] , but our estimates were slightly larger. Unlike our work, that study of self-reported BMI included only age and no other BMI-related covariates associated with standard SES and obesity [bib_ref] Implication of socio-economic status on the prevalence of overweight and obesity in..., Manios [/bib_ref]. Another study of the same occupational cohort found a higher odds of weight gain (≥5 kg) with increasing frequency of economic difficulties after conventional SES adjustment, but again without accounting for smoking status, living arrangement or other health behaviours [bib_ref] Multiple socioeconomic determinants of weight gain: the Helsinki Health Study, Loman [/bib_ref]. The Whitehall II study of financial hardship and coronary events in middle-aged men also found that a higher economic difficulties score was associated with a higher BMI and waist-hip ratio measured objectively, but the age-adjusted association did not account for conventional SES [bib_ref] Self-reported economic difficulties and coronary events in men: evidence from the Whitehall..., Ferrie [/bib_ref]. To our knowledge, no other studies of financial hardship have reported on central obesity for us to compare our findings. Existing evidence supports the notion that social inequalities in obesity differ between the sexes with SES differences being associated more strongly and consistently with BMI in women [bib_ref] Consortium TE-P: Tackling the social and economic determinants of nutrition and physical..., Pomerlau [/bib_ref] [bib_ref] Central and total obesity in middle aged men and women in relation..., Langenberg [/bib_ref] [bib_ref] Socioeconomic inequalities in health in 22 European countries, Mackenbach [/bib_ref]. Sex differences have also been reported among the few studies examining financial hardship and obesity, suggesting independent associations are stronger in men [bib_ref] Multiple dimensions of socioeconomic position and obesity among employees: The Helsinki Health..., Laaksonen [/bib_ref] [bib_ref] Multiple socioeconomic determinants of weight gain: the Helsinki Health Study, Loman [/bib_ref]. Notably, those results were reported in a younger population comprised of civil servants. The present study of a population-based sample of older British adults revealed contrasting results. Conventional SES proxies appeared to be more strongly and consistently associated with men's higher odds of obesity which does not have a clear explanation. By contrast, the main associations between financial hardship and obesity were larger than SES and stronger in women. However, after adjusting for conventional SES, sex differences observed in the magnitude of associations depended on the type of hardship and obesity measure. This observed reversal between the sexes in the strength of socioeconomic disparities in obesity might point to gender-based differences in the experience of financial hardship. For example, men and women have differential vulnerabilities to financial hardship as women report not having enough money for food twice more often than men [bib_ref] Gender differences in health: a Canadian study of the psychosocial, structural and..., Denton [/bib_ref] [bib_ref] Sociodemographic factors and attitudes toward food affordability and health are associated with..., Bihan [/bib_ref] , and they also have differential power in intra-household economics and division of labour [bib_ref] The intra-household economics of voice and exit, Katz [/bib_ref] [bib_ref] Economic dependency, gender, and the division of labor at home, Brines [/bib_ref]. Several potential mediators might explain our finding of a link between financial hardship and obesity. Financial hardship is a powerful stressor and sociologists have shown the utility of coping and social support in explaining differences between socially and economically demarcated groups in effects of financial hardship [bib_ref] The sociological study of stress, Pearlin [/bib_ref] [bib_ref] The structure of coping, Pearlin [/bib_ref]. Coping behaviours, involving the manipulation of goals and values, were found to be effective for minimizing adverse effects of household financial strain, and to be used to a greater extent by socially advantaged groups, namely men, the educated, and the affluent [bib_ref] The structure of coping, Pearlin [/bib_ref]. Other potential mediators include psychological resources such as self-esteem and sense of mastery [bib_ref] The sense of mastery as a mediator and moderator in the association..., Pudrovska [/bib_ref]. Structural factors range from consumer prices of goods and services (e.g. food, transportation) [bib_ref] Reduced food access due to a lack of money, inability to lift..., Burns [/bib_ref] , and neighbourhood access to healthy foods and safe spaces for physical activity, to employment [bib_ref] Overweight and obesity: the role of education, employment and income in Spanish..., Rodriguez Martin [/bib_ref] [bib_ref] Psychosocial and socio-economic factors in women and their relationship to obesity and..., Rosmond [/bib_ref] and cultural norms and social meanings reinforced through media and advertising [bib_ref] Food choices in later life, Lumbers [/bib_ref]. Since financial hardship was a stronger correlate of obesity than conventional SES, excess weight and abdominal fat may be more directly influenced by mechanisms related to spending power and material resources, including lack of sleep from financial worries and physiological responses to hardship-related stress, than by non-material factors such as social roles, cultural norms, and knowledge. Both chronic stress and insufficient sleep have independent associations with obesity [bib_ref] Stress and obesity: the role of the hypothalamic-pituitary-adrenal axis in metabolic disease, Bose [/bib_ref] [bib_ref] Meta-analysis of short sleep duration and obesity in children and adults, Cappuccio [/bib_ref] [bib_ref] Do stress reactions cause abdominal obesity and comorbidities?, Björntorp [/bib_ref]. Although people of all ages may encounter financial hardship, adults in older age groups are at greater risk of increased financial hardship which commonly results from events they are more likely to experience such as divorce, death of spouse, or involuntary job loss [bib_ref] The sense of mastery as a mediator and moderator in the association..., Pudrovska [/bib_ref] [bib_ref] The sociological study of stress, Pearlin [/bib_ref]. Our results suggest that monetary and coping interventions may be useful in efforts to reduce obesity among over-50s. Formal mediation analyses of stress-related indicators are warranted to examine physiological mechanisms of influence between financial hardship and obesity in older women and men. Future research should also explore how both social and economic aspects of an individual's life circumstances interact to produce combined effects on obesity as called for in the public health research and policy literature. Nevertheless, prevention of obesity in over-50s would benefit more from an increased focus on their experience of financial hardship in addition to their education or income levels. # Conclusions British over-50s reporting greater levels of financial hardship were more likely to have excess weight and abdominal fat. Likelihood of obesity was more strongly correlated with financial hardship than conventional markers of SES. Thus, financial hardship indicators provided additional explanatory power beyond education, social class or home-ownership in understanding variation in prevalence of obesity in over-50 women and men. Our findings confirm that it is not sufficient to solely consider education, social class or home-ownership when examining the role of socioeconomic factors in the prevention of obesity or in weight support among older adults. Rather, public health policies and strategies need to support older people in terms of their more contemporaneous economic concerns. Interventions and practice standards to reduce or prevent obesity might include coping and monetary strategies and a focus on meeting bill payments might be a suitable target for approaches to address obesity. ## Additional file Additional file 1: . The process of sample selection from the EPIC-Norfolk cohort. [fig_ref] Table 2: Odds ratios of general obesity across levels of financial hardship in women... [/fig_ref] : Characteristics of self-reported financial hardship for over-50s in the EPIC-Norfolk cohort. [fig_ref] Table 3: Odds ratios of central obesity across levels of financial hardship in women... [/fig_ref] : Odds ratios of general and central obesity in women and men (≥50 years) across levels of social class, education, and housing tenure in the EPIC-Norfolk cohort. : Odds ratio of general obesity across levels of financial hardship in women and men (≥50 years) in the EPIC-Norfolk cohort. : Odds ratio of central obesity across levels of financial hardship in women and men (≥50 years) in the EPIC-Norfolk cohort. [table] Table 1: Odds ratios of general and central obesity in women and men (≥50 years) across levels of social class, education, and housing tenure in the EPIC-Norfolk cohort [/table] [table] Table 2: Odds ratios of general obesity across levels of financial hardship in women and men (≥50 years) in the EPIC-Norfolk cohort [/table] [table] Table 3: Odds ratios of central obesity across levels of financial hardship in women and men (≥50 years) in the EPIC-Norfolk cohort [/table]

Amide proton transfer weighted (APTw) imaging based radiomics allows for the differentiation of gliomas from metastases We sought to evaluate the utility of radiomics for Amide Proton Transfer weighted (APTw) imaging by assessing its value in differentiating brain metastases from high-and low grade glial brain tumors. We retrospectively identified 48 treatment-naïve patients (10 WHO grade 2, 1 WHO grade 3, 10 WHO grade 4 primary glial brain tumors and 27 metastases) with either primary glial brain tumors or metastases who had undergone APTw MR imaging. After image analysis with radiomics feature extraction and post-processing, machine learning algorithms (multilayer perceptron machine learning algorithm; random forest classifier) with stratified tenfold cross validation were trained on features and were used to differentiate the brain neoplasms. The multilayer perceptron achieved an AUC of 0.836 (receiver operating characteristic curve) in differentiating primary glial brain tumors from metastases. The random forest classifier achieved an AUC of 0.868 in differentiating WHO grade 4 from WHO grade 2/3 primary glial brain tumors. For the differentiation of WHO grade 4 tumors from grade 2/3 tumors and metastases an average AUC of 0.797 was achieved. Our results indicate that the use of radiomics for APTw imaging is feasible and the differentiation of primary glial brain tumors from metastases is achievable with a high degree of accuracy.AbbreviationsAPTwAmide Proton Transfer weighted ML Machine learning TA Texture analysis GLRLM Gray-level run length matrix GLSZM Gray-level size zone matrix NGTDM Neighbouring gray tone difference matrix GLDM Gray-level dependence matrix CEST Chemical Exchange Saturation Transfer Amide proton transfer weighted (APTw) imaging represents a novel contrast media free molecular MR imaging technique that has recently shown promise in characterizing and differentiating brain neoplasms as well as malignancies in other body regions 1-7 . The APTw signal originates from amide protons in endogeneous proteins and peptides in the parenchyma. In tumor tissue, the content of mobile proteins and peptides is increased thus resulting in increased APTw signal intensity values 8,9 . www.nature.com/scientificreports/ With the exception of one study [bib_ref] Amide proton transfer imaging in predicting isocitrate dehydrogenase 1 mutation status of..., Han [/bib_ref] , these previous investigations utilized standard histogram analyses techniques at the most to analyse the APTw signal of the tissue at hand thus only scratching the surface of the information that can potentially be extracted from radiological images [bib_ref] Histogram analysis of amide proton transfer imaging to identify contrastenhancing low-grade brain..., Park [/bib_ref] [bib_ref] Applying protein-based amide proton transfer MR imaging to distinguish solitary brain metastases..., Yu [/bib_ref] [bib_ref] Amide proton transfer MR imaging of endometrioid endometrial adenocarcinoma: association with histologic..., Takayama [/bib_ref]. With recent advances in the field of machine learning (ML), radiomics techniques allowing for the extraction of high-dimensional mineable data from medical images have been developed and introduced to medical imaging thus enabling in-depth tissue classification and characterization [bib_ref] Subacute and chronic left ventricular myocardial scar: accuracy of texture analysis on..., Baessler [/bib_ref] [bib_ref] Radiomics: Images are more than pictures, they are data, Gillies [/bib_ref] [bib_ref] A radiomics-based comparative study on arterial spin labeling and dynamic susceptibility contrast..., Hashido [/bib_ref] [bib_ref] Prognostic value of texture analysis from cardiac magnetic resonance imaging in patients..., Mannil [/bib_ref] [bib_ref] Prediction of malignant glioma grades using contrast-enhanced T1-weighted and T2-weighted magnetic resonance..., Nakamoto [/bib_ref]. In this proof-of-concept study we aimed at assessing the potential of radiomics and ML for APTw imaging. To this extent, we used radiomics on APTw images to differentiate WHO Grade 2, 3 and 4 gliomas from brain metastases. # Materials and methods This study received institutional review board approval (Cantonal Ethical Committee Zürich, Switzerland) and was performed in accordance with all guidelines and regulations defined by the institutional review board. All subjects gave written informed general consent. ## Subjects. In this retrospective study we included 48 patients (mean age: 61 years, range: 37-83 years) diagnosed with either low-or high grade gliomas (10 patients with WHO Grade 2 tumors, 1 patient with WHO Grade 3 tumor and 10 patients with WHO Grade 4 tumors (= glioblastoma)) or brain metastases (MET; 27 patients, with 15 lung, 1 breast, 9 melanoma and 2 kidney as primary sites of origin). Initially 64 consecutive patient studies acquired between august 2018 and april 2020 at a single tertiary institution were reviewed. Then the following exclusion criteria were applied: < 18 years, lack of histological analysis of neoplasm (6 patients), unavailable MRI data (1 patient), treatment prior to MRI, lesions with a diameter of under 10 mm 16 (3 patients), failure to compute APTw signal intensities during image reconstruction (6 patients). In the case of multiple metastases all lesions were used for analysis (if they fulfilled size requirements) and were pooled 17 thus resulting in one data point per patient. A flowchart can be found in the supplementary material. MR imaging. Subjects were examined on a clinical 3 T scanner (Achieva, Philips Healthcare, Best, the Netherlands) with an eight channel receive-only head coil array. The following sequences were acquired: precontrast transverse diffusion-weighted imaging (DWI), 3D double inversion recovery (DIR), 3D T1-weighted (T1w) turbo field echo (TFE) and postcontrast 3D fluid attenuated inversion recovery (FLAIR), 3D T1 black blood turbo spin echo (TSE) or 3D T1w m-Dixon TFE, APTw and 2D T2w TSE. [fig_ref] Table 1: APTw MRI sequence parameters [/fig_ref] shows the sequence parameters for the APTw sequence. We used a clinically approved APTw sequence 18-20 acquired in transverse oblique orientation parallel to the intercommissural line. 16 slices with a slice thickness of 3.85 mm were acquired. The first slice was centered at the inferior border connecting the rostrum and the splenium of the corpus callosum [bib_ref] Amide proton transfer contrast distribution in different brain regions in young healthy..., Sartoretti [/bib_ref] [bib_ref] Amide proton transfer weighted imaging shows differences in multiple sclerosis lesions and..., Sartoretti [/bib_ref]. Details on how APTw imaging contrast was generated can be found in the supplementary material. ## Radiomics feature extraction and image analysis. aptw and postcontrast t1w or flair digital Imaging and Communications in Medicine (DICOM) files were loaded into the open-source software platform 3D Slicer (v. 4.10.2) and were aligned geometrically. Subsequently, two readers (TS and ES each with 3 years of experience) manually segmented the neoplasms on overlayed images according to contrast enhancement and solid parts of neoplasms [bib_ref] Differentiating brain metastases from different pathological types of lung cancers using texture..., Li [/bib_ref]. Segmentation was performed on all axial slices for 3D segmentation of either T1w postcontrast or FLAIR images superficially overlaid onto the APTw images [fig_ref] Figure 1: Representative image examples of 4 patients who presented with different neoplasms [/fig_ref]. Specifically, readers outlined neoplasms on pseudo-images that were generated by accurately overlaying APTw with the now geometrical identical structural images. These pseudo-images contain the APTw imaging information but visually reflect www.nature.com/scientificreports/ T1w postcontrast or FLAIR images. Intensity discretization was performed to a bin width of 25. Gray level cooccurrence matrix (GLCM) features were computed at 4 inter-pixel distances. Then 110 radiomics features were extracted with the built-in pyRadiomics package implemented into 3D Slicer 22 . Most features are in accordance with those described in the Imaging Biomarker Standardization Initiative (IBSI) [bib_ref] The image biomarker standardization initiative: Standardized quantitative radiomics for high-throughput image-based phenotyping, Zwanenburg [/bib_ref]. Radiomics features corresponded to seven different matrices/feature classes: First-order statistics/ histogram matrix, shape-based features, gray-level cooccurrence matrix (GLCM), gray-level run length matrix (GLRLM), gray-level size zone matrix (GLSZM), neighbouring gray tone difference matrix (NGTDM), and gray-level dependence matrix (GLDM). A detailed overview and description of radiomics features can be found elsewhere 22,24 . Radiomics features-dimension reduction. Dimension reduction was performed in two steps. First, radiomics features of both readers were compared by means of intraclass correlation coefficients (ICC). ICC values of greater than 0.8 were interpreted as excellent agreement [bib_ref] Subacute and chronic left ventricular myocardial scar: accuracy of texture analysis on..., Baessler [/bib_ref] [bib_ref] The measurement of observer agreement for categorical data, Landis [/bib_ref]. Radiomics features with ICC values below this threshold were discarded from further analysis, as shown previously [bib_ref] Correlation of texture analysis of paraspinal musculature on MRI with different clinical..., Mannil [/bib_ref]. In a second step, a classifier attribute evaluation filter (CfsSubsetEval) of the open source software package Weka (WEKA, version 3.8.3, University of Waikato, Hamilton, New Zealand) were applied on the training data to evaluate the worth of an attribute. This method measures the significance of attributes on the basis of predictive ability of attributes and its degree of redundancy. The subsets which are having less intercorrelation but are highly correlated to the target class are selected for further analyses. The remaining radiomics features were then used to train ML classifiers. Combinations of the weighted radiomics features were used then to distinguish metastases from glial primary brain tumours. For the consecutive subanalyses of distinguishing glioblastomas from other glial brain tumors and metastases, we performed a prior principal component analysis to cover approximately 95% of variance in the original dataset. Machine learning. For ML analysis, open-source software (WEKA, version 3.8.3, University of Waikato, Hamilton, New Zealand) was used. For prediction of histopathology, a commonly-used ML algorithm implemented in the open source WEKA package was tested with handpicked hyperparameters: Multilayer perceptron, which uses backpropagation to learn a multi-layer perceptron to classify instances with a learning rate of 0.3 and a momentum of 0.2. For further subanalyses we used a random forest classifier. All results were tenfold cross validated to overcome overfitting. # Results Dimension reduction. After dimension reduction regarding reproducibility and attribute evaluation, eight out of 110 radiomics features remained for further analysis [fig_ref] Table 2: Radiomics features after dimension reduction [/fig_ref]. # Subanalysis ii. In the subanalysis of primary brain tumors combined with metastases [fig_ref] Figure 3: Cell plot of eight standardized Radiomics features visualize similarities between glioblastomas, other... [/fig_ref] , the random forest classifier was able to distinguish these entities with an average sensitivity of 62.5%, a specificity of 74.9%, a recall of 0.625, F-measure 0.628, and an area under the curve in receiver operating characteristics of 0.797 after stratified tenfold cross validation. # Discussion In this proof-of-concept study we assessed the utility of radiomics for APTw imaging. To this end, we used radiomics features and machine learning algorithms to differentiate glioblastomas from gliomas and brain metastases. Our results indicate that the application of radiomics to APTw imaging is feasible and allows for the differentiation of these brain neoplasms. APTw is a novel molecular MRI technique that relies on endogenous cellular proteins in vivo to generate contrast. APTw imaging belongs to the chemical exchange saturation transfer (CEST) imaging group and is the only form of CEST imaging that has yet achieved FDA approval. The APTw signal is theoretically caused by two major sources: Firstly, the intracellular water-exchangeable amide proton content in the cytoplasm and secondly the base-catalyzed exchange rate at physiological pH range [bib_ref] Improving the detection sensitivity of pH-weighted amide proton transfer MRI in acute..., Heo [/bib_ref]. Incidentally however, the APTw signal is not pure and may be contaminated by a variety of sources [bib_ref] Amide proton transfer CEST of the cervical spinal cord in multiple sclerosis..., By [/bib_ref] [bib_ref] Reduction of transmitter B1 inhomogeneity with transmit SENSE slice-select pulses, Zhang [/bib_ref] [bib_ref] Correction for artifacts induced by B(0) and B(1) field inhomogeneities in pH-sensitive..., Sun [/bib_ref] [bib_ref] Towards the complex dependence of MTR, Zu [/bib_ref] [bib_ref] Accuracy in the quantification of chemical exchange saturation transfer (CEST) and relayed..., Zhang [/bib_ref] [bib_ref] Insight into the quantitative metrics of chemical exchange saturation transfer (CEST) imaging, Heo [/bib_ref]. Specifically, the water longitudinal relaxation time (T1) may influence the APTw signal. T1 effects (T1 recovery and T1 related saturation) may influence the APTw signal linearly or in a more complex manner depending on the level of direct water saturation effects, the field strengths of the MR scanner, irradiation power and whether non-steady-state or steady-state acquisitions are performed. Furthermore. The APTw signal may also be affected by semi-solid magnetization-transfer (MT) effects and other nearby CEST and relayed nuclear Overhauser enhancement (rNOE) saturation transfer effects. Ultimately, APTw intensity values may also be impacted by B1 effects which can be triggered by an imperfect distribution of the irradiation power across the brain. Currently, APTw is mainly used for brain tumor imaging. With malignant brain tumors exhibiting a high degree of protein content, the APTw signal increases steadily with the amount of protein content relative to the surrounding parenchyma. This has been successfully leveraged for differentiating and grading tumors according [bib_ref] APT-weighted MRI: Techniques, current neuro applications, and challenging issues, Zhou [/bib_ref]. Specifically, a recent meta-analysis listed the sensitivity and specificity of APTw for differentiating high grade from low-grade glial tumors as 88% and 91% respectively [bib_ref] Amide proton transfer-weighted MRI in distinguishing high-and low-grade gliomas: A systematic review..., Suh [/bib_ref]. Furthermore, based on this principle, high grade regions can be identified within histologically heterogenous brain tumors, thus allowing for more accurate sampling during stereotactic biopsies. Additionally, APTw has also been successfully employed for monitoring tumor response to therapies such as radio-/chemotherapy or high-intensity focused ultrasound. Most importantly, APTw enables the differentiation of recurrent tumor from treatment effects such as radiation necrosis. Lastly, APTw has also been successfully used for identifying genetic markers in gliomas, such as the MGMT or IDH status [bib_ref] APT-weighted MRI: Techniques, current neuro applications, and challenging issues, Zhou [/bib_ref]. While APTw has already proven to be a valuable addition to the field of tumor imaging, the development of innovative approaches to further leverage the potential of APTw is highly desirable. Radiomics has become a popular method for extracting more data from radiological images thus enabling in-depth study of the tissue at www.nature.com/scientificreports/ hand [bib_ref] Radiomics: Images are more than pictures, they are data, Gillies [/bib_ref] [bib_ref] Prognostic factors of disease recurrence in breast cancer using quantitative and qualitative..., Lee [/bib_ref]. Therein numerous studies have shown that radiomics approaches based on various imaging modalities (i.e. T1w, T2w, FLAIR, DWI, ADC, SWI, DTI) enhance brain tumor imaging [bib_ref] Radiomics: Images are more than pictures, they are data, Gillies [/bib_ref] [bib_ref] Classifying brain metastases by their primary site of origin using a radiomics..., Ortiz-Ramón [/bib_ref] [bib_ref] Differentiating brain metastases from different pathological types of lung cancers using texture..., Li [/bib_ref]. Here we further enhance the spectrum of radiomics in terms of 3D texture analysis (TA) for brain tumor imaging by applying it to APTw imaging. Our approach yielded a high degree of accuracy in differentiating different types of brain tumors. After dimension reduction all first order TA features were excluded from further analyses due to high intercorrelation. A total of eight features remained for further analysis: (1) Dependence Non-Uniformity Normalized (DNN), which measures the similarity of voxel dependence throughout the image, an indirect measure of homogeneity. (2) LargeDependenceHighGrayLevelEmphasis (LDH-GLE), which measures the joint distribution of large dependence of higher gray-level values. (3) Low Gray Level Emphasis (LGLE), which measures the distribution of low gray-level values, and (4) SumAverage, which measures the relationship between occurences of pairs with lower intensity values and occurences of pairs with higher intensity values. All four aforementioned features are derived from the GrayLevelDependenceMatrix (GLDM), which are used to quantify gray level dependencies in a medical image by taking into account neighbouring voxels. The remaining features were associated with the size of the neoplasms. While VoxelVolume and Mesh-Volume are directly correlated with size, ZoneVariance measures the variance in zone size volumes. Similarly, ZonePercentage takes into account the number of zones and number of voxels within a defined region of interest, which represents the coarseness of texture. While gliomas were found to exhibit a larger overall size than metastases, which was reflected in these features, they may have no true biological value as metastases and gliomas can present with various sizes. A major limitation of the current study is its retrospective, single centre design and its small sample size with heterogeneous spectrum of pathologies. It should however be noted, that due to the novelty of the imaging modality (i.e. clinical approval of the sequence occurred in 2018) a larger dataset is not available at the time. Furthermore, larger datasets are likely to decrease the risk of overfitting the machine learning classifiers. We counteracted these limitations by implementing tenfold cross validation of our results. Moreover, the quality of segmentations may strongly influence radiomics results [bib_ref] Experimental texture analysis in glioblastoma: A methodological study, Hainc [/bib_ref]. Two readers segmented the neoplasms and subsequently features with low interreader agreement were excluded thus reducing bias. However, we acknowledge that there may be more sophisticated methods of performing segmentations and of reducing reader bias. Lastly, our study was performed on a single MR scanner, which limits the generalizability of our findings. The influence of different acquisition parameters, sequences, field strengths, and MR scanners on 3D TA features in APTw images remains to be investigated. In conclusion, we show that radiomics allows for the differentiation of various brain neoplasms on APTw images. The current work justifies the further study and development of radiomics for APTw imaging in an effort to widen the applicability and utility of APTw imaging for various diseases and anatomies. ## Data availability Data can be made available upon reasonable request to the corresponding author. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. [fig] Figure 1: Representative image examples of 4 patients who presented with different neoplasms. FLAIR or T1w postcontrast images are provided in the top row, while the corresponding APTw images are shown in the bottom row. Machine learning. The Multilayer Perceptron classifier yielded a sensitivity of 81.3%, a specificity of 81.1%, a recall of 0.81, F-measure 0.81, and an area under the curve (AUC) in receiver operating characteristics (ROC) of 0.836 (Fig. 2) in distinguishing primary brain tumors (glial tumors and glioblastomas) from metastases. Subanalysis I. In the subanalysis of primary brain tumors, the random forest classifier was able to distinguish glioblastomas from other glial cell tumors (WHO I-III) with a sensitivity of 90.5%, a specificity of 90.4%, a recall of 0.905, F-measure 0.905, and an area under the curve in receiver operating characteristics of 0.868. [/fig] [fig] Figure 3: Cell plot of eight standardized Radiomics features visualize similarities between glioblastomas, other gliomas and metastases. Stark differences can be observed in the values of glioblastomas and metastases. [/fig] [fig] Figure 4: Relative visualization of the eight standardized texture analysis features for glioblastomas, other gliomas and metastases. Overall lower values are observed for metastases compared to glioblastomas and other gliomas, respectively. [/fig] [table] Table 1: APTw MRI sequence parameters. [/table] [table] Table 2: Radiomics features after dimension reduction.Figure 2. Receiver-operating-characteristics (ROC) of the machine learning algorithm to correctly identify primary brain tumors and metastases. The overall area-under-the-curve (AUC) was 0.836. [/table]

First Report of the Commissioners Appointed to Inquire Whether Any and What Special Means May Be Requisite for the Improvement of the Health of the Metropolis; with Minutes of Evidence ## G8 On the Causes and Diffusion of Cholera. [July, were not attacked in undue proportion; corps liaving free intercourse with infected districts wholly escaped; some portions of the principal town also escaped, although not isolated from the affected parts. In addition, it is mentioned that the inhabitants of villages, when attacked, universally left their homes and travelled into the jungle, with the result of stopping the disease ; although it is argued that, under the privations and exposure of such a flight, the villagers must have been more predisposed to the attacks of the disease, and the poison of the disease must have been heightened in its contagious property, had it really possessed it. But the most striking instance is recorded in the Report, by Mr. Tliom, of the epidemic which attacked the 86th regiment at Kurrachee, in Scinde, in 1846.* The fearful mortality of this epidemic, which destroyed in a few days 700 fighting men and several thousand civilians, has made a profound impression on the profession and on the public. In our first Number we quoted from Dr. Milroy an account of the attack, and to this account we may refer our readers. It appears, however, that there has been some misconception, which ought to be corrected. It has been stated, that the epidemic which visited Kurrachee was an isolated attack, occurring altogether separately from any general epidemic affecting the neighbouring districts. This is a mistake. Long before the outbreak in Scinde, cholera had been traversing in various directions Affghanistan, Ilindostan, and the outlying countries. It had been spreading gradually from Madras towards Bombay and the coast of Malabar. "Long before its visit to Kurrachee," writes Mr. Thom, "we heard of its appearance in the Madras Presidency, and subsequently at Poonah, Bombay, and Alnnedabad, Deesa, and finally arriving by the coastward to Scinde, where it' first broke out at the sea-coast, and from thence gradually extended upwards to Hydrabad and Sehwan, where its further progress appears to have been stopped, as it never reached Sukkur or Upper Scinde." (Report, p. 8.) The presumed isolated and sudden attack at Kurrachee, was not merely evidently a part of the general epidemic, but cases of undoubted cholera had occurred at intervals for some months before, proving the gradual development of the epidemic constitution. It has also been said, on the authority of a Bombay paper, that the attack commenced at a particular hour, after the appearance of a black cloud and a tremendous thunderstorm. It now appears, however, that, not only had the disease prevailed for a fortnight in the native town, but that, for two or three days previously, cases had been coming into the 86th hospital, and that more than twenty cases had been admitted before the storm. " An absurd cause," says Mr. Thom, " passed current in camp, and has been reechoed by the Bombay papers; viz. that the disease broke out after c a black cloud' and dust-storm, which took place at five o'clock p. m. on the 14th; while, in reality, twenty cases had been admitted, and arrangements had actually been made to move our regiment, before this not uncommon phenomenon terrified the crowd. As it was next morning before the awful condition of our corps was generally known in camp, a few futile imaginations attributed the disease to what they had seen, and others accepted this, because it was full of mystery." Mr. Thorn gives the following account of the outbreak of the disease, which, excepting in its terrible rapidity, coincides with the general mode of attack : "It was equally apparent," lie says, " that there was nothing contagious in the nature of the disease; for, instead of a few cases appearing first, and then the disease gradually spreading, it suddenly burst forth in a few hours in every European regiment, whether in camp or barracks, in every tent, and in every house; and it was at its acme in forty-eight hours after; when, instead of spreading further, it gradually and steadily declined. Now, it appears that, for some days, or even weeks, a few cases had appeared in the native town of Kurrachee, but there, also, at the same period, the malady became suddenly general over the whole place." (Report, p. lo.) If its outbreak was thus opposed to the idea of contagion, so also was its future progress. The native town of Kurrachee, where the disease first appeared, is about a mile from the cantonment. In June, 1846, two native regiments were quartered in barracks at the point of the cantonment nearest to the town; some way further removed from the town were the officers' quarters ; still further off were the barracks of H. M. 60th ; and to leeward of all, most remote from the town, and half a mile from the 60th barracks, were II. M. 86th regiment and the Bombay Fusiliers, encamped on a low plain. For about a fortnight, cholera was prevailing to some extent in Kurrachee itself, and between this place and the nearest point of the cantonment, occupied by the native regiments, there was daily and hourly communication. But when the attack commenced in the cantonment, it did not commence in these Sepoy lines, as must have been the case had it been propagated by contagion, but it passed over all, and "burst forth with unparalleled fury in the 86th regiment, quartered at the most leeward and remote point. It then turned back in the teeth of the wind, and broke out in the 60th barracks as suddenly as in camp ; and a few days later it recrossed the officers' lines, and appeared among the native troops nearest to the town. These facts," says Mr. Thom, On the Causes and Diffusion of Cholera. [July, Mr. Thorn also states, that "medical officers and attendants did not suffer more from the disease than others, although fatigue caused a greater numerical proportion to fall sick with diarrhoea."* It appears almost tiresome to multiply instances in which an attack of cholera could not have arisen from the ordinary operation of a contagious virus; but we will cite one more, which seems to us peculiarly strong. In 1842, II. M. 9tli Lancers proceeded up the Ganges in two divisions, en route to Cawnpore. The regiment had just arrived from England, and the men were in rude health. The left wing marched on the 22d September ; an unfavorable time for a voyage up the Ganges, on account of the rapid subsidence of the inundations, after the heavy rains of the monsoon. As they proceeded, a few cases of fever and of dysentery occurred, and on the 25th of October they had the first case of cholera. Admissions from cholera soon became general, while the fever cases disappeared. The cholera was of the worst kind, with few spasms, and no great amount of vomiting and purging. It reached its height in about four days. It was, at the same time, frightfully prevalent in the villages on the banks of the Ganges ; and, on arriving at Monghyr, they found the bazaars deserted, or depopulated by the disease, which had raged for some months. The mortality on board the squadron of boats was great, and the medical officers recommended the speediest possible transit through the infected districts. Accordingly, every exertion was made to pass rapidly up the river, and in about twelve days the disease began to decline in severity. This change was coincident with their arrival in a country not suffering from cholera in so great a degree. A few days later they emerged altogether from the affected district, and at the same time the cholera completely left them. There was no evidence of contagion ; the boats, 30 or 40 in number, were equally attacked, and the sick were necessarily mixed up with the other patients on board the hospital boat, yet the disease did not spread to these latter, or to the attendants. But the important point is, that about a month after this, the right wing and head-quarters left Calcutta, and proceeded in the same way up the Ganges. The voyage was an exact counterpart of that of the other wing?they were attached by cholera at the same point, pushed rapidly on, and lost it where the first division lost it. The severity of the disease, the mortality, &c., were exactly the same. The disease still continued to prevail in the villages on the banks.f We shall not occupy more space by details on this subject,?our readers may possibly think we have already been too prolix. Let us simply repeat our conclusions, that cholera does not ordinarily spread by contagion, that is, by generation of its poison by the human body only ; that it recognises other laws and very different modes of increase; and, as is proved both by theory and practice, that it cannot be arrested and bound in by quarantines and cordons. We have already said, however, that it is a question of a different kind, * Ibid., page 375. The proportion of deaths among the " medical subordinates" of the 86th Regt. was only 4 out of 40, or at the rale of 100 to 1000 of strength. ## 1848.] On the Causes and Diffusion of Cholera. whether the poison of cholera can ever be given off from an infected person .?there is no reason why it should not; there is some real evidence to prove that it sometimes is. But then this mode of increase is certainly very rare; many excellent observers have never met with a case, although intimately acquainted with the disease; and many of the recorded cases are, we are prepared to show, quite inadequate to warrant the conclusion drawn from them. It is an undecided point, on which no man is warranted in giving at present a positive opinion. Fortunately, however, it is a point of little practical value ; for of what consequence is it, if, once in a thousand times, or, it may be, in fifty thousand times, reproduction of the poison occurs in this way? No art can guard against it; no precautions avert it. And it would be the extreme of absurdity to look at cholera only from this narrow point, and to legislate for one case rather than for nine hundred and ninety-nine. It is a scientific curiosity ; not a thing for statesmen and legislators. And this appears to be the view taken of it by the Sanitary Commissioners. They seem to have considered that their Report, intended to be the basis of preventive measures, should contain only recommendations which were practicable, and principles which could be used ; they did not wish to theorise till all became uncertain, and to refine till limitations and distinctions were blended into obscurity by qualifications and provisos. They offer to the Government a certain and well-ascertained fact; the usual mode, say they, in which the cholera spreads, is not from man to man ; and, consequently, against this disease other appliances and other precautions are to be used, than measures which are applied only to one multiplying source, and that the most uncommon, viz. the human body. At the same time, although it is advisable not to discuss with a nonprofessional audience scientific points of such extreme difficulty as this, viz. whether a poison which ordinarily spreads without acting on the body, does in rare instances multiply itself in this way,?still, it must not be supposed that we underrate the importance of this inquiry. It is, both as regards cholera and as regards other morbid poisons, particularly those of plague and yellow fever, one of extreme interest. Well-weighed and well-authenticated facts are necessary before it can be affirmed or disproved,?at present, the weight of the argument is on the affirmative side. There is another point in the propagation of cholera which requires deeper examination. There can be no doubt that cholera often spreads, if it finds its conditions of increase, over a disti'ict which may be nearly uninhabited, and that it thus arrives at one place from another, without any intercourse whatever having taken place. But, in some cases, the poison has certainly appeared to be transported by a body of men?it lias become "portable," to adopt a term which has been used, Ave observe, by Dr. Watson. As a good example, we would quote the fact of regiments in India, which have been attacked at a certain place, continuing to be attacked during their march through an unaffected district,* and, at * Orton (a contagionist) writes ; ? In many cases it is distinctly proved, that the towns and villages through which the infected corps passed were quite free from the epidemic at the time?the diseased body passed on in a state of most intense suffering, bearing with them the seeds of disease and death, while the peaceful inhabitants of the country were enjoying perfect health." (On Cholera, 2d edition, p. 31)5.) It must be remembered, also, that marching heightens immensely the predisposition to the disease. On the Causes and Diffusion of Cholera. [July, a period of time too remote to allow us to attribute it merely to an incubative period, unusually prolonged. These cases are not common, but tliey do occur; and tlie explanation does not seem to us to be very difficult. Wherever the poison of cholera meets with its conditions, it will propagate itself; these conditions seem to be furnished in a high degree by the effluvia and emanations of camps or bodies of men ;* the poison once introduced among such a body, finds in the moving camp the necessary conditions, as easily and as abundantly as in the stationary city. Those who know the multifarious crowd, which, under the name of camp followers, tracks a regiment on the march in India, will not for a moment doubt that such an uncleanly and impure assemblage must inevitably furnish to the poison ample materials for its support. We are willing to admit that there may be an actual transport of poison in this way, and no doubt some of the stories of contagion have thus arisen. But, of course, such a mode of transmission as this, comparatively rare as it is, cannot be confounded with contagion, that is, with the increase of the poison by its action on the body. The two things are quite different; and though it should be hereafter proved that a poison may increase in both ways, they are totally distinct. A custom is common in India, which may at first sight seem to contradict this explanation of the conveyance of cholera. It is not an uncommon thing, when a corps is severely attacked in barracks or camp, to shift ground from day to day, aDd so, as it were, to elude the enemy. This practice has really'been followed by good results ; after a few days' marching and countermarching the disease has disappeared. Now, it might appear strange, that cholera should at one time be got rid of by marching, and that at another time it should be carried for a long distance. But the explanation is very easy : ?when a corps moves out of barracks to avoid cholera, it takes no baggage ; it is in light marching order; it has few camp followers; the marches are short, and do not fatigue the men ; there are plenty of tents, and room to pitch them; there are none of the circumstances attending a regular march, when the distances are great, the halting-grounds perhaps low and damp, and the camp is pitched as close as possible, and is surrounded by an innumei'able crowd of camp followers, who sometimes reach the astonishing number of eight or ten for every fighting man. It may be urged, that, in a case of this kind, when a regiment conveys the poison, and might thus introduce it into a place where it can find its conditions of increase, quarantines and cordons might be advantageously employed. This we are not disposed to contest, although we should be puzzled to find instances proving the benefit of such measures. And, on the other hand, we are overflowed with examples, in which regiments, suffering terribly from cholera, have been permitted to enter a place, and have not communicated the disease to the inhabitants. Thus, in 1843, the right wing of H. M. G3d regiment was attacked with cholera on the march from Madras to Bellary; on arriving within eight miles of the latter place they were forbidden to enter, and therefore encamped outside, butchauged their ground from day to day. The heat was intense, the thermometer stood at 110? to 115? in the tents, and the air was perfectly motionless; ## 1848.] On the Causes and Diffusion of Cholera. in two days the disease liad increased so much, that the surgeon reported, in the strongest terms, that unless the men were put into suitable buildings, the entire wing would be annihilated?accordingly, they were permitted to enter the fort at Bellary ; the men were comfortably accommodated, and, to use the words of the surgeon, "the disease at once assumed a different character." Before marching in, they had lost 7G non-commissioned officers and men out of a strength of 600 ; afterwards there were only 7 more deaths. The disease did not spread in the town or fort, and no ill consequences whatever followed.* This example is only one of many which we might quote; we have taken it because it is very instructive, as it proves how really amenable cholera is to sanitary measures. The disease was raging furiously till the men were placed in proper hygienic conditions; then it was at once arrested. It is a good illustration of what correct principles of prevention will hereafter do, in mitigating the ravages of cholera. As to the employment of quarantines in the case where a large body of men, affected With cholera, approach a place, it must be left to individual discretion. If the place is in good sanitary order, and if the new comers could be comfortably put up in well-ventilated, and not crowded, quarters, they might be at once admitted. If the place is one in which cholera often prevails, quarantines may be advisable, and, at any rate, all measures should be on the side of caution. But, in thus admitting a possible use of quarantines, we are aware that we are rather deducing a rule from uncertain and ill-ascertained premises, than from the examples and records of experience. To judge by these latter only, we should deny all efficacy to quarantines ; when we advocate their use in the case referred to,we go on the uncertain ground of the possible and the contingent. The Swedish Commissioners appear to attach greater importance to this mode of propagation than we do. ## 1848.] On the Causes and Diffusion of Cholera. 75 unnecessary tlius formally to announce them?have been proved by a multitude of observations, both in tliis country and in India. To some of these observations we shall have occasion presently to refer. It must not be supposed, however, that, in addition to the immense influence of impure and humid air, the poison of cholera is not affected by other circumstances, which require much investigation. Thus, there is no doubt that a great influence is exerted upon it by soil;?it often adheres to the soil, and refuses to cross roads and narrow rivers?it has been supposed to avoid volcanic soils more than others?and it is a remarkable fact, that, in 1832, it is said to have made quite the circuit of Auvergne, which is altogether volcanic, but to have never penetrated into it. Sandy soils are also unfavorable to its spread ; and this has been attributed to their absorption of water, and the consequent comparative dryness of the atmosphere. But to the real or supposed influence of these several circumstances, we must return on another occasion; and we shall now proceed to analyse the evidence which the Commissioners bring forward, to prove the influence of impure and contaminated air on the spread of cholera. " The disease wliicli may be taken," write the Commissioners, " as the type of the entire class of epidemic diseases tlmt infest this country, is typhus fever. The habitat of typhus is that of the class; and the conditions which favour the spread of this disease, and which convert it into a pestilence, and those which locate to a great extent in these very places all other pestilences that come, and which give them their fearful fatality, are, as far as we have any knowledge of them, precisely the same. It is now universally known, that in the metropolis, as in every town and city, the places in which typhus is to be found, from which it is rarely if ever absent, and which it occasionally decimates, are the neglected and fdtliy parts of it; the parts unvisited by the scavenger, the parts which are without sewers, or winch, if provided with sewers, are without house drains into them; or which, if they have both sewers and house drains, are without a due and regulated supply of water for washing away their filth, and for the purposes of surface clcansmg and domestic use. The evidence that the track of typhus is everywhere marked by the extent of this domain of filth, lias been so often adduced that it is needless to repeat it; but the evidence that, during the prevalence of cholera, this was also everywhere the precise track of this pcstilence, is not so well known." In the second Report, issued after the late epidemic of influenza, the Commissioners state that not only are the tracks of typhus and cholera identical, but that the same districts were those in which influenza manifested itself most severely. It is necessarily understood that these remarks of the Commissioners are not to be interpreted quite literally. It cannot be meant to be asserted that cholera or influenza only prevails where typhus is; for such an assertion is contradicted by daily experience. There is a great difference in the volatility of these poisons, and in the rapidity of their diffusion; cholera is no more limited in this country to the localities where typhus the two divisions above referred to, the proportion of deaths to attacks was as 1 to 2*1 in the most unhealthy, and as 1 to 1*9 in the most healthy section. On the point the Commissioners remark : "Piffercncc of social grade less exempts the individual from the attacks of cholera than of fever; and cholera more often, and apparently more capriciously, bursts its usual boundaries, and attacks the inhabitants of comparatively healthier districts, amongst whom it then proves little less mortal than when it ravages its accustomed haunts. If, as is justly remarked by the Registrar-General, in the present social condition of the civilized world, the vast populations of different and distinct nations arc intimately united,?if it be true, that, were the health of India sound, Europe might be safe, and hear no more of the epidcmic which is now traversing Russia,?if the lives of thousands depend on the condition of the Pariahs of Jess ore,* much more in one and the same town and city, must the health of the wealthiest portion of the inhabitants depend on the salubrity of the poorest."f (First Report, p. 28.) The districts we have cited as being those chiefly affected both by typhus and cholera, are, it is needless to say, disgracefully distinguished by a total disregard of sanitary regulations. Thus, the streets of St. Olave and St. Thomas, a district which stands first on the list of unhealthy places in the table referred to, are stated by the medical officer, "to be a disgrace to the civilized world." In Whiteeliapel, we are informed, " that the great majority of its confined and crowded streets, courts, and alleys remain without air, water, or any arrangements for the removal of its accumulated filth." (Second Report, p. 28.) The whole district of Bermondsey is reported "to be intersected by open ditches of the most offensive character, and abounding in several parts with 'fever nests.' " (Ibid., p. 28.) In Lambeth, where cholera also prevailed severely, it is stated that "there are numerous open ditches of the most horrible description; and, in general, the cleansing, paving, and water supply, and consequently the internal cleanliness, is either totally wanting or grievously defective." We are extremely concerned to find how little has been done in the shape of sanitary improvement in these parishes since 1832. In Lambeth, Bermondsey, and Whiteeliapel, hardly anything has been amended :? Wandsworth and numerous other places remain without a single sewer. The Sanitary Commissioners remark that,? Throughout England generally, the same influences of impurity and humidity of the air produced the same result. Thus, in Manchester and in the Lancashire towns generally, it is stated, in the evidence by Mr. We may here correct a slight error. Cholera appeared In several places, nearly at the same time, in the provinces of Dhacca, Sylhet, Chittagong, and Behar. It was months before it reached Jessore; but from this place the first official report was made. + Dr. Stan's pamphlet, which is well and ably written, appears to be chiefly intended to show the necessity of sanitary regulations. It is written as much for the public as for the profession, and gives a good and sensible view of this part of the question. [July, Roberton, to have manifested itself cliiefly along water-courses, including docks, wharfs, districts occasionally flooded, &c., and with peculiar violence at the outlet of drains.* In Sunderland, Leeds, Liverpool, Musselburgh, Edinburgh, Easter-Ross, and other places, the same fact was universally true?the greatest ravages of cholera occurred in places notoriously in a bad sanitary condition. On the continent, the same law holds equally true. Perhaps in no town in Europe was cholera so fatal as in Paris, and perhaps in 1832 there was no town of so lai'ge a size and with such pretensions to civilization and refinement, which was so celebrated for its effluvia and its emanations. The internal arrangements of every house were of the most miserable kind ; each house was dirty, ill ventilated, ill supplied with water, and wretchedly accommodated with privies and sewers. The single house was the representative of the city; the houses collectively repeated, on a gigantic scale, the nuisances of the separate habitation. In 1832 cholera commenced in the "Quartiev de la Mortellerie," and in other places similarly badly situated and peopled by a miserable population. The mortality greatly preponderated in the low unchained localities. We had believed that Paris had greatly improved in point of cleanliness since 1832; but M. Bureaud-Riofrcy thinks otherwise. "It would seem, at first sight," says lie, " that one need little fear epidemics in our grand cities. At Paris, we admire the houses so splendidly built, the apartments so luxuriously furnished; we admire the richness of the furniture, the gilding, the glasses, the mirrors, the bronzes, the statues, the vases, and most recherche woods; everything would seem to exclude the idea of insalubrity. Yet, in spite of this brilliant exterior, Paris is perhaps the most unhealthy of capitals.?Paris wants air, water, cleanliness, above all, privies and sewers. It wants air in this sense, that almost all the houses are narrow, and the ceilings arc low; in a Parisian apartment, we feel imprisoned. It wants water, because this is sold at an extravagant price; water is six times as dear in Paris as in London, where everything is so dear. Paris wants cleanliness, for hygienic measures are incomplete or powerless. Each house, and in every house, each flight, are the ' foyers' of vapours more or less fetid. The remains of food, the water of washings corrupted and surcharged with decomposing and fermenting organic matters, the emanations of workshops, the residue of manufactures, the accumulation of all cxcretions cast into the street., and turned and returned a hundred times daily by the seekers for rags ; the dung deposited in heaps by the wayside, and diluted by the urine of a population little accustomed to restrain itself; this accumulation of ordure or of excretions in fermentation alters, in a very great degree, the purity of the air of this capital, which believes and proclaims itself to be at the head of the civilization of the world." If this be so, and if, as there seems every reason to believe, a period of great social distress is impending over France, it is as well for Paris that the cholera has halted in its march, else the second visitation might be more fatal even than the first. The beneficial result of good sanitary measures has been very apparent at Moscow during the present epidemic. In 1830, the disease extended to every part of the town; in 18-17, it was confined chiefly to the districts south of the river, while in the other districts the cases have been few and scattered. The Russian physicians attribute this to the fact, that the population south of the river consists of the poorer * First Report, p. 3. ## 1848.] On the Causes ancl Diffusion of Cholera. classes, wlio subsist on miserable or raw kinds of food, and have only the impure river water to drink ; while on the north side, the inhabitants are not only of the better class, but have most excellent drinking water, which is brought from springs 18 wersts from the city, by waterworks which have been erected since 1830. In Gallicia, the disease has spared the German villages, which are distinguished for their cleanliness,?while in other places it has been very fatal. It is reported that the colony of Sarepta, noted for the cleanliness of its inhabitants, and for its salt springs, has escaped in 184/ as it did in 1830. If we pursue the same course of inquiry into Asia, it is found that, with certain modifications, the same rule may be laid down, and that the greatest ravages of cholera occur in places notoriously deficient in hygienic precautions. But it is at once evident that the rule must be qualified to a certain extent, and that the habitat of cholera cannot be so clearly foretold in India as in England. It is, we think, certainly established, that humid and contaminated air is the medium in which the poison chiefly propagates. But then it is evident that the contamination may be of different kinds. In this country the impurities are chiefly connected with our towns, and consist of the effluvia which stream up from dense masses of people paying little attention to cleanliness and to the removal of decaying substances. In India there is not only this source of impurity ; there are also various exhalations from the soil. From jungle and marsh lands, from alluvial and cotton soil, from rice and indigo fields, from the muddy and slimy banks of the broad oriental streams, are poured into the air the various miasmata, which, in a greater or less degree, can cause the increase, as under extraordinary atmospheric conditions, they can perhaps cause the development, of the poison. In that country the necessary humidity of the air is abundantly supplied by the monsoons; and the burning sun, it may well be supposed, volatilizes into greater subtlety the gases of decaying organization. We expect then beforehand to discover a greater prevalence of the disease, and a more uniform diffusion. No town, hardly a village, situated in the lowlands, can be supposed to be so perfect in its sanitary conditions, as to be equally free from cholera with a town moderately well drained in temperate Europe, And yet, there is here also abundant evidence to prove, that a bad or good sanitary condition produces a corresponding result of greater or less prevalence of the disease. This point was most clearly recognised by one of the best and earliest writers on Indian cholera, Mr. Jameson, the Editor of the Bengal Report. He was struck with the circumstance, that in 1817, the town of Muttra, situated forty miles higher up the Ganges, and more remote from the approaching pestilence, than Agra, was yet attacked first, and suffered more severely, both as to severity of symptoms and to actual mortality. Mr. Jameson remarks that, "Muttra is a filthy and crowded town, Agra, a dry and airy town." After his attention had been once drawn to this circumstance, Mr. Jameson took every opportunity of collecting information on this point. He found th^t in Sylhet the influence of situation was extremely well marked, and that the natives had universally recognised the fact, that the villages most unhealthy and exposed to the malaria from marshes and lakes suffered most. The same circumstances were found to be attended by the same results throughout [July, the whole of India; and a great deal of valuable evidence on this point is contained in the works both of Jameson and Orton. We cannot afford space at present to enter into the evidence on this point, but we believe few people would credit the influence which in India these circumstances, which are to a great extent easily removable, have upon cholera. Hereafter we shall return to it; at present we must content ourselves with continuing the analysis of Mr. Thorn's Report, and try to discover the causes of the terrible attack at Kurrachee, in Scinde, in 1846. The climate of Kurrachee has only been known for a few years, but in that time every European regiment that lias been stationed here, has been more than once decimated by cholera, often within a few days." (Ileport, p. 10.) What, then, are the causes of this great insalubrity ? Do we find here the same influences of humid and impure air, which in Europe and in other parts of India play so prominent a part in the diffusion of cholera? Apparently we find them in a high degree ; the climate of Lower Scinde is remarkable for its great humidity. " It is curious," says Mr. Thom, " that rain hardly ever falls at this place, yet the air is for six or eight months very humid, and contains more aqueous vapour suspended in a cubic foot than that of Sierra Leone or the Mauritius, an island surrounded by a hot ocean, and subject to heavy rains. The evil of the climate, in ray opinion is, because the atmosphere will not deposit its load of vapour in the form of rain, to cool the surface of the earth, and to lower the dew-point." (Report, p. 11.) In addition to this great humidity, the thermometer in June and July stands as high as 88? to 94? Fahrenheit, at 3 p.m., and has a daily range of not more than 7?. Immediately before the outbreak in June 184G, these characteristic features of the climate existed in an intense degree. The thermometer was unusually high, being 90? to 92? in good houses, and in the tents of our soldiers it rose to 90?, 98?, and 104?. Secondly, the quantity of moisture in the atmosphere was greater than I ever saw it before in any part of the world, or at any season, the dew-point being at 83?, and the thermometer in the shade at 90?, the lowest range ; even tins gives 12'19 grains of vapour in each cubic foot of air. The mean heat in the 24 hours was such as to suspend an unusually large proportion of vapour in the air, always near, but rarely or ever reaching, the point of deposition. Even at the equator, with the sun overhead, I never saw the point of deposition above 78?." The winds were westerly or from the sea, and were unusually light, instead of being, as in the two previous years, strong, steady, and cool. With regard to impurity of the atmosphere, the country about Kurrachee is dry and bare, almost entirely destitute of vegetation, marshes, or jungle, with the exception of a few stunted Euphorbia? thinly scattered over the desert; and the town is about sixty miles from the Indus, and therefore probably removed from any influence in that direction. So far, we do not see much evidence of decaying organization ; but in the town itself, and in the cantonment, are found abundant so\irces of effluvia.* * It is necessary to state here, that Mr. Thom does not adopt the hypothesis of a specific agent for cholera, but believes that the atmospheric conditions of intense heat and humidity produce the disease. We shall hereafter discuss this point; at present we describe the conditions which Mr. Thom would consider as exciting causes, as furnishing a part only of the conditions of the atmosphere necessary for the diffusion of the poison. ## 1848.] On the Causes and Diffusion of Cholera. ## 81 The streets are narrow, crooked, without free current of air, and are very filthy ; and a dirty bazaar is situated a few hundred yards to windward of the barracks. As Mr. Thom remarks, these circumstances were not the causes of cholera, as the disease had disappeared before they were remedied ; but they must be allowed to have had great influence in favouring the necessary condition of the atmosphere. Such, in June 1846, was the condition of Kurrachee: a still, hot atmosphere, overloaded with moisture; a dirty town and cantonment, the emanations from which were not precipitated by showers of rain. Slowly, in its mysterious course, the poison was spreading itself towards it, across the peninsula from the Madras Presidency, which it had ravaged the year before. How was the English force prepared to receive it ? In what state, as to health and condition, were the men ? In what state, as to sanitary arrangement, was their cantonment ? As to this latter point, the arrangement was certainly very bad. The barracks were built in rows ; the canteen, schoolroom, and. other high buildings, were in front of all, and nearest the sea ; then the rooms for each company were ranged behind them ; by this arrangement, the sea breeze was totally arrested by the front line, or if it travelled interruptedly and in languid puffs to the second, it never reached the third. ? Ventilation in the barrack-rooms was not apparently conducted on sound principles, and the soldiers were accustomed, at the time of the attack, to close both the doors and windows at night.* But if the barracks were badly arranged in 1846, the tents in which the 86th regiment and Bombay Fusiliers were quartered, appear, from one or two expressions of Mr. Thorn's, to have been still more badly placed, and still more crowded. But on these points, as Mr. Thorn's hypothesis leads him to attach little importance to hygienic conditions, he is not so explicit as we could wish. We must here give an account of the force at the time stationed at Kurrachee. (a) There were stationed in barracks, On the Causes and Diffusion of Cholera. [July, 3. lGth Bclooch Battalion?820 strong. Tliey lost 93 men, or 113*4 per 1000 of strength. Or to take the different items together. 1. Those quartered in barracks were 1739 strong. They lost 121 men, or at the rate of 69'10 per 1000 of strength. 2. Those quartered in tents were 1855 strong. They lost 321 men, or at the rate of 172'8i per 1000 of strength. 3. Those quartered in mat huts were 2796 strong. They lost 195 men, or 69'20 per 1000 of strength.* It thus appears that the two classes in barracks and mat houses had about an equal mortality; while in the regiments in tents, the mortality was considerably more than doubled. And liere we may inquire, in the first place, to what point the mortality of the whole force could have been reduced, with men in good bodily condition and under perfect sanitary arrangements. We believe that, under such circumstances, it would have been reduced to almost nothing. For example, the fearful loss of 637 soldiers out of 6380, or nearly 100 per 1000 of strength, strikes one with astonishment, when we compare it with the officers' loss. There were 200 officers, and only 3 deaths,f or at the rate of 15 per 1000 of strength; so that if the 86tli regiment had suffered only an equal loss with the officers, there would have been 16 deaths instead of 238. In the same way, the soldiers' wives suffered a loss of 23 out of 159, or 144-6 per 1000 of strength; while out of 42 ladies there was not a single case of cholera. How far, then, would it be practicable to reduce the mortality to the point named ? To answer this question, we must inquire into the causes which, in India in general, and in this attack at Kurrachee in particular, have caused such a disproportionate mortality among the men as compared with the officers. In the tables above given, we included Europeans and Asiatics, but, for the purpose of simplifying the inquiry, it will be advisable to leave the latter people at present out of consideration. Three English regiments, four batteries of artillery, and a troop of horse brigade, composed the European force. The artillery and the horse brigade, being comparatively small bodies of men, may be also left out of the account, so that there only remain the three European regiments to consider. These regiments agreed in the following particulars : 1. They were composed of men of the same nation. 2. Their internal organization, duties, and habits were presumably the same. 3. Their diet and clothing were absolutely the same. 4. They were subjected to exactly the same influences of climate ; they felt the same temperature, breathed the same air, and were exposed to the same winds. And yet the 60th Rifles lost by cholera only at the rate of 76 per 1000 * This table is compiled from tlie staff-surgeon's return, at p. 20 of the Report. f Of these three officers, "one died of congestion of the brain, subsequent to the attack of cholera; another was in so weakly a state, that any disease must have carried him off; so that only one case remains without palpable modification." (Report, p.27.) ## 1848.] On the Causes and Diffusion of Cholera. 83 of strength ; the Bombay Fusiliers lost at the rate of 108 per 1000 of strength ; and the 86tli regiment at the rate of 218 per 1000 of strength. How is this astonishing difference to be accounted for 1 It can not be accounted for by the following circumstances : 1. By any fancied exposure to contagion on the part of the latter regiment; because, as we have already proved, contagion has nothing to do with these attacks, and if it had, the 86tli regiment had in this instance less intercourse with the town of Kurrachee than the GOtli Rifles. 2. By any peculiar misconduct on the part of the most affected regiment. Mr. Thom states that the 8Gth were not more intemperate than the other two corps. 3. By any peculiar maltreatment of the sick, as this, of course, would not account for the number of admissions, and it is a curious circumstance that the ratio of deaths to admissions of cholera were only 39 per cent, in the Bombay Fusiliers, 58 per cent, in the 86th regiment, and as much as 63 per cent, in the 60th Rifles, the most healthy regiment of the three. The cause of the disparity between the GOth, on the one hand, and the Fusiliers and the 86th, on the other, must be sought for in some common condition of the two latter regiments. The first discoverable cause is, that, while the Rifles were in barracks, the other two corps were encamped in tents, "on alow, hot, and arid plain." Ten or twelve men "were cooped up in a 14 foot square tent."* Here, exposed to the burning heat of the sun, with the thermometer at 96? or 100? Fahrenheit, they remained from sunrise to sunset, unable to move out, in a still sultry air, and altogether deprived of the influence of the sea breeze. Such was evidently one cause; a deficiency in proper accommodation led to the death of hundreds of men. Twelve men were breathing in a tent hardly large enough for one, the tent itself carefully secured from all means of ventilation, and the season unfortunately one in which the winds fell peculiarly light. In any point of view, this situation was liygienically bad. As regards the poison, it must have found in that close and contaminated air, with the humid atmosphere all round it, the very conditions most favorable for its rapid increase. As regards the human beings on which that poison acted, they must, in a variety of ways, have been rendered peculiarly susceptible to its influence. These things are clear as daylight, they cannot be gainsaid; an immense increase of mortality was then caused by circumstances which could have been most easily removed.^ But of the men in tents, one regiment suffered much more than the other. What additional causes were then active here ? The Bombay Fusiliers and the 86th regiment agreed with the 60th Rifles in having the same duties, dress, diet, habits, &c., and in being exposed to the same atmospheric agencies; they differed in the situation of their camp, hence a cause of difference of health; but the Fusiliers and the 86th agreed in * Report, p. 26. + It is, of course, understood that we make no objection to the use of tents, if numerous and properly pitched. Several times, when men have been crowded in barracks, small, old, and uncomfortable, as too many of them unfortunately are in India, and cholera has broken out, it has been checked by putting the men under canvas, and moving them about; but then, in this case, the very principles are followed out, for which we are contending,?ventilation and cleanliness are favored by the change. [July, the situation of the camp, and yet between tliese corps was a striking difference of mortality. IIow was this ? To answer this question we must briefly allude to one of the most important points in the whole history of cholera; the nature of the predisposition which permits the action of the specific cause. This point is not only important, but it is most difficult; because we are often at a loss to know whether the severity of an epidemic is owing to the intensity of the poison, or whether the state of the system is merely unusually favorable to its action. For the same causes may increase the intensity of the agent, and augment at the same time the susceptibility of the subject. But in the present case, the intensity of the choleraic virus must have been nearly the same for the 80th regiment and for the Bombay Fusiliers, as they were encamped side by side, and equally under canvas, The Bombay Fusiliers had the advantage of being less strong than the 86tli; and it clearly appears, from the observations of Dr. Lorimer, that the liability to attacks of cholera increases, cseteris paribus, in an arithmetical ratio to the number of men. This could not have had much influence, however; and we are therefore compelled to look for the mortality of the 8Gth, not in the virulence of the poison, but in some cause which increased the susceptibility of the men. What are the causes which, both in England and in India, give a predisposition to cholera? 1. As regards strength or weakness, it is not mere debility* as has been asserted by many, and especially by the writer of one of "the works we are reviewing. The leading idea of M. Bureaud-Riofrey's work is the endeavour to explain the action of cholera by proving a previous debility on the part of those attacked. We doubt this exceedingly : first, because the evidence is very unsatisfactory; secondly, because if a person, no matter how robust, live in the choleraic district, he may be attacked ; and thirdly, because in India there is positive evidence that the sick in hospital are not attacked in undue proportion. 2. As regards age, the data are not yet sufficiently accurate to enable us to lay down any rule. It has been always noticed that infants and children are comparatively exempt. In the 86th regiment, only 1 child out of every 14 was seized, and 1 in 18? died; while the mortality among the women was 1 in 6, and among the men 1 in 4. Mr. Thorn thinks that there is an increased susceptibility between the ages of 18 and 35 ; but we think that his numbers in the higher ranges are too few to warrant the generalization of his observation. 3. As regards sex, women generally suffer less than men, and this is obviously owing to diminished exposure. Among the women of the 86th, there was great exposure, and a proportionably increased mortality. 4. As regards previous disease, or previous habit of body, constitution, or temperament, nothing certain is known. Diseases which diminish oxygenation, as emphysema, chronic bronchitis, obstructive heart diseases, have been supposed to cause the predisposition. But the evidence is * Mr. Thom says on this point: " Throughout the regiment it was the most powerful, muscular, and robust men that most speedily and generally fell victims to the malady ; a fact that, with us, admits of no doubt. Any one who ever saw the splendid men in our flank companies, would appreciate the truth of this." Dr. Parkes says: " Impaired health is not a great predisposing cause, nor is robust health an effectual safeguard?the peculiar predisposition is independent of these circumstances." (Researchesinto the Pathology of Asiatic Cholera, p. 199.) ## 1848.] On the Causes and Diffusion of Cholera. 85 deficient. Bowel complaints probably predispose, particularly those having a choleraic character; but then these are perhaps more rightly considered as the early stages of the disease, and not as mere phenomena unconnected with, except in so far as they lead to, the developed disease. 5. As regards habits, intemperance has been considered a great predisposing cause ; but here, too, the evidence is not stringent. In this country, it must be remembered that the poor, the badly fed, the badly clothed, the crowded, and the dirty, are generally intemperate. It is from these classes that the chief victims of cholera come; but are we to overlook the bad food, the bad air, the excretions both of lungs and skin impeded by want of cleanliness, and to fix only on a particular habit as the cause of the evil 1 The predisposition arises in such a case from composite causes, not from a single custom. At Ivurrachee, the men of the 86tli regiment were not more intemperate than the Bombay Fusiliers,* or these than the 60th Rifles. It has been found also that in India, teetotallers do not show a greater diminution in the per centage of attacks, than can be accounted for by the fact, that they are often steady men and non-commissioned officers, and therefore placed under more favorable circumstances than the privates. We do notr however, deny that intemperance does in some measure predispose; but it is not a powerful cause in producing the constitutional state which permits the action of the poison. Mr. Thom makes some very good practical remarks on the system of moderate dram-drinking, in which soldiers indulge in India ; but this is common to all regiments. 6. As regards diet ; the influence even of this is doubtful. Europeans and natives in India pursue the most diverse plans, and yet all suffer; the Hindoo even more than the Englishman. The use of fruits, or of rich dishes producing diarrhoea, may occasionally have some effect; but we have no doubt that too much influence has been attributed to this cause. 7. As regards mental causes, the fear of the disease has been considered a predisposing cause ; and we may safely allow this to have an effect in a few remarkable cases. 8. It is also apparent that, looking at the point from the most elevated situation, climate has little to do with the predisposition to cholera. For this disease has prevailed in every climate and in every season. It has ravaged towns in Russia, with the thermometer below zero ot Fahrenheit; in temperate Europe its attacks have on some occasions equalled in violence the death-blasts which depopulate Asia. We must consider, it appears to us, the predisposition as a peculiar unrecognised state of the body, resulting from the composite effect of age, strength, sex, previous health, temperament, diet, habits, and climate upon the system. Let us then inquire if the 86th at Ivurrachee had been subjected to any influence which may have heightened this predisposition over that of the GOth Rifles, or the Bombay Fusiliers,?regiments composed of materials so similar, and one of them at least placed in an analogous^ituation. It has been long known that the state of the body produced by prolonged marching has an astonishing effect in increasing the predisposition. Mr. Orton, who discusses this point minutely, adduces much important evidence ; but adds that, although his " statements are abundantly suf-* Report, p. 25. On the Causes and Diffusion of Cholera. [July, ficient to show the increased prevalence of the disease arising from marching or travelling, they give no idea of the dreadful extent to which this simple, and apparently harmless, circumstance is capable of operating to the destruction of human life." (On Cholera, Second Edition, p. 383.) Dr. Lorimer,* in his excellent Report, proves, by a series of very conclusive tables, that the number of attacks of cholera occurring on the march, increases regularly according to the number of miles, and to the number of days. The liability to attack increases so rapidly, that at the end of the 3d month the attacks of cholera occurred in the ratio of 61 per cent, of all the marches performed by the Madras Infantry ; while the attacks in all the native part of the Madras service were 46 per cent, in marches over 600, and under 800 miles, and 75 per cent, in marches over 800, and under 1250 miles. Whatever be the causes of this susceptibility, there is no doubt that it is in part attributable to the great fatigue and exposure; since the officers, who are mounted, suffer comparatively little, and cavalry suffer less than infantry.j* To this powerful influence the 86th regiment was exposed in 1846. "During four months," says Mr. Thorn, "the men were under canvas, and moved by forced marches over 600 miles to Bhawulpore, 300 back to Sukkur, then for 9 or 10 days cooped up in fearfully crowded steamers on the river Indus, and after this marched 60 miles more into Kurrachee. In accomplishing this, the men had broken sleep, long marches, and few halts. The ordinary distances daily gone over, varied from 12 to 19 miles. The men were usually roused at one or two o'clock in the morning, in order to strike tents and get to the next encamping ground by seven, a.m. to avoid the morning sun. On arrival, they had generally to lie down wet with perspiration, till the tents and baggage came up. At the same time, while undergoing this fatigue, they were exposed to extreme vicissitudes of temperature." (lleport, p. 25.) During this march the regiment was healthy, and reached Kurrachee towards the latter end of April; the attack commenced on the 14tli of June. Although in most cases the attack of cholera commences in a corps while yet marching, and ceases when it gets into comfortable quarters, yet, in the case of the 86tli, after such a march, it may well be supposed that the influence would not speedily be dissipated. This unlucky regiment had been previously, it appears, peculiarly unfortunate. "It landed in India in July 1842, and remained at Bombay in the wet monsoon of that year, and within a few months lost 100 men almost entirely from cholera.^ * Report on the Epidemic Cholera as it has appeared among the Native Troops of the Madras Army, on the line of march from one station to another, from 1820 to 1844. By Alex. Lorimer, m.d. Published by Order of Government. Madras, 1846. t When we state that marching has so much effect, we must admit that this general rule is subject to exceptions. The following is, perhaps, an apparent, rather than a real exception. In 1839, the right wing of H. M. 13th Light Dragoons marched from Bangalore to Bellary; during their halt at Uellary they suffered severely from cholera; returning to Bangalore, they joined the left wing, and the regiment marched to Madras. During this second march, cholera broke out. The left wing, which had been stationary at Bangalore, was 320 strong; it lost 35 men, or at the rate of 109 per 1000 of strength. The right wing, which had performed the fatiguing march to Beliary, only lost 4 men out of 208, or at the rate of 19 per 1000 of strength. (Report on Cholera in the 13th Light Dragoons, by Dr. Mouat, contained in one of the early volumes of the Madras Medical Journal.) It is very probable that the first attack of cholera had carried off the men most susceptible to the disease. It is Well known that a regiment has ceased to suffer from cholera, simply from the exhaustion of proper subjects, while the virus has remained unaltered, as is proved by a fresh corps arriving and immediately commencing to furnish cases. In Mr. Orton's work are many instances of a similar kind. J It is an interesting point, which we mention here, as we may not be able to return again to it, <hat, at Kurrachee, out of 50 men who had suffered from cholera at Bombay, 21 were attacked, and 9 died, or at the rate of 180 per 1000 of strength. On being sent to the cool station of Belgaum, there was much dysentery at first, and just as the corps was getting healthy, it was removed to Scinde, where at Hydrabad, in 1844, it was again cut up by a summer's residence in that place, and lost 80 men in a few months. In 1845 it had a year's quiet and restoration in barracks at Kurrachee." (Report, p. 24.) Even after tlieir long march in 1846, the men were allowed no rest: ? "After months of active work," writes Mr. Thom, "a forced indolence, the usual animal diet, and large libations of bad arrack, did their work. Surely these were predisposing causes, and, as if not sufficient, brigade field-days once or twice a week were persisted in up to within a few days of cholera appearing in June. Had these field-days been in the morning, little mischief might have occurred, but they were in the evenings, so that after the lassitude of a long day in a hot tent, the men had to commence preparations for parade at 4 p.m. when the thermometer stood above 90?." Predisposing circumstances with a vengeance; when will rulers learn wisdom, and believe that men are not made of iron? We recommend this passage to the consideration of the authorities at the Horse G uards; we should like to know what is there thought of field-days with the sun overhead, and the temperature in the shade at 90?. The Bombay Fusiliers, on the other hand, were in a much better state of health; they had not long before arrived in Scinde, and though they had been to Sukkur, they went there and returned by steam, and only marched from Latta to Kurrachee, a distance of 60 miles. The 60tli Rifles, again, had arrived in Scinde only six months previously, and had gone at once into barracks without making a single march. It therefore follows that, both from our knowledge of the general laws of cholera, and also from the circumstances attending this very epidemic, we are entitled to draw these conclusions : 1. That the excess of mortality of the 8Gth regiment over the Bombay Fusiliers was attributable to the bad condition of health in the men of the former corps, consequent on hard service and long marching. 2. That the surpassing mortality of both these corps, compared with the 60th Rifles, was attributable to want of good hygienic arrangements, whereby the poison of cholera was allowed to increase in virulence, and the predisposition of the individuals exposed to its influence was also increased. But we must now consider this mortality in the 60th Rifles a little more closely. In this corps, out of a strength of 980, 118 were attacked, and 75 died. Could this mortality have been reduced to that of the officers? Of 200 officers, 9 were attacked and 3 died; and of the 9 attacked, 4 belonged to the Bombay Fusiliers, and had been living in tents. So that, had all the officers lived in detached well-ventilated houses, the per centage might have been reduced still more. The houses of the officers were between the 60th barracks and the native lines, and therefore the exemption was not owing to more healthy locality. It is unlikely that the hygienic condition of soldiers in India can ever be made equal to that of the officers ; but still the inequality may be reduced. The barracks of the Rifles were ill ventilated and crowded. " I feci persuaded," says Mr. Thorn, "that this corps would have had very little sickness, had they been less crowded in barracks, and their rooms ventilated by [July, better arrangements, such as a medical board recommended at the moment the disease was breaking out, and had beer been issued instead of ardent spirits." (Report, p. 28.) Of this we also are persuaded, and for this reason. The mortality among the natives in the town of Kurrachee was 1 in every 10 of population, that is to say, 1500 died of cholera in 6 weeks, out of a population of 15,000. This town, we are informed by Mr. Thorn,? " Consists of mud houses, with mere crannies as windows, or means of ventilation, while the houses are built so closely together, and the streets, barely wide enough to allow a loaded camel to pass, are so very tortuous, and inaccessible to currents of air, that all ventilation must be arrested, unless during a perfect gale of wind." (Report, p. 18.) A new bazaar has lately been built at some distance from the town, in the cantonment, and is laid out in large " compounds, divided by wide streets, straight and at right angles to each other, and the houses and stores are well built, so that the general ventilation of the place is secured." The natives, however, and the servants of the officers have built their " narrow close huts" between the stores ; and yet, in spite of this, ventilation is so much improved, that the mortality among these men, exactly of the same class as those in the native town, was only 1 in 30, or less than this. (Report, p. 18.) Compare now these different rates of mortality : 1. The natives in the town lost 1 out of every 10 of population. 2. The natives in the bazaar lost 1 out of every 30. 3. The 60th Rifles, the healthiest of the three regiments, lost 1 out of every 13. 4. The officers lost 1 out of every 66. Now we said we agreed with Mr. Thom, although we never saw the 60th barracks, for this reason. How comes it that the mortality in the 60th Rifles, so much less even than that in the other two regiments, was so fearfully above that of the natives in the bazaar, which was situated close to them ? We cannot reply,?because they were Englishmen, and predisposed to the disease,?for two reasons. Because, if so, how was it the English officers escaped; and why did the natives in the town have a still higher mortality ? It is a fair inference, which we do not see how to avoid, that the same cause which raised the mortality in the town, and diminished it in the bazaar and officers' quarters, gave it its standard in the barracks ; and this cause was the relative purity or impurity of air. If this be the case, is it not disgraceful that our soldiers in India should be placed under such hygienic conditions ? Does it not call for immediate inquiry, why one of the finest regiments in the service sees itself decimated by cholera, while close to it a body of natives suffer little more than a third of its loss ? Before closing this history of the Kurrachee epidemic, we must glance at one or two important points yet unnoticed. The remaining European force, to which we have not yet alluded, was composed of (a) a troop of Horse Brigade. These men had made the march to Bhawulpore, but being mounted, suffered little fatigue; they were placed in good barracks on tlieir return, and lost only 5 men out of 135, or at the rate of 37 per 1000 ## 1848.] On the Causes and Diffusion of Cholera. 8y of strength ; (b) 4 batteries of Artillery, stationed in good barracks : they lost 37 men out of 3/5, or 98'6 per 1000 of strength, giving a mortality over that of the 60th Rifles. To account for this, Mr. Thorn mentions that 3 of these batteries had just returned, we presume with the 86th, from a march of 1000 miles. We regret that we are not informed of the amount of loss in the battery which had remained at Kurrachee. If, however, it was really the march which produced the increased mortality among these men, it gives a kind of measure of the influence which marching exerts. Thus, even after a march of 1000 miles, these men in good barracks, suffered considerably less than the Bombay Fusiliers who had not marched at all, but who were exposed in tents. The mortality among the native regiments varied considerably, as will be seen by reference to the table already given. The highest is that of the Beloocli battalion, viz. at the rate of 113 per 1000 of strength, or over that of the Bombay Fusiliers ; these men were in huts, and had returned from a march of 1000 miles. The 12th regiment Bombay Native Infantry lost at the rate of 67 per 1000 of strength; they had also returned from the same march, and were partly in barracks, partly in mat huts. "NYe do not know to what to attribute this diminished mortality, unless to the circumstance of one wing being in barracks ; but we should not like to assume this, as, unfortunately, we are not told the relative loss of this wing. But in the case of the other regiment, B. N. I., which lost only at the rate of 36 per 1000 of strength, we are entitled to attribute it to the fact of their having been stationary at Kurrachee for six months. It is also a curious circumstance, as regards intensity of disease, that the native regiment, as well as the European, which lost fewest men, yet lost most compared with the number of admissions ; thus, in the Belooch battalion, the deaths to admissions of cholera were 47 per cent.; in the 12th B. N. I. the deaths to admissions were 50 per cent., while in the stationary and healthy regiment, the deaths to admissions were 55 per cent. Although we attach so much importance to the purity or impurity of the air, as the only mode of explaining completely the differences of mortality in men under similar circumstances, we must also note that Kurrachee, independent altogether of cholera, must be an unhealthy station. This arises probably from the extraordinary humidity and heat of the climate. Among other diseases, scurvy is commonly seen at Kurrachee; in fact, till lately, lime-juice was issued to all the troops in LoAver Scinde. And it is very interesting to learn, that scurvy became very prevalent after this terrible burst of cholera. Spongy gums, livid spots on the body, and great muscular weakness, occurred in many men who had not had cholera, and also in many convalescent from this disease. The 3d B. N. I., the freest from cholera of the native regiments, suffered more severely from scurvy than any; but in all the regiments the scorbutic diathesis was more or less marked. This is not the first time that scurvy has been noticed to follow cholera ; the 22d regiment in Scinde, after losing 150 men from cholera, had a great many cases "of scurvy.* After prevailing at Kurrachee, the cholera spread towards Upper Scinde; but it never reached Sukkur, having apparently died away in the dry air of this province. It was moderately fatal in Hydrabad. Cholera is common at Latta on the Indus. * 1. At Kurrachee, among the most favoured class of Europeans, as far as regards hygienic condition, viz. the officers and ladies, the mortality was trifling. 2. Among the most healthy of the three European regiments, the mortality was enormously increased over this ratio, apparently from bad accommodation. 3. The mortality was still further heightened among the other two regiments, 1st, by an insalubrious camp, and, 2dly, in the 86th regiment, by a low condition of health of the corps, brought on by causes well known to predispose to cholera. 4. There is good reason to believe that the mortality of the latter classes might be nearly reduced to that of the former; in other words, that the varying mortality in these several bodies of men is explicable only on the principle, that the poison of cholera derived its terrible power chiefly or entirely from the accessory circumstances which attended its attack. When these were absent, the poison became powerless. Examined in this way, this epidemic at Kurrachee loses all its terrors. We see no longer the terrible burst of a mysterious plague, which seemed to be ushered in by whirlwinds and thunderstorms, and, like these mighty powers, to perform its work of destruction, unrestrained by human efforts. On the contrary, we see that it is our ignorance which has' given it power, our carelessness which has prepared its easy prey. Although the necessity of service could not prevent the long march to Bliawulpore, we might have counteracted the effect of that march, instead of aiding it; although barracks could not have been prepared, we need not have crowded twelve men into a tent; although discipline must be preserved, we might have had some compassion on the men who had marched 1000 miles, and not have buckled them up for a field-day twice a week. We do not, for a moment, attach the chief blame to the authorities at Kurrachee; this frightful mortality does not lie at their door alone; we must all take our share of reproach. The medical profession is the most to blame ; because it has not hitherto sufficiently recognised the simple causes which give such astonishing activity to morbid poisons. We have contemplated epidemics through an atmosphere of prejudice, which has distorted their form, and obscured their real proportions. But we are now beginning to learn; and if we do not deceive ourselves, we are, at last, on the right path to disarm of its malignity one of the most terrible plagues which lias ever ravaged the earth. We have reassumed our proper character of preventers as well as curers of disease. We can only spare a few lines to inquire into the means by which the Sanitary Commissioners propose to remove or lessen the humidity and impurity of the air, which aid the development of typhus, influenza, and cholera. In the metropolis, of course, the first thing is to ensure a more uniform and effectual system of drainage. Upon the practical details of this point, the Commissioners have accumulated much valuable information ; our present inquiry, however, will not permit us to go into the analysis of this part of the Report. We regret to say that the difficulties in the way of a revision of the Metropolitan Sewerage, are neither few nor small: but we trust, most sincerely, that energy and determination may vanquish the opposition of ignorant, apathetic, or interested men; and that the contest for pure and "wholesome air may eventually triumph. Since the visitation of influenza, the Commissioners have directed their attention to the suburban districts. They discover, in the first place, that three of the suburban districts suffered as great mortality from influenza, as the most crowded part of London. Inquiring into this circumstance, they come to the following conclusions : the general characteristics of the places where the inhabitants suffered least from influenza, are dryness, warmth and shelter from the humid atmosphere; model lodging-houses, well-regulated prisons, and places of a like kind, having enjoyed a marked exemption. In the Westminster House of Correction, only 6 prisoners out of 700 were attacked. In the Pentonville Model Prison, the average of attacks among the prisoners was 1 in 6, among the attendants who sleep outside, 1 in 2\. In none of the prisons of the metropolis did a single death occur from influenza; but of course this observation is to be qualified by the fact of the very young and very old being excluded from the prisons. It also appears that the deaths from influenza in the clay districts of the metropolis, compared with "gravelly districts within a few miles of London," were as 3 to 1. Proceeding upon their belief, that influenza prevailed most in humid and exposed cold situations, the Commissioners call attention to the immense quantity of undrained or halfdrained marsh land in the neighbourhood of London, particularly on the eastern and Essex side. The Essex and adjoining marshes contain about 3500 acres, or 5 square miles of undrained land, and upwards of 200 acres of ditches, many of them very offensive from the addition of sewage water. It appears, indeed, that even when the marshes are attempted to be drained, this is done in the most primitive way by open ditches; in one district in the Poplar Marsh, out of 520 acres, there are 21 acres of ditches ; in the marshes of Greenwich, upon an area of 450 acres of land, there are 13 miles of ditches, or 1 acre of ditch to 24 acres of land. In the districts of the Surrey and Kent Commissions of Sewers, there are 70 miles, or upwards of 60 acres of ditches. The Sanitary Commissioners bring forward evidence to prove that in places drained within the last few years, the temperature has been raised, fogs have disappeared, and influenza, marsh fevers, rheumatism, and neuralgic pains have much diminished. They point out also the ineffective mode of the present system of draining, and the great loss of good land arising from the use of ditches; they therefore recommend, on the evidence of those wellknown drainage authorities, Mr. Smith of Deanston, and Mr. Josiali Parkes, the abolition of ditches and the employment of covered drains. Earnestly do we hope that the principles proclaimed by the Sanitary Commissioners, which, with necessary modifications of execution and detail, are applicable to all countries, may be encouraged in that land, from which issues the disease that has stimulated the labours of the present Commission. If, as we believe, a wise recognition of the conditions of existence of the poison of cholera can lead, in some measure at least, to their removal, and to a proportionate diminution in the mortality from this terrible scourge, then we hold it incumbent on the great Company, whose magnificent dominions are the garden of the earth, to avert from the Hindoo, in his close and fetid village, from the Englishman, in his On Ike Causes and Diffusion of Cholera. [July, confined, crowded, and ill-constructed barrack, some portion of tlie evil, which has acquired magnitude and strength from our ignorance and neglect. The Company cannot alter the laws of Nature; they cannot prevent India from being the country, more than all others, subject to cholera, on account of its marshes, forests, plains, and rivers, its heavy rains, its burning sun ; but they can do much to weaken the force of the poison, and to strengthen the frame against its influence. But we must pass on to a very brief consideration of the next division of our inquiry. III. NATURE OF THE SPECIFIC CAUSE. The phenomena of great pestilences occur on so vast a scale, their consequences are so stupendous, their immediate action so impregnated with an almost universal suffering and fear, that men have found nothing to compare them with, but the most terrible and awful convulsions of Nature. The earthquake and the volcano seemed appropriate metaphors for diseases which changed the destinies of nations, and produced greater political and social changes than have flowed from the contentions or passions of man. In the middle ages, the comet or the volcano soon passed from a simile into a reputed cause;?in those times, when the social condition of the mass of the people of Europe was in the most degraded and miserable state, diseases assumed an extent of spread, a malignity of action, and a variety of type, unknown in these happier days;?coincidences between the outbreaks of such plagues, and the diseases, we are still as far as ever from accounting for them. These conditions are not the primary and specific agencies; they are only the accessory causes which give them power. What, then, is the nature of the indispensable cause of cholera? We think it a fair assumption that there is a specific cause or agent, because all the atmospheric causes that have been assigned may be excluded either in one epidemic or another, and yet the disease remains with unchanged characters. It is not great heat with moisture which causes cholera, because it has prevailed in cold countries in the period of their greatest cold; it is not excessive moisture, because it has prevailed with every degree of this, short of absolute dryness ; there is no evidence that it is any electrical condition of the air, for this, at present, cannot be proved to undergo any change during an epidemic. But other and stronger arguments still, support the hypothesis of a specific virus, which we shall presently adduce. The Sanitary Commissioners do not touch on the nature of the specific cause of cholera ; this was not in their province?they dealt only with conditions. Mr. Thorn, on the other hand, devotes much space to a discussion on this point, and we shall now give his argument in as condensed a form as we can. skin, which, in hot climates, is vicarious of the urine, is checked by the diminution of evaporation in the moist atmosphere. This state of things, then, produces the " choleric diathesis." The most intense condition of this state of things merges the choleric diathesis into an actual attack of cholera. To the obvious objection that "a stagnant, hot, and moist atmosphere" does not prevail in all the countries which cholera has visited, Mr. Tliom replies that? " Afghanistan, Persia, Turkey, Southern Russia, and Central Europe form a [July, chain of countries which arc exposed to alternations from cold and dry, to hot and moist currents of wind, resembling those of the Indian peninsula, but in a modified degree, except in extraordinary hot years, when the similarity will be perfect." And at these times, therefore, we presume cholera will, according to the hypothesis, prevail in these places. Such is the hypothesis* which is qualified in tlie next page, in rather an illogical manner. Alluding to the difference in mortality at Kurrachee, in the several corps, Mr. Tliom remarks that? " It will be neccssary to direct attention to the circumstances, peculiar to certain classes, which in one case gave the malady an unexampled virulence and fatality, and in another, complete immunity, and such as happily are, in a considerable degree, under our control. The general atmospheric causes cannot be prevented; but, in all probability, it very rarely happens that these are in such an intense degree as to produce severe and prevalent cholera, without the usual accessories, which appear to have lent a far more dangerous character to it than even the purely primary causes alone." (Report, p. 10.) So that here the accessory causes which we have already detailed are absolutely raised in importance above the primary causes. But leaving this, the hypothesis appears to us in all points very doubtful:?it is not proved that in hot climates carbon accumulates in the system ; it is not proved that the liver excretes it;f it is not proved that, even if it accumu-that it has seemed to those not acquainted with the details to be capable of arising only from atmospheric causes) there is good reason to infer the presence of a specific virus, undergoing, from peculiar atmospheric conditions, a development unusually rapid, in the general history of cholera are to be found much stronger and indeed conclusive arguments in favour of such a view. Our space will allow only a very short enumeration of these : 1. Cholera has prevailed in a variety of atmospheric conditions; not merely in those presumed to be capable of retaining carbon in the system. 2. It has often been absent when the presumed atmospheric causes have been present in their greatest intensity. 3. It seldom commences or extends like an atmospheric disease; in illustration of this, we may compare it with influenza, which either arises altogether from atmospheric causes or possesses a specific poison of extraordinary volatility. Influenza appears at once over wide tracts of country, it attacks numerous individuals at the same moment; it is not perceptibly checked by seas; it is not bound to the soil, or connected with its exhalations, for it has attacked a ship's crew in the middle of the Atlantic; on the other hand, cholera travels slowly, often preferring the lines of communication between countries, probably because it finds there its conditions of increase most abundantlyit often adheres to the soil, and is checked by rivers or open spaces ; when it enters a place, it selects almost always a particularly damp and dirty locality ; it attacks a few individuals, then increases for a certain number of days, and then declines, or, if the conditions be favorable, it remains located in a place for months, and during this time it may ravage only a comparatively small section of a town. 4. This localization of cholera has always appeared to us the strongest argument in favour of the specific poison. Every one has seen cholera attack one side of a street, or one end of a town, and leave the other untouched, although atmospheric causes are common to the whole district. How is it explicable, except on the hypothesis of a specific poison, that a regiment in India shall be attacked with cholera in one locality, and shall throw it off, by marching a few hundred yards to a better locality ? Or how can we explain the fact that the disease does not pursue straight lines, and will sometimes oscillate, so to speak, in a country, passing through it, and then returning through the portions it had left untouched, as in the Island of Salsette, in 1818? Or how can we account for a small section of land remaining often unattacked, although round it the disease is raging, the inhabitants in each place being of the same nation and having similar customs, as in the cases of Surdrep in the Sunderbund, Kristofsky at St. Petersburgh, or the Faubourg Leopoldstadt at Vienna? Or take the march of regiments in India.?How, on the supposition of atmospheric causes, can we account for one wing of the 9th Lancers, as in the case formerly given, taking cholera at a certain locality, and losing it at a certain locality, and a month afterwards, the other wing arriving at the same districts, commencing to suffer, and ceasing to suffer at the same places ? What, indeed, is a common mode of attack in regiments on the march in India ? A regiment is in perfect health till a certain date; it then hears that the next march will bring it into a district suffering from cholera: as the district must be got through, the commanding officer pushes hurriedly on. In two or three days, however, cases appear, not in the regiment itself, but in that motley and ill-regulated assemblage in the rear, which, under the name of camp-followers, attend the march. It may be very fatal among these people before it attacks the fighting men, or it may be confined to them. Now in such a case, the atmospheric conditions may be absolutely the same for the infected and non-infected districts, and for the two classes of men. 5. Another argument is derived from the necessity of including cholera among the class of epidemic diseases, all of which arise from peculiar and specific poisons. G. The progress of a case of cholera, its regularity, its similarity of feature to every other case, even the general uniformity of its mortality, point to a special agent. 7. Again, if cholera be ever contagious and propagated from one body to another, this can arise only from a specific cause. Additional arguments and illustrations crowd upon us, but our space forbids us to enlarge upon them; our readers will doubtless be able to supply many more. Let us repeat that we are content to ground the argument for a specific virus on this single plea; that in a certain town cholera will occupy one section of it, will prevail here and here only for months, and will last through several seasons, thereby clearly indicating that its existence is independent of general atmospheric causes, although its diffusion is influenced by them. Holding this opinion firmly, we make two qualifications: ?First, we have argued the point on the supposition that atmospheric causes must be general, that is, must be diffused over a general extent of country?but in this we may be wrong ; if in a given small space, either from humidity, from emanations from the soil, or from the relative bearings to each other of river and land, there may be some disorder in the atmospheric combinations, some allotropic condition of the oxygen (as that which produces ozone), produced either from the action of heat, electricity, or some similar agent, then the argument for a specific virus must be defended on other grounds. But of such a local change in the atmosphere there is no evidence.?Secondly. It is just possible that the cause may be generally diffused, and yet the inhabitants of a special locality only be affected, because, from the circumstances of that locality, only those individuals are possessed of the requisite predisposition ; but such an explanation, on many accounts, is improbable, and indeed at variance with well-known phenomena. But now, admitting, for the sake of the argument, this hypothetical virus,?what is it ? what is its nature ? is it organic or inorganic 1 is it a chemical agent produced by the catalytic action of an organic or inorganic power ? or does it spring from germs which have a regular existence, and a certain life and death ? To answer these questions, it is evident that we must not look only at cholera. Cholera is but one of the links in that vast chain which connects all epidemic diseases, whether of man, of animals, or of plants. The inquiries which eventually will solve these difficult problems must have reference to an immense series of processes, of which cholera is both a section and a type. The theme is too extensive for our limits, as ## 1848.] On the Causes and Diffusion of Cholera. it is too stupendous for our powers. On the present occasion, we shall content ourselves with giving a simple abridgment of Dr. Cowdell's views on the " fungous origin of cholera," reserving as far as we can a critical review of them for another occasion. But we may remark,? 1. That it is almost impossible this agent should be a gas, else why \ does it not diffuse itself according to Graham's law 1 why does a road limit it, or why, like marsh miasmata, does it sometimes sweep along the lowest stories of a range of buildings, and refuse to rise many feet from the ground.* 2. That the power of reproduction apparently possessed by the agent of cholera, in common with all the poisons of epidemics, looks more like the property of an organic than an inorganic substance. It is hardly conceivable, that a mere chemical agent should multiply itself by acting on the living body. Dr. Babington, in the preface to Hecker, seems to doubt the fact of such a development. "We are constantly furnished with proof," lie says, " that that which affects life is not itself alive; and, whether we look to the earth for exhalations, to the air for electrical phenomena, to the heavenly bodies for an influence over our planet, we can perceive nothing which resembles the regular succession of birth, growth, decay, death, and regeneration, observable in organized beings." But surely in smallpox, with its wonderful regularity of course, its incubation, its maturescence, and its decline, we see something like a fixed term of power and activity. How, except by regular growth, can we suppose the poison of smallpox, which we choose for illustration, as being the most definite of epidemics, to acquire suddenly, in the change from a cool and dry air to a hot and moist one, a most astonishing increase in the extent of its spread, and the violence of its attack ? But not to spend more time at present on these difficult questions, we will pass to Dr. Cowdell's hypothesis of the cryptogamic nature of the cause of cholera. Feeling dissatisfied with the common and indefinite notions of epidemic agents, as well as with that hypothesis which attributes pestilences to microscopic animalcul0e,f Dr. Cowdell was led to inquire what kind of analogy existed between the origin and progress of cholera, and the phenomena attending the development and diffusion of the lowest orders of the vegetable kingdom. Pursuing this conception, he found an apparent agreement between the origin, increase, and effects of the two classes of agents ; and he has now furnished us with an analogical argument in favour of their identity. The same conception has been discussed by Henle and others, and in a manner somewhat similar ; but this does not at all detract from the merit of Dr. Cowdell s ingenious and interesting hypothesis. We need not spend much time over the first five chapters, which are occupied with an analysis of the history, nosology, nature, cause (as far as contagion is concerned), and treatment, of cholera?the writings of * A good instance of this is related by Livingstone, in speaking of cholera in China. A number of individuals, sleeping on the yZoors of apartments, were attacked with violent cholera; others, in the same apartments, sleeping in beds, wholly escaped. had he done so, he would have omitted many statements to which he now attaches credence. In the sixth chapter, the more original part of the work commences. The author first inquires what is the nature of fungi, and quotes a description of them from Lindley and Berkeley. The chief points insisted upon, are the simple form of their organization, which consists chiefly of cells and fibres ; the minuteness and subtlety of their sporules, and their rapid growth; thus, in a single individual of Reticularia maxima, Fries has counted above 10,000,000 sporules, and some species have been known to grow several inches in the course of a night. They are frequently "meteoric, that is, springing up after storms, 01* only in particular states of the atmosphere," (Lindley). Their rapidity of growth, and their small specific gravity, ensure a very extensive diffusion, even if we admit that they cannot be spontaneously generated by special conditions of light, heat, earth, and air ?a point for which many of the highest authorities have contended. After these preliminary observations, Dr. Cowdell proceeds to arrange his argument under five heads, which we take in order. 1st. The aptitude of fungi for entrance and localisation in the human body. The author starts with the remark, that the protein compounds of the body are nearly identical in composition with fungin, the basis of these low vegetable organisms. Therefore, the elements of the blood would be easily assimilated by beings of a fungoid or protophytic organization.* He then alludes to the fact that we have evidence of fungi vegetating in living animals, and quotes from Dr. Carpenter the fact of fungous growth in the bodies of a species of polistes, the wasps of the West Indians, which are occasionally seen flying about with plants of their own length projecting from the surface, which have arisen from fungi introduced through the lateral breathing pores: the disease called "muscardine" which attacks silkworms in the south of France, and which is due to the growth of a minute fungus within the body, and which fungus can be inoculated, is also adverted to. Dr. Cowdell then observes that even in beings of the highest organization fungi have been found ; as in the fluid in pyrosis, in the abdominal exudations of puerperal fever, in the exudations of dysentery, &c. He then quotes from the British and Foreign Medical Review, Berg's observations on thrush in children. It is hardly necessary to remind our readers that Berg describes the white coating of thrush as composed of thickened epithelium-cells, a molecular albuminous deposit, and a parasitic fungus, consisting of nucleated cells of various sizes, which afterwards develope into fibres; there maybe either a preponderance of these cells (sporules), or a preponderance of ramifying stems or fibres, according to circumstances. Berg believes that these sporules may float in the air, and find a fitting nidus in other living systems ; he has also succeeded in inoculating thrush. Admitting the truth of all these * We are not certain, however, that we should not expect the poison of cholera to be something absolutely dissimilar and antagonistic to the organic compounds. It is the opposition of properties which produces the greatest results. statements, Dr. Cowdell believes that in some at least of these cases, in the exudations of puerperal fever, for example, these fungi or their germs must have passed through the blood. The only remarks we have to make on this chapter are: 1, that the observations are very few in number; 2, that the relative connexion of the disease and the fungi in these and other cases, as in favus, has not yet been determined. The disease may merely give them their habitat, as, in spite of Berg's experiments, we believe to be the case in some forms of thrush, in which the local disease of the mouth is but a sign of a general disease, which may also exist altogether without aphthae. At any rate, the point is not yet cleared up. Having thus proved that fungi may exist in the body of man, Dr. Cowdell proceeds to consider? 2. The known effects of fungi.?He first alludes to the "vegetating fungi," such as the dry rot of wood, the contagious "ropiness of bread," &c.; he then passes on to yeast, and adopts the opinion that the fermentation is owing to the growth of the Torula cerevisise. He also believes that all ferments will be found to depend on the presence of an agent, analogous to the yeast-plant. He quotes Mr. Graham's account of the peculiar putrefaction of the Wurtemberg sausages which become poisonous, and, being then taken into the stomach, probably enter the blood, and impart, by some catalytic power, their peculiar action to the constituents of that fluid. He thinks it probable that this is owing to a fungus; and concludes that all these changes, and perhaps even more recondite transformations, such as occur in animal nutrition, are accomplished by the catalytic agency of organized agents,?thus raising the importance of fungi in the economy of Nature to a very high point. ## 3. The capability of fungi to produce the phenomena of pestilential cholera.?The author does not profess to be able to be very complete in this part of his argument. " It will not be expected," he says, " that we should be able, from merely analogical proofs, to supply the minutiae by which the doctrine of a fungous origin of pestilential cholera would be perfectly explanatory of all the known symptoms of that disease." (p. 142.) He first calls attention to the fact that all animals and plants are equally composed of cells, and have nearly the same ultimate composition. He then asks :? " Why may not the germs of a fungus or alga be absorbed into the blood, and be so absorbed at a time when an extraordinary impulse has been communicated by external concurring conditions, probably meteorological, the blood, meanwhile, being in such a bio-chemical (electrical) condition, as to be able to oppose little resistance to the reproduction of fungi within it ? And as it has been shown that these can live and grow within the human body, and in the fluid of it, we may naturally expect to find some of the effects described as fermentation taking place within the blood itself." He then goes on to remark that in cholera, almost all writers believe that the first impression of the materies morbi is upon the blood. Now it appears probable, that the well-recognised poisonous fungi act also upon the blood, and in many cases produce symptoms resembling cholera; such as in the case of the Amanita muscaria and citrina, the Hypophyllum sanguineum, and ergot, which is said to produce " a gangrenoid state of the blood," &c. Ergot is next considered with its active agent, a fungus named by Quekett "Ergotaetia abortifaciens," Perhaps the most On the Causes and Diffusion of Cholera. [July, important argument in this chapter is derived from the action of the Amanita muscaria. A certain quantity of this fungus produces a peculiar drunkenness; this passes off in a very few hours, apparently from the active principle being excreted by the urine, for the urine becomes so highly impregnated with it, as to produce intoxication if drank by another individual. The effects of a single fungus, eaten by one person, have been propagated through five individuals, by the second drinking the urine of the first, the third of the second, and so on. So that here there really seems to be an increase in the quantity of the fungus during its passage through the living system. The author also adduces other arguments, which, as we are not certain we could correctly interpret, we give in his own words. After alluding to the disease called Ergotism, and to the experiments of Mr. Quekett, and to Wiggers' analysis of ergot, he continues? " Besides the disease here described as ergotism, symptoms of a peculiar kind have not unfrequently been observed to follow the medicinal use of ergot In Mr. Quekett's experiments of inoculating healthy plants with the sporidia of ergotsetia, we have an instance of contagion; while, in the viscous exudation from the flower, we have, perhaps, a phenomenon representing the viscosity of the peritoneum, or the albuminous exudation on the mucous membrane of the alimentary canal in pestilential cholera, the latter corresponding probably also in nature and in similitude of cause to the albuminous, aphthous crust, constituting thrush. In Wiggers' analysis of the excrescence of ergot, the undoubted product of a fungous growth, we have seen that there was a large proportion of fixed oil; in the account of the Wurtembere sausages, we have read that the increased greasiness was one of the most remarkable of the visible alterations which had taken place in this variety of food, while the consequent wasting of the muscular fibre and of all the constituents of the body similarly composed, may be almost paraphrased by Dr. Christison's description of similar symptoms in gangrenous ergotism. In some infectious diseases, oiliness of the blood has been commonly remarked, as in plague and cholera. An abundance of fat has also been found in the faeces discharged in abdominal typhus." (pp. 158-9.) Now we must say we think this line of reasoning rather far-fetched ; it is, in fact, an instance of the great fault of the treatise, a desire to push analogical argument to extremes. Why should we thus violently connect together the fact of oil existing in the ergotsetia and the Wurtemberg sausages, with the doubtful observations of oil being in abnormal quantity in the blood of plague or cholera patients, or in the fseces in cases of "abdominal typhus?" On the whole, in this chapter, Dr. Cowdell is not so happy as in the former part of his work. The arguments he has adduced from the diseases produced by well-known fungi taken in large quantity, are not, it appears to us, applicable to pestilences. Dr. Cowdell then arrives at his next proposition. 4. The consideration of the circumstances which probably concur to produce from the germs of fungi the effects of pestilential disease.? Starting with the opinion that fungi spring up in particular states of the atmosphere, the author remarks that in all pestilences certain remarkable meteorological changes have preceded and attended them. With regard, more particularly to cholera, he refers to the attack at Kurrachee, which we have already discussed, and gives the erroneous account of it, to which we have referred as derived from the Bombay Times. He then takes Kurrachee and Jessore in Bengal as good examples of the places, which may be presumed to be most prolific in fungi. In both places, he says, the moist climate, the alluvial soil, the incredible quantity of vegetation, giving rise to great electrical disturbance, would probably give occasional impulse to the wonderful reproductive power of these beings.* He then alleges that the state of electricity, which evidently exerts a great effect over the production of fungi, may have an effect also in predisposing to, or shielding the human body from, their influence ; as an illustration of his meaning, he refers to the adherence of certain vegetable powders to the lines traced by a magnet on wood, while from other parts of the wood the powder is easily blown away. The eleventh chapter of the book is on the " identity of remedies recommended in pestilential cholera with anti-fungic agents." Bichloride of mercury, zinc, copper, tartar emetic, aromatic substances, volatile oils, are all anti-fungic agents, and all have been found useful in cholera. Dr. Cowdell justifies his use of this argument, which seems to us peculiarly weak, by a reference to the diagnostic effect of colchicum in gout, or of bleeding in certain diseases, in which the good effect of the remedy is held to justify the diagnosis. But in cholera, unfortunately, no remedy is unequivocally and specifically useful. Looking back now to the whole course of the argument, we are inclined to come to the following conclusions: 1. That the phenomenon of a rapid cell-growth appears to be a good type of what we may suppose to be the mode of increase of the specific virus, presuming it really to exist. 2. That the simple fungi appear, from their rapid growth and their known diffusible properties, to be the most likely class in which to seek for agents multiplying themselves in this way ; and that the remarkable catalytic power of these organisms renders it not unlikely that, if absorbed into the blood, they might, by germinating there, produce changes somewhat analogous to fermentation. bable, based on stronger arguments, and on more scientific foundations, than tlie animalcular theory of cholera. The great difficulty under which Dr. Cowdell has laboured, has been want of sufficient material; his hypothesis, if true, is as yet insusceptible of determination; it cannot be proved or disproved ; it must abide its time ; still, with the scanty number of facts yet known, Dr. Cowdell has made a strong argument, and has written an interesting and instructive work. We should like him to apply his hypothesis to all epidemics. He would perhaps find yellow fever and plague still more to his purpose than cholera. But our space warns us that we must hurry on to our fourth heading: IV. RELATION OF ENGLISH AND ASIATIC CHOLERA. We may, first of all, give a short epitome of the contents of a clever little book by Dr. Spencer Thomson, on British Cholera. We cannot, however, at this time enter so fully into the subject of English cholera as its importance deserves. Dr. Thomson introduces his subject with a few observations, in which he points out the importance of the disease, and the method he has pui*sued in investigating it, which has been mainly by an attentive study of the earliest periods of the disease, before the advent of vomiting and purging, which he considers to be merely the natural terminations of the affection. Curiously enough, Dr. Thomson takes somewhat the same view of English as Mr. Thom of Asiatic cholera, and believes it to depend upon the accumulation of carbon in the system, athough he has not proved this by chemical analysis. Dr. Thomson's observations were chiefly collected during the summer of 1846?a season remarkable for great and unusual heat; English cholera, as is well known to all, having been in the autumn of that year unusually prevalent. The Registrar-General's returns, ending September 1846, show an excess of mortality over the corresponding quarter of 1845, of 15,227 deaths. In London, the average mean temperature for this quarter was 63*10, being an excess of 6? over the corresponding quarter of 1845. The excess of deaths in London was 1567; and of these no less than 1303 were from cholera, diarrhoea, and dysentery. Therefore the author concludes that the high range of temperature, and the prevalence of cholera, stand related to each other as cause and effect. The mode of the relation is thus explained : almost all the waste carbon of the system appears, if Liebig be correct, to be got rid of by the lungs, where it combines with oxygen?a certain balance being always maintained between the one element and the other; our readers are too well acquainted with Liebig's views, to render it necessary for us to go into detail. But as the quantity of inspired oxygen varies with the temperature and density of the air, the amount of watery vapour, the number of inspirations, &c., in hot seasons and in hot climates, so, if carbon be still introduced in the same quantity as in cold climates and seasons, it must accumulate in the system. Whether this be the case or not, the symptoms said to be present in early periods of cases of cholera, such as overpowering drowsiness, headache, dusky skin, high-coloured urine, and catamenia, are such as would result from an excess of carbon ; and, in a mild degree, are very similar to those which result from poisoning by charcoal, or are seen in some forms of jaundice, in cyanosis, &c. Even intense cold and drowning produce effects evidently of an analogous kind. The author then remarks, that, although heat is the great primary cause, its action is greatly assisted by certain subordinate causes. He first alludes to diet, quotes Liebig's well-known opinions on this point, and insists strongly on the pernicious effect of alcohol in hot seasons and countries, as it not only increases the necessity for oxygen, but also diminishes the excretion of carbon. He then enters on the subject of the purity of the atmosphere, particularly as to the presence or absence of carbonic acid, which, with other compounds of carbon, he believes to be generated in large quantity from sewers, cesspools, dunghills, &c., during summer. Lastly, the author alludes to the function of the skin, and dwells on the increased necessity of keeping this in a fit and proper state during seasons of elevated temperature. We may sum up the author's hypothesis in a few words. Great heat diminishes the excretion of carbon by diminishing the volume of air and the number of inspirations. In addition, this state of things may be aggravated by the ingestion of more carbon with the food than can be burnt off, and by the fact of the air, during hot seasons, being contaminated, particularly in large towns, by the evolution of different compounds of carbon. The symptoms of English cholera are described with care, particularly the premonitory stage, which we shortly detail. This stage, it is stated, may last for weeks before those violent symptoms of vomiting and purging ensue, which are usually considered to be the disease. "All the premonitory symptoms indicate derangement of the liver; there is general feeling of lassitude, and inaptitude for exertion; headache, especially after meals, heavy sleep at night, and constant, insuperable drowsiness in the daytime; mouth clammy, dry, and tasting disagreeable and bitter; a painful sense of fulness and distension, extending from the epigastrium into the hypochondrium; pain constant or intermittent between the scapulsc, extending up the back of the neck; bowels irregular, occasional griping; urine generally deeply tinged, and depositing urate of ammonia, of various shades of colour, or deep-coloured uric acid; skin dusky, not yellow." (pp. 10-11.) The author states, that, in all the cases of bilious cholera these premonitory symptoms occur; at length the accumulated carbon finds its outlet in the violent bilious vomiting and purging which is the natural cure of the disease. We need not describe the severe attacks, as we observe nothing novel in their description. In the chapter on Treatment, the author refers particularly to a proper diet, and advocates the use of fruits, as prophylactic measures. If medicine is necessary, mercurials, which often act with some violence, may be taken. In the severest forms, when the disease simulates Asiatic cholera, opium is the l-emedy he chiefly uses. His other measures we need not detail. The author next enters upon the connexion between Asiatic and British cholera, and argues the question with much skill. As this is more germane to our subject than the previous chapters, we shall enter into it a little more minutely. N Before two diseases can be compared, it is necessary to have some tolerably definite opinions about the etiology and pathology of both. If we are not sufficiently acquainted with a disease, to distinguish primary On the Causes and Diffusion of Cholera. [July, from secondary symptoms, if we raise incidental phenomena to the rank of constant and necessary occurrences, and if at the same time the causes are obscure, and the anatomical signs unusually indistinct, we are almost certain to fall into some error in comparing such a disease even with one which has only a superficial resemblance to it. Now Asiatic cholera is certainly only partly described; we understand something about it, and we know the probable direction of future discoveries ; but we are not yet sufficiently acquainted with its pathology to speak without reservation on a single point. We shall, therefore, merely allude to the more obvious differences between it and the disease commonly called British cholera. 1. The causes seem to be different in the two diseases. Nothing is more certain than that Asiatic cholera spreads and extends itself; it does not appear at any particular time of year, and is limited to no locality. English cholera is remarkable for the tendency to occur only at the end of summer ; it occurs then, as Sydenham says, as regularly as swallows in spring. This character has always appeared to us to favour the opinion that Sydenham's description applies not to Asiatic but common English cholera, which, like all other epidemic diseases, was more severe in his day, from the bad hygienic condition of the population.* English cholera, moreover, does not travel, and is evidently connected with increased temperature, in some way or other. 2. The symptoms of the two diseases are in most cases strikingly dissimilar. Thus as to the manner in which they commence : the attack of Asiatic cholera is sometimes sudden ; if preceded by a premonitory period, this is of short duration; whereas it appears, if Dr. Spencer Thomson's observations be correct, that in English cholera there is a long period, during which the system is becoming more and more out of health under the influence of hygienic conditions, and not from the incubation of a specific poison, until the outbreak takes place, which is the cure of the disease. Again, in the future progress of Asiatic cholera, it has been clearly proved that the vomiting and purging are only secondary symptoms, and in the purest forms of the disease may be absent. In addition to the evidence already before the public, we may detail in this place Mr. Thorn's observations on the forms of disease seen at Kurrachee in 1846? observations which correspond exactly with those made by preceding writers. "1. Among the first 100 cases which occurred," writes Mr. Thorn, "many died in a few hours, and some in less time; one man, I am told, went off in less than an hour. In these vomiting and purging were not always present. Sudden collapse, ending in profuse sweating, were the most prominent symptoms?in fact, as if asphyxia had already taken place. It was often found that the pulse had ceased at the wrist, the eyes were turned up, and the voice hollow and feeble, before the natural hue had fully given way to that horrible lividity which is so characteristic of the disease?so instantaneously was the power of life arrested. Spasms of the muscles were very generally, but not always, present, in these early and more severe forms of the disease; in fact, not a few sunk almost without much suffering or complaint, but lay down to die with an apathy scarcely credible. " 2. The next class of cases was those in which the first seizure was equally sudden, and the collapse preceded the vomiting and purging. There were sudden * Although we have not entered into the question as to the nature of the cause of British cholera, our own opinion inclines to the belief that this disease does not arise from heat only, but from that and some other conjoint causes, connected particularly with our sanitary deficiencies and our peculiar climate. faintness, prostration of strength, restlessness and anxiety, accompanied by vertigo, deafness, loss of vision, alteration or liollowness of the voice, weak and slow respirations, performed convulsively or in sighs. These were followed bv nausea, vomiting, and purging of conjee-water stools, sensation of burning heat at the prsecordium, intense thirst, and desire for something cool. The circulation became impeded, especially in the extreme vessels and capillaries, and still more in the vena porta;; the collapse rapidly progressed, the expression was of extreme anxiety, amounting to agony, accompanied by restlessness and jactitation, the features bccame shrunk and cadaverous, the lips and skin assumed a livid colour, and the whole body, bathed in profuse cold sweats, soon yielded to dissolution. The respiration was slow, feeble, and irregular, being performed with convulsive starts, and with very little dilatation of the chest to receive any quantity of fresh air. Perhaps nothing was more uniform than the desire to be exposed to fresh air or a current of wind, and to have every covering removed from the body. The spasmodic action of the muscles was most severe in the first stages, and was not confined to the extremities, but affected every voluntary muscle in the body. "3. Another class were attacked with vomiting and purging of rice-water stools and copious sweating; cramp of legs, arms, and abdominal muscles, rapidly producing prostration of strength, and collapse, with all its train of symptoms, were consecutive on the discharges. " 4. Lastly, as the disease was subsiding, cases began to exhibit signs of reaction. In general, these cases were much more manageable than the others; and when the purging and vomiting were once arrested, a reaction in fever took place."* He also states that, out of the 400 cases in the 86th regiment, " 30 were followed by relapse once, and about 8 of these by a second, which generally proved fatal." We should extremely like to have the clinical detail of these cases. Mr. Thom also remarks that " the greater proportion recovered without any consecutive fever, this I was not prepared to expect; the number attacked with fever, which was of a low congestive typhoid type, was as 1 in 8."-f This description agrees closely with the general opinion as to the real nature of the disease, which consists in a more or less complete arrest of the circulation, with escape of some of the blood-constituents from free surfaces, in the inverse ratio to the degree of arrest of the circulation, and consequently to the intensity of the disease. The vomiting and purging are then, in Asiatic cholera, only secondary symptoms; in English cholera, they mark the utmost severity of the disease : when English cholera kills, it is by the exhausting nature of these discharges, and not from the action of some agent, whose highest influence is quite unattended by discharges in any notable degree. The nature of the discharges is, too, in many cases, quite different. Bilious purging never occurs in Asiatic cholera; in fact, the liver appears but little in fault, whereas in English cholera we certainly look to some deranged action of the liver, as well as of the gastro-enteric membrane generally, as being the precursors of the disease. We shall only devote a few words to the subject of our fifth heading. * Report, pp. 30-1. t ibid., p. 41. that they were badly situated, badly ventilated, very much overcrowded, and were filled with the worst cases from the poorest and most miserable population. But we conceive the Commissioners have made out no case against good hospitals in good districts, in which cases could be received before the latter stages, when removal, if not immediately dangerous, is eventually hurtful. We conceive it to be almost impossible, that during an epidemic the poor could be attended at their own houses, without an enormous staff of medical men and nurses. In an hospital there are, both for the attendants and the patients, all the advantages of combination. Besides, we hold it to be an important indication to remove cholera patients from the locality in which the poison which has attacked them still exerts upon them its fatal influences ; and even apart from this, those who know the confined and baneful dwellings of the London poor, will deem it necessary for the favorable issue of cholera, as of almost any disease, to insure them, in the first place, one of the important necessaries of life and health,?an abundant supply of pure and wholesome air. How can this be accomplished in the present day without cholera hospitals? But we must draw this article, already too long, to a close. We will conclude it with a single remark. The true philosophy of the science of medicine is the knowledge of the causes of disease. Or, if these causes be too subtle and refined for our gross senses, it is the knowledge of the several conditions, external or internal to the body, which give those causes power. In the future history of medicine, we shall see men returning to the principles promulgated by its earliest founders. They will perceive that the treatment of the fully formed disease is at the same time the most difficult, and the least useful part of their noble profession. They will learn to arrest the evil at the fountain-head, and not to dam the current swollen by a thousand tributaries. And if the principles which we have analysed in this article be correct, it will not be the least triumph of this philosophy, that it has indicated the true mode in which the great epidemic of our time can be most easily and most effectually controlled. It bars out the disease, not with quarantines and cordons sanitaires, but with a cleanly people and uncontaminated air. The evil which springs from the bosom of Nature only needs for its removal an observance of the rules which Nature herself reveals.

Phosphorylation of the Canonical Histone H2A Marks Foci of Damaged DNA in Malaria Parasites Plasmodium falciparum parasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage; therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate under such conditions. In higher eukaryotes, double-strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X, which marks the site of damaged DNA. We show that in P. falciparum that lacks the H2A.X variant, the canonical P. falciparum H2A (PfH2A) is phosphorylated on serine 121 upon exposure to sources of DNA damage. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the nonphosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and that over time, as the parasite activates the repair machinery, this phosphorylation is removed. Finally, we demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay in P. falciparum.Citation Goyal M, Heinberg A, Mitesser V, Kandelis-Shalev S, Singh BK, Dzikowski R. 2021. Phosphorylation of the canonical histone H2A marks foci of damaged DNA in malaria parasites. mSphere 6:e01131-20. https://doi . IMPORTANCE Plasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the bloodstream and begins proliferating inside red blood cells, where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such conditions are also the driving force for acquiring genome plasticity that enables them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied for these parasites. The paper addresses our recent discovery that P. falciparum that lacks the histone variant H2A.X phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms. KEYWORDS malaria, Plasmodium falciparum, DNA damage, DNA repair, H2A phosphorylation, double-strand break P lasmodium falciparum is the protozoan parasite responsible for the deadliest form of human malaria. This parasite is estimated to infect 200 million to 300 million people worldwide each year, resulting in approximately half a million deaths, primarily of young children. P. falciparum replicates within the circulating red blood cells (RBCs) of an infected individual, and its virulence is attributed to its ability to modify the erythrocyte surface and to evade the host immune attack. During their intraerythrocytic development, Plasmodium parasites replicate their haploid genomes multiple times through consecutive mitosis cycles called schizogony, which makes them particularly prone to errors during DNA replication. In addition, blood-stage parasites that live in a highly oxygenated environment produce potent DNA-damaging agents while digesting hemoglobin and are exposed to oxidative substances released from immune cells. Therefore, Plasmodium parasites that are exposed to numerous sources that can damage their DNA must have evolved efficient mechanisms to protect their genome integrity. Orthologues to many of the proteins involved in the DNA damage response (DDR) are encoded in P. falciparum genome, including those involved in homologous recombination (HR), microhomology-mediated end joining (MMEJ), and mismatch repair machineries. However, these mechanisms have not been extensively studied for these parasites. It appears that malaria parasites utilize both HR and an alternative end joining pathway to maintain their genome integrity. Thus, in the absence of a homologous sequence in their haploid genome that can serve as a template for HR, blood-stage parasites primarily repair double-strand breaks (DSB) using the alternative microhomology-mediated end joining mechanism. In mammals, a single double-strand break of the DNA triggers the DDR that rapidly leads to extensive ataxia telangiectasia mutated (ATM) kinase-dependent phosphorylation of the core histone isoform H2A.X to form phospho-H2A.X (g-H2A.X), which marks the site of damaged DNA. However, the P. falciparum genome lacks an orthologue of the H2A.X variant, and it encodes only two H2A variants, the canonical P. falciparum H2A (PfH2A; PF3D7_0617800) and PfH2A.Z (PF3D7_0320900), which was shown to be associated with a subset of active promoters. Previous histone phosphorylation analysis suggested that PfH2A could be phosphorylated on serine 121. We were interested to determine whether in P. falciparum phosphorylation of PfH2A might be correlated with DNA damage. We show that these parasites phosphorylate the canonical PfH2A on serine 121 in response to DNA damage and that the phosphorylated PfH2A is recruited to the damaged foci. In addition, the ability to specifically detect the dynamics of this phosphorylation using an anti-g-H2A.X antibody provides a useful marker for studying DNA damage mechanisms which allowed us to establish a direct DNA repair assay in P. falciparum. # Results In model systems, phosphorylation of H2A.X is elevated following exposure to DNA-damaging agents and is commonly used as a marker for double-strand breaks. The phosphorylation of serine 139 found on a conserved SQ (serine-glutamine) motif of mammalian H2A.X serves as a differential epitope for detection of the phosphorylated form, known as g-H2A.X. Plasmodium parasites do not contain a gene encoding the H2A.X variant in their genome, but instead they express the canonical H2A and the H2A.Z variants (PF3D7_0617800 and PF3D7_0320900, respectively). In the absence of a good marker for DNA damage in Plasmodium, we were interested to test whether the antibody that recognizes g-H2A.X in mammals could be used as a marker for DNA damage in P. falciparum. As a first step, we aligned the two Plasmodium H2A variants with human H2A.X and noted that only the canonical PfH2A has a long C-terminal tail containing the SQ motif, which is conserved among Plasmodium species, while no SQ motif was found in PfH2A.Z (see in the supplemental material). This SQ motif is conserved among the canonical H2A proteins of several protozoan parasites, such as P. falciparum, Giardia lamblia, and Trichomonas vaginalis, that lack H2A.X orthologues. Similarly, the budding yeast Saccharomyces cerevisiae lacks the H2A.X orthologue, and instead, its canonical H2A was found to be phosphorylated on serine 129 (SQ motif). This phosphorylation is detected by the anti-g-H2A.X antibody, and thus, the phosphorylated form of S. cerevisiae H2A is often referred to as g-H2A.X. Interestingly, in contrast to Plasmodium spp., the apicomplexan parasite Toxoplasma gondii has an H2A.X variant (TgH2A.X) in addition to its canonical H2A . As expected, in silico structural prediction of PfH2A suggests that the SQ motif is found in its C9-terminal tail and that this motif is likely to be an ATM kinase phosphorylation site , In silico analyses of putative ATM kinase-specific phosphorylation site (conserved SQ motif) in PfH2A. (A) Multiple-sequence alignment of amino acid sequences of PfH2A and some of H2A variants from human, budding yeast, and protozoan parasites using ESPript 3. Similar and identical amino acids are boxed and marked with a red background. The conserved C9-terminal SQ motif is underlined with asterisks. Database accession numbers or gene names appear in parentheses. Abbreviations: PfH2A, P. falciparum histone H2A; HsH2AX, Homo sapiens histone H2AX; ScH2A1, Saccharomyces cerevisiae histone H2A1; TgH2A1, Toxoplasma gondii histone H2A1; TgH2AX, Toxoplasma gondii histone H2A.X; TvH2A, Trichomonas vaginalis histone H2A; GiH2A, Giardia intestinalis histone H2A. (B) 3D homology model of PfH2A (developed using structure homology-modeling server SWISS-MODEL) showing the core histone domain and that the extended C-terminal tail contains the conserved S 121 Q 122 motif. (C). In silico prediction of ATM kinase-specific phosphorylation sites in PfH2A (using KinasePhos, version 2.0). The sequence-based amino acid coupling-pattern analysis and solvent accessibility of PfH2A suggest serine 121 as the most prominent ATM kinase-specific phosphorylation site. H2A Phosphorylation and DDR in Plasmodium similar to the serine 139 of the mammalian H2A.X, which is the major residue phosphorylated in response to DNA damage by the ATM kinase. To test if PfH2A is indeed phosphorylated in response to exposure of the parasite to a source of DNA damage, we exposed tightly synchronized ring-stage NF54 parasites to X-ray irradiation. We chose to irradiate early-stage parasites that do not replicate their DNA and do not have hemozoin, and therefore, the detected DNA damage should be mostly due to the exogenous source. We used terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays as direct evidence that exposure of the parasite to 6,000 rads caused DNA damage, which was detected in most parasites' nuclei . We then used the g-H2A.X antibody for immunofluorescence assays (IFA) and were able to detect strong signals within the parasites' nuclei after exposure to X-ray irradiation . This observation was further confirmed using g-H2A.X antibody on proteins extracted from parasites exposed to increasing levels of irradiation, which showed a corresponding elevation in the levels of g-PfH2A , left, and . Similarly, exposing the parasites to hydrogen peroxide (H 2 O 2 ), another source of DNA damage, caused an increase in the levels of g-PfH2A recognition , right). To ensure that the anti-g-H2A.X antibody specifically recognized the phosphorylated form of PfH2A and did not cross-react with the nonphosphorylated form, we incubated the extracted proteins with calf intestine phosphatase (CIP), which removes phosphate residues. The CIP treatment specifically abolished immunoblot detection using the anti-g-H2A.X antibody, while the nonphosphorylated PfH2A was detected at similar levels in parasites exposed to increasing X-ray levels . In addition, when we initially probed with the anti-g-H2A.X antibody after irradiation, we observed increasing levels of phosphorylation; however, when the blot was stripped, treated with CIP, and reprobed, the anti-g-H2A.X antibody signal disappeared, while detection of the canonical PfH2A was unchanged . Together, these data suggest that PfH2A is phosphorylated in response to DNA damage and that the antig-H2A.X antibody is specific to the phosphorylated form of PfH2A. To further confirm that the phosphorylation detected by the anti-g-H2A.X antibody, following parasite exposure to DNA damage, is indeed phosphorylation of the canonical PfH2A, we extracted and purified histones from parasites that were either exposed or not exposed to X-ray irradiation. Immunoblot analysis of the total histone extract showed an increase in the level of the phosphorylated form of PfH2A following irradiation, while the total levels of PfH2A were similar and B). We further exposed parasites to X-ray irradiation and performed immunoprecipitation (IP) of total PfH2A using an anti-H2A antibody. The IP fractions were subjected to immunoblotting with the anti-g-H2A.X antibody, which demonstrated significant enrichment of the phosphorylated form of PfH2A in the elution . This fraction was subjected to trypsin digestion followed by mass spectrometry analysis, which identified phosphorylation on serine 121 of PfH2A . The specificity of the anti-g-H2A.X antibody to the phosphorylated form of PfH2A allowed us to image its nuclear distribution compared with that of the nonphosphorylated PfH2A in irradiated parasites. Immunofluorescence assay using anti-H2A and antig-H2A.X antibodies indicated that while the canonical PfH2A is spread throughout the nucleoplasm, its phosphorylated form is found at distinct foci. To further validate this observation, we performed superresolution stochastic optical reconstruction microscopy (STORM) imaging, which enabled us to image the nuclear distribution of the two forms of PfH2A in detail at the nanoscale level. This analysis clearly demonstrated the differential distribution of the two PfH2A forms in the nucleoplasm. The nonphosphorylated PfH2A is indeed spread throughout the nucleoplasm, while the phosphorylated form is much less abundant and is found at distinct nuclear foci. Thus far, the process of DNA repair in Plasmodium was mostly studied indirectly by measuring the recovery of parasites in culture after exposure to a source of DNA damage (10). In addition, repair mechanisms were studied directly by creating a transgenic inducible DSB system by integrating an I-SceI cleavage site into the P. falciparum genome and sequencing of the repaired locus after induction of the I-SceI endonuclease (4). Our data strongly suggest that phosphorylation of PfH2A could be used as a specific and immediate marker for damaged DNA in P. falciparum. Therefore, we were interested in examining the dynamics of this phosphorylation over time after exposure FIG 2 DNA damage in P. falciparum causes histone H2A phosphorylation. (A) DNA fragmentation imaging by TUNEL assay of RBCs infected with NF54 P. falciparum parasites exposed to X-ray irradiation (6,000 rads) showing nuclear foci of damaged DNA. (B) Immunofluorescence analysis of Xray-irradiated parasites (6,000 rads), using anti-g-H2A.X (S P Q) antibody, shows foci of phosphorylation signal in the nucleus. (C) Western blot analysis, using anti-g-H2A.X(S P Q) antibody, of protein extracts from parasites exposed to increasing levels of X-ray irradiation (left) and from parasites treated with increasing concentrations of H 2 O 2 (right). (D and E). The anti-g-H2A.X (S P Q) antibody specifically recognizes phosphorylated PfH2A and does not cross-react with nonphosphorylated PfH2A. Proteins from parasites, which were irradiated with increasing doses of X-ray radiation (control [no irradiation] and 1,000 and 6,000 rads), were subjected to Western blot analysis using either anti-g-H2A.X (S P Q) antibody or anti-H2A antibody. The membrane was either incubated with calf intestine phosphatase (CIP) before incubation with the antibodies (D) or incubated with the antibodies, stripped, treated with CIP, and reincubated with the antibodies (E). Anti-aldolase antibody was used as a loading control. The anti-g-H2A.X (S P Q) antibody detected increasing levels of protein associated with the increasing levels of irradiation only without CIP treatments, while the anti-H2A antibody detected constant protein levels even after CIP treatment. ## Fig 3 Histone extraction followed by mass spectrometry shows that serine 121 of PfH2A is phosphorylated upon exposure to X-ray irradiation. (A) SDS-PAGE analysis of histone extraction and purification from X-ray-irradiated (6,000 rads) and untreated parasites. (B) Western blot analysis of total histone extracted from untreated and X-ray-treated (6,000 rads) parasites using anti-g-histone H2AX (S P Q) and anti-PfH2A antibodies. (C) X-ray irradiated parasites (6,000 rads) were subjected to immunoprecipitation (IP), using anti-H2A antibody, followed by Western blot analysis using both anti-g-H2A.X (S P Q) and anti-H2A antibodies. (D) Trypsin digestion followed by mass spectrometry analysis identified phosphorylation of serine 121 of PfH2A in the irradiated parasites. to X-ray irradiation, hypothesizing that it could be exploited to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms. We exposed parasite cultures to different levels of X-ray irradiation and measured the levels of PfH2A phosphorylation over time. We observed that the levels of PfH2A phosphorylation, which had increased immediately after irradiation, decreased already 3 h after irradiation to levels that were similar to those prior to irradiation. These data imply that during this period, the parasites were able to activate their DNA repair machinery, and thus, these dynamics could be used as a valuable tool to study DNA repair in malaria parasites. # Discussion In any living organism, the ability to repair damaged DNA is key for maintaining genome integrity. This DNA damage repair (DDR) machinery should be extremely efficient in organisms, such as Plasmodium parasites, that are continuously exposed to numerous intrinsic and exogenous sources that may damage their DNA. In addition to its crucial role for the parasites' basic biological functions under these conditions, efficient DDR machinery contributes to the parasites' ability to expand their antigenic repertoire and to maintain mutations that enable them to resist drug treatment. However, although many regulators of DDR were identified encoded in the Plasmodium genome, the mechanisms for DDR in these parasites remained understudied and poorly understood. A major obstacle for advancing our knowledge on DDR machinery in Plasmodium is the lack of good molecular markers for damaged DNA and the inability to perform an accurate assay that directly measures the kinetics of DNA repair. Here, we show that exposure of P. falciparum parasites to X-ray irradiation and H 2 O 2 , which cause double-strand breaks (DSB), leads to increased phosphorylation of the canonical PfH2A. We found that although Plasmodium has no H2A.X variant, the canonical PfH2A is phosphorylated on the SQ motif found in its C9-terminal tail and that the phosphorylated PfH2A could be differentiated from the nonphosphorylated form of this core histone protein. Since PfH2A is phosphorylated following exposure to DNA-damaging agents, the quantitative measurement of phosphorylated PfH2A can act as a sensitive molecular marker for DNA damage in P. falciparum. Most of the approaches employed to date to study DNA damage in Plasmodium rely on measuring the relative instability of DNA damage products under alkaline conditions (comet assay)or on the relative expression of DNA damage and repair genes (reverse transcription-quantitative PCR [qRT-PCR]). In addition, thus far, the ability of P. falciparum parasites to repair DNA damage has been estimated by the rate of recovery of parasite populations exposed to DNA-damaging agents, i.e., the time it takes for these populations to reach approximately 5% parasitemia (usually 10 to 20 days) (10). Any delay in recovery was then interpreted as a malfunction of the repair machinery, which is, of course, indirect evidence reflected 2 weeks after the actual repair has happened. In this manner, the analysis of PfH2A phosphorylation kinetics can fulfill the need for a direct, simple, sensitive/quantitative, and reproducible way of measuring DNA damage and repair kinetics in Plasmodium in a timescale of minutes to hours, which better represents the velocity of the repair machinery. In higher organisms, phosphorylation of histone variant H2A.X is a highly specific and sensitive molecular marker for monitoring DNA damage and repair. However, in some organisms, other histone H2A variants undergo phosphorylation in response to exposure to DNA damage. For example, in Drosophila melanogaster, H2A.Z is phosphorylated in response to DNA damage instead of H2A.X, and in the budding yeast Saccharomyces cerevisiae, which does not encode an H2A.X variant, the with different doses of X-ray radiation (i.e., 500, 1,000, and 6,000 rads) and put back in culture (3 h and 6 h) to allow them to repair their damaged DNA. Protein extracts from these parasites were then used for Western blot analysis with anti-g-H2A.X (S P Q) antibody and anti-aldolase antibody as a loading control. The ability of the parasites to repair their damaged DNA is demonstrated by the rapid reduction in the levels of phosphorylated PfH2A found 3 h after irradiation. Lanes C, parasites that were not exposed to irradiation and used as control. (B) Semiquantitative densitometry analysis of the Western blot presented in panel A. Shown is quantification of the changes in the ratio between the signal detected by the anti-g-H2A.X and anti-aldolase antibodies before irradiation, immediately after irradiation, and 3 h and 6 h after irradiation. The density of each band is presented as a proportion of the total signal obtained. canonical H2A is phosphorylated at the serine found near its C terminus at an SQ motif, similar to what we report here for P. falciparum. This also appears to be the case in protozoan species in which histone H2A.X is either missing or replaced by other histone variants. A marked example is in Trypanosoma brucei and other trypanosomatids as well, in which histone H2A undergoes phosphorylation at a threonine residue (Thr 130) instead of serine in response to DNA damage. Interestingly, in the apicomplexan parasite Toxoplasma gondii, which does contain an H2A.X variant, the canonical H2A (also named H2A1) was also proposed to be phosphorylated at a C-terminal SQ motif as a response to DSB. This may suggest the possibility of functional redundancy among these variants that could be exploited through evolution for functional replacement by the canonical H2A when the H2A.X variant is missing. This is somehow supported by the high level of conservation of the SQ motif in the canonical H2A of other protozoan and in particular in other Plasmodium species that face similar exposure to sources of DNA damage, such as P. falciparum. Interestingly, lower eukaryotes prefer high-fidelity HR as the mechanisms to repair DSB, while higher eukaryotes prefer nonhomologous DNA end joining (NHEJ). We noted that both P. falciparum and S. cerevisiae, which phosphorylate their canonical H2A proteins in response to DSB, use HR for repair, while T. gondii, which encodes and phosphorylates its H2A.X variant, uses mainly NHEJ, similar to higher eukaryotes. One can speculate whether the preference for NHEJ might have evolved in association with the H2A.X variant. In higher eukaryotes, histone H2A.X is known to be phosphorylated by members of phosphatidylinositol 3-kinase family (PI3K) namely, ataxia telangiectasia mutated (ATM) kinase, ATM Rad-3-related kinase (ATR), and DNA-dependent protein kinase (DNA-PK). However, to the best of our knowledge, only one PI3K was identified in Plasmodium (24), while other members of this family are not well characterized. Interestingly, in the apicomplexan parasite T. gondii, an ATM kinase orthologue (TGME49_248530) was proposed to be involved in TgH2A.X phosphorylation. Incubation of cultured parasites with a known ATM kinase inhibitor (KU-55933), which was tested as a potential anti-T. gondii agent, caused cell cycle arrest and was able to inhibit phosphorylation of TgH2A.X. The single PI3K which was previously identified in P. falciparum (PF3D7_0515300) shows some sequence conservation with T. gondii (TGME49_248530) and human (NCBI:protein database accession number AAB65827) ATM kinases. However, this P. falciparum PI3K (PF3D7_0515300), which was shown to be important for hemoglobin digestion, was localized to the parasite plasma membrane (PM), parasitophorous vacuole membrane (PVM), and food vacuole (FV) but did not appear to be localized to the nucleus. Thus, the Plasmodium ATM kinase homologue that phosphorylates PfH2A has yet to be identified. Overall, the identification of PfH2A phosphorylation as a marker for DNA damage and the ability to quantify and time the appearance and disappearance of this marker in response to exposure to sources of DNA damage open new possibilities for understanding the mechanisms of DNA damage and repair that contribute to the persistence and pathogenicity of these important pathogens. # Materials and methods Parasite culture. All experiments were conducted on the human malaria NF54 parasite line. The parasites were cultivated at 37°C in an atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen at 5% hematocrit in RPMI 1640 medium, 0.5% Albumax II (Invitrogen), 0.25% sodium bicarbonate, and 0.1 mg/ml of gentamicin. The parasites were synchronized using Percoll/sorbitol gradient method in which infected RBCs were layered on a step gradient of 40/70% Percoll containing 6% sorbitol. The gradient was subsequently centrifuged at 12,000 Â g for 20 min at room temperature. The late-stage synchronized parasites were recovered from the interphase, washed twice with complete culture medium, and placed back in culture. The percent parasitemia was calculated by SYBR green I DNA stain (Life Technologies) using a CytoFLEX (Beckman Coulter) flow cytometer. Bioinformatics analyses. The full-length sequences of histone H2A and its variants were obtained from different species based on sequence similarity. Multiple-sequence alignment of PfH2A (PF3D7_0617800) and other histone H2A variants from different species was performed using CLUSTALW and further analyzed using ESPript3 program. A homology model of the PfH2A was built by comparative modeling using the crystal structure of histone H2A (Protein Data Bank entry 1EQZ, chain A) by using the SWISS-MODEL server. The structure visualization of the PfH2A three-dimensional (3D) model was performed using the PyMOL program. DNA damage of parasites by X-ray irradiation and H 2 O 2 . DNA damage in the parasites was performed by X-ray irradiation using a PXi precision X-ray irradiator set at 225 kV and 13.28 mA. In brief, 2% ring-stage NF54 parasites were exposed to different doses of X-ray irradiation (1,000, 3,000, and 6,000 rads). After irradiation, parasites were either collected immediately (i.e., 15 min after irradiation) or put back in culture with fresh medium for further analysis at different time points as mentioned elsewhere. The level of DNA damage was measured by in situ DNA fragmentation (TUNEL) assay and phosphorylated H2A by Western blotting as described above. To check the hydrogen peroxide (H 2 O 2 )-mediated DNA damage, ring-stage-infected RBCs (;2%) were treated with different concentrations of H 2 O 2 (0 to 10, 50, 100, and 400 mM) for 1 h at 37°C. Parasites were collected from RBCs by saponin lysis, and the level of DNA damage was measured by phosphorylated H2A following Western blotting as described above. In situ DNA fragmentation (TUNEL) assay. Tightly synchronized ring-stage parasites (NF54) were fixed for 30 min in freshly prepared fixative (4% paraformaldehyde and 0.005% glutaraldehyde). After fixation, cells were rinsed three times with phosphate-buffered saline (PBS) and incubated with permeabilization solution (0.1% Triton X-100 in PBS) for 10 min on ice. The cells were washed twice with PBS and one time with wash buffer supplied with TUNEL assay kit bromodeoxyuridine (BrdU) red (Abcam; catalogue number ab6610). TUNEL assay was performed as per manufacturer guidelines. Briefly, following a washing, 50 ml of TUNEL reaction mixture (DNA labeling solution) was added to each sample. The cells were incubated for 60 min at 37°C with intermittent shaking. The cells were then washed three times with rinse buffer (5 min each time) and resuspended in 100 ml of antibody solution for 30 min at room temperature. The cells were then washed three times with PBS, mounted using Invitrogen Molecular Probes ProLong Gold antifade reagent with 49,6-diamidino-2-phenylindole (DAPI), and imaged using a Nikon Eclipse Ti-E microscope equipped with a CoolSNAPMyo charge-coupled-device (CCD) camera. Western blotting. Infected RBCs were lysed with saponin, and parasites were pelleted down by centrifugation. The parasite pellet was subsequently washed twice with PBS and lysed in 2Â Laemmli sample buffer. The protein lysates were centrifuged and the supernatants were subjected to SDS-PAGE (gradient, 4 to 20%; Bio-Rad) and electroblotted onto a nitrocellulose membrane. Immunodetection was carried out by using rabbit anti-g-H2A.X (S P Q) primary antibody (generated using a peptide containing the S P Q motif [catalogue number 9718S, 1:1,000; Cell Signaling]), anti-H2A antibody (Abcam; catalogue number ab88770, 1:1,000), and rabbit polyclonal anti-aldolase antibody (1:3,000). The secondary antibodies used were antibodies conjugated to horseradish peroxidase (HRP), goat anti-rabbit (Jackson ImmunoResearch Laboratories; 1:10,000). The immunoblots were developed in EZ/ECL solution (Israel Biological Industries). Immunofluorescence assay. Immunofluorescence assay (IFA) was performed as described previously, with minor modifications. In brief, iRBCs were washed twice with PBS and resuspended in a freshly prepared fixative solution (4% paraformaldehyde [Electron Microscopy Sciences] and 0.0075% glutaraldehyde [Electron Microscopy Sciences] in PBS) for 30 min at room temperature. Following fixation, iRBCs were permeabilized with 0.1% Triton X-100 (Sigma) in PBS and then blocked with 3% bovine serum albumin (BSA; Sigma) in PBS. Cells were then incubated with primary rabbit anti-g-H2A.X (S P Q) (Cell Signaling; catalogue number 9718S, 1:300) and anti-H2A (Abcam; catalogue number ab8870, 1:100) antibodies for 1.5 h at room temperature and washed three times in PBS. Following this, cells were incubated with Alexa Fluor 488 goat anti-rabbit (Life Technologies; 1:500) or Alexa Fluor 568 goat anti-rabbit (Life Technologies; 1:500) antibodies for 1 h at room temperature. Cells were washed three times in PBS and laid on polytetrafluoroethylene (PTFE) printed slides (Electron Microscopy Sciences) and mounted in ProLong Gold antifade reagent with DAPI (Molecular Probes). Fluorescent images were obtained using a Plan Apo l 100Â oil immersion lens (numerical aperture [NA] = 1.5; working distance [WD] = 130 mm) on a Nikon Eclipse Ti-E microscope equipped with a CoolSNAPMyo CCD camera. Images were processed using NIS-Elements AR (4.40 version) software. STORM imaging and analysis. Stochastic optical reconstruction microscopy (STORM) imaging was performed as described recently (29) using anti-H2A (Abcam; catalogue number ab88770, 1:150) and rabbit anti-g-H2A.X (S P Q) (Cell Signaling; catalogue number 9718S, 1:300) as primary antibodies. Alexa Schematron Fluor 594 goat anti-rabbit antibody (Life Technologies; 1:500) was used as a secondary antibody. Parasite nuclei were labeled with YOYO-1 (1:300; Life Technologies) for orientation and were not subjected to STORM. STORM was performed with a Nikon Eclipse Ti-E microscope with a CFI Apo total internal-reflection fluorescence (TIRF) Â 100 differential inference contrast (DIC) N2 oil objective (NA, 1.49; WD, 0.12 mm) as described. For each acquisition, 10,000 frames were recorded onto a 256-by 256pixel region (pixel size, 160 nm) of an Andor iXon-897 electron-multiplying CCD (EMCCD) camera. Superresolution images were reconstructed from a series of the least 5,000 images per channel using the N-STORM analysis module, version 1.1.21, of NIS Elements AR v. 4.40 (Laboratory imaging s.r.o.). Total-histone extraction. Total histones were extracted using an acid extraction method as described previously, with minor modifications. All steps were performed at 4°C in buffers containing protease and phosphatase inhibitors to protect the enzymatic interference with posttranslational modifications (PTMs). In brief, 200 ml of parasite cultures (;10% parasitemia) were saponin lysed and washed with PBS containing protease and phosphatase inhibitors. To prepare the intact nuclei, the cell pellet was resuspended in 1 ml of lysis buffer (20 mM HEPES [pH 7.8], 10 mM KCl, 1 mM EDTA, 1% Triton X-100, and 1 mM dithiothreitol [DTT]) and incubated for 30 min on rotator at 4°C. Following cell lysis, the intact nuclei were washed and pelleted by centrifugation at 10,000 Â g for 10 min at 4°C. The nuclei were resuspended in 400 ml of 0.4 N H 2 SO 4 or 0.25 N HCl. The nuclei were incubated on a rotator overnight, and supernatant containing the acid soluble histone fraction was collected after centrifugation at 16,000 Â g for 10 min.

Growth Differentiation Factor 15 Predicts Chronic Liver Disease Severity # Introduction Chronic liver disease has high global mortality rates. However, clinical outcomes are diverse due to liver remnant synthesis, causes, and the occurrence of hepatocellular carcinoma, making prognosis difficult to predict. In patients with chronic liver disease, it is critical to have a clear understanding of hepatic fibrosis and its severity because it could help clinicians predict patient prognosis and choose a liver transplant date. Over the past 20 years, many prognosis prediction models have been suggested, but each had various limitations, creating the need for a simpler and more accurate biomarker. From the clinical point of view, distinguishing between compensated and decompensated cirrhosis when predicting patient prognosis is important. However, current models including Child-Turcotte-Pugh (CTP) and Model for End-stage Liver Disease (MELD) scores cannot distinguish between compensated and decompensated cirrhosis.Growth differentiation factor 15 (GDF-15) is a transforming growth factor β (TGF-β) protein involved in infection, fibrosis, and apoptosis pathways in the presence of tissue damage or disease. GDF-15 is also known as TGF-PL (placental), macrophage inhibitory cytokine-1, prostate derived factor, placental bone morphogenetic protein, and placental growth factor-β, and its mRNA is known to be distributed particularly in the liver but also in the kidneys, heart, and lungs. In the presence of hypoxia, anoxia, inflammation, short-wave length exposure, and tissue injuries, the GDF-15 gene is expressed by activated macrophages and GDF-15 protein expression increases. [bib_ref] Divergent molecular mechanisms underlying the pleiotropic functions of macrophage inhibitory cytokine-1 in..., Mimeault [/bib_ref] The mechanisms behind this are not clearly known, but they are thought to be related to inflammation by CCR2-mediated chemotaxis and known to be involved with connective tissue fibrosis. [bib_ref] Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage..., De Jager [/bib_ref] This study aimed to investigate the effectiveness of GDF-15 for predicting liver disease severity and fibrosis as well as the applicability of biomarkers in patients with chronic liver diseases, which are primarily caused by inflammation and fibrosis. # Materials and methods ## Patients A total of 101 patients admitted for medical check-ups between August 2013 and December 2013 were included in the control group, while 145 patients with chronic liver diseases were included in the patient group and identified as having chronic hepatitis, compensated cirrhosis, or decompensated cirrhosis. Among the patients with chronic liver diseases, patients with hepatocellular carcinoma were excluded from the study. Patients on anti-inflammatory drugs such as aspirin and steroids, diagnosed with other cancers, or with autoimmune diseases such as gout and rheumatic arthritis were also excluded from this study because these factors could affect GDF-15 concentrations. In addition, considering the traditional technology that could use GDF-15 as the biomarker for infections, patients with damaged liver cells from past infections in the past were tested, and the result showed that the GDF-15 blood concentrations were uniformly increased. Hence, patients with toxic or acute hepatitis were also excluded from this study. Decompensated cirrhosis was defined as the patient having complications such as variceal bleeding, ascites, encephalopathy, and jaundice. [bib_ref] Compensated cirrhosis: natural history and prognostic factors, Ginés [/bib_ref] Compensated cirrhosis was identified when patients did not display any of the aforementioned complications. # Methods Once informed consent is obtained, demographical characteristics, clinical characteristics, and laboratory results of each group were investigated and analyzed and the patients were followed on an outpatient basis. Blood was collected from patients who agreed to genetic testing. The study was reviewed and approved by Chungnam National University School of Medicine, Institutional Review Board. The blood concentration of GDF-15 measured by enzymelinked immunosorbent assay (ELISA) using a human GDF-15 ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). Body mass index (BMI), aspartate aminotransferase (AST), alanine aminotransferase (ALT), cirrhosis discriminant score (CDS), Child-Pugh score, and MELD score were measured to compare them with other indexes. BMI was calculated using the formula (weight [kg]/height [m 2 ]). The blood specimens were centrifuged, and AST and ALT activity and blood concentration analyses were performed of the supernatant. The blood analysis kit was then used according to the manufacturer's instructions. CDS measured the number of platelets in the blood specimen and the prothrombin time. The obtained score was added accordingly to the score system and then classified. The Child-Pugh score was based on the sum of the scores from following systems: (1) serum bilirubin <2 mg/dL (score 1), 2 to 3 mg/dL (score 2), >3 mg/dL (score 3); (2) serum albumin >3.5 g/dL (score 1), 2.8 to 3.5 g/dL (score 2), <2.8 g/dL (score 3); (3) prothrombin time international normalized ratio (PT INR) <1.70 (score 1), 1.71 to 2.20 (score 2), >2.20 (score 3); (4) no ascities (score 1), adjustable ascites (score 2), nonadjustable ascites (score 3); (5) no hepatic encephalopathy (score 1), easily treated hepatic encephalopathy (score 2), noneasily treated hepatic encephalopathy (score 3). MELD score was calculated by the equation of 9.6×ln (creatinine mg/dL)+3.8×ln (bilirubin mg/dL)+11.2×ln (INR)+6.4. # Statistical analysis The resulting values are expressed as mean±standard deviation and the statistical analyses were performed using SPSS for Windows version 21 (IBM Corp., Armonk, NY, USA). The pvalues <0.05 were considered statistically significant. Patient gender, disease causes, and clinical information were compared using Fisher exact test, while the ×2 chi square test was used for categorical data. The correlation among GDF-15, CDS, MELD score, and CTP score was replaced by the log value and analyzed using Spearman correlation coefficients. Determinates of GDF-15 levels were tested using a univariate binary logistic regression analysis. All variables, which were tested significant in the univariate model, were inserted in a multivariate binary logistic regression model with inclusion method. Results of binary logistic regression analysis were presented as odds ratio and 95 % confidence interval (CI). To confirm the effectiveness of GDF-15 in diagnosis, the receiver operating characteristic (ROC) curve was used to determine the area under the ROC curve. The cutoff value was set up to find the sensitivity and specificity as well as the predictability for positives and negatives. # Results ## Clinical characteristics of the patient group A total of 246 blood samples collected for this study from 101 healthy adults and 145 patients with chronic liver disease were used, and the participants' basic characteristics are shown in [fig_ref] Table 1: Characteristics of Study Participants [/fig_ref]. The control group consisted of 55 men and 46 women (n=101). The ratios of men to women in the chronic hepatitis, compensated cirrhosis, and decompensated cirrhosis groups were 28/26, 26/18, and 18/29, respectively, and no significant difference was seen among groups (p=0.741). The average age of the control group was 46±9 years, while those of the chronic hepatitis, compensated cirrhosis, and decompensated cirrhosis groups were 45±13, 59±10, and 56±12 years, respectively, showing that those in the control group and chronic hepatitis group were significantly younger (p<0.01). Causes of chronic liver disease in the 54 patients with chronic hepatitis consisted of hepatitis B virus (n=39), hepatitis C virus (n=12), and alcohol intake (n=3), while causes of compensated cirrhosis included hepatitis B virus (n=22), hepatitis C virus (n=7), and alcohol (n=7), and other autoimmune diseases or unknown causes (n=8) and causes of decompensated cirrhosis included hepatitis B virus (n=8), hepatitis C virus (n=8), alcohol (n=27), and other (n=4), respectively. The average BMI of the control and chronic liver diseases groups were 24.2±2.94, 24.1±3.7, 22.8±5.9, and 24.1±3.6, respectively; there were no statistically significant differences among groups (p=0.382). The average AST level was higher in the liver disease group than in the control group, but there was no major difference seen among the liver disease subgroups. The average ALT level was higher in the chronic hepatitis and compensated cirrhosis groups than in the control and decompensated cirrhosis groups. The CDS, CTP score, and MELD score, which can predict liver fibrosis and severity in patients with cirrhosis, were 6.7±1.6 versus 8.7±1.7, 5.3±0.7 versus 8.2±1.9, and 8.7±2.7 versus 13.8±4.7, respectively, significantly higher in the patients with decompensated cirrhosis (p<0.01). In a multivariate analysis, GDF-15 levels were independent predictors of chronic liver diseases [fig_ref] Table 2: Multivariate Analysis of Blood GDF-15 Concentration Using a Multivariate Logistic Regression Test [/fig_ref]. ## Blood gdf-15 concentration in patients with chronic liver diseases When the blood GDF-15 concentration was measured within the 95% CI of each patient group, the average value in the control group was 490 ng/L, but that value rapidly increased in the chronic liver disease patients to >1,000 ng/L. In particular, GDF-15 concentration in the decompensated cirrhosis group was 3,483 ng/L, which was clearly higher than that in the chronic hepatitis patients at 1,232 ng/L and that in the compensated cirrhosis patients at 1,861 ng/L, indicating a statistically significant difference (p<0.01). 3. Correlation between serum marker assessment (MELD score, CTP score, and CDS) in liver fibrosis and severity prediction and GDF-15 When the significant serum markers from the analysis of correlation between the factors that could predict fibrosis and liver disease severity and GDF-15 were ranked from the highest bivariate Spearman rank correlation coefficient to the lowest, the results were as follows: Child-Pugh score (r=0.474), CDS (r=0.447), and MELD score (r=0.415) (p<0.01). Furthermore, Child-Pugh score and MELD score showed the highest positive correlation of 0.873. ## Auc value of gdf-15 in predicting chronic liver disease severity The AUC value of GDF-15 in the ROC curves for chronic hepatitis, compensated liver cirrhosis, and decompensated liver cirrhosis patients were 0.656, 0.886, and 0.984, respectively [fig_ref] Figure 2: Receiver operating characteristic [/fig_ref]. ## Sensitivity, specificity, and predictability for positives and negatives of gdf-15 in predicting severity in chronic liver diseases On the ROC curve, the value with highest specificity and rapid decrease in sensitivity was set as the optimal cutoff value. When the optimal cutoff value was set as 574 ng/L for patients with chronic hepatitis, the predictability of GDF-15 had a sensitiv-ity of 53.7%, specificity of 79.2%, predictability for positives of 58.0%, and predictability for negatives of 76.2%. When the optimal cutoff value was set at 760 ng/L for patients with compensated liver cirrhosis, the predictability of GDF-15 had a sensitivity of 75.6%, specificity of 92.1%, predictability for positives of 81.0%, and predictability for negatives of 89.4%. When the optimal cutoff value was set at 869 ng/L for patients with decompensated liver cirrhosis, the predictability of GDF-15 had a sensitivity of 97.9%, specificity of 94.1%, predictability for positives of 88.5%, and predictability of negatives of 99.0% [fig_ref] Table 3: Diagnostic Accuracy of GDF-15 for Chronic Liver Disease [/fig_ref]. # Discussion Some new facts were discovered through this study. First, patients with chronic liver diseases had higher GDF-15 concentrations than patients in the control group, and the patients with more severe liver diseases showed proportionally higher GDF-15 values. To date, studies have reported that a high serum GDF-15 value was related to lymph node metastasis and a low survival rate in endometrial cancers [bib_ref] Elevated plasma growth differentiation factor-15 correlates with lymph node metastases and poor..., Staff [/bib_ref] and increased incidence of oral leukoplakia, squamous cell carcinoma, and prostate cancer. [bib_ref] Elevated level of serum growth differentiation factor 15 is associated with oral..., Yang [/bib_ref] [bib_ref] Growth differentiation factor-15 upregulates interleukin-6 to promote tumorigenesis of prostate carcinoma PC-3..., Tsui [/bib_ref] Additionally, it was reported as the predicting factor of reoccurrence of colorectal cancer 8 and the independent predicting factor of the mortality from cardiovascular diseases. [bib_ref] Prognostic value of growthdifferentiation factor-15 in patients with non-ST-elevation acute coronary syndrome, Wollert [/bib_ref] [bib_ref] Growth differentiation factor-15 as a prognostic marker in patients with acute myocardial..., Khan [/bib_ref] In addition, GDF-15 was reported to be related to abdominal obesity and insulin resistance, and it was reported as the predicting factors of insulin resistance. [bib_ref] Growth differentiation factor 15 predicts future insulin resistance and impaired glucose control..., Kempf [/bib_ref] There are few papers on plasma levels of GDF-15 in chronic liver disease, but Liu et al. [bib_ref] Association of serum level of growth differentiation factor 15 with liver cirrhosis..., Liu [/bib_ref] reported that GDF-15 increased the sensitivity and specificity of diagnosis of hepatocellular carcinoma accompanied with cirrhosis when measured with AFP. In addition to this paper, Si et al. [bib_ref] Growth differentiation factor 15 is induced by hepatitis C virus infection and..., Si [/bib_ref] reported that GDF-15 was associated with the pathogenesis of hepatitis C virus. But so far, no studies to date have investigated the blood GDF-15 concentration in chronic liver diseases. This study is the first to confirm that patients with more severe chronic liver diseases had proportionately higher GDF-15 values. Second, the increase in GDF-15 seemed to be caused by fibrosis rather than the hepatocellular damage by the infections in chronic liver diseases. To prove this, patients who had cancer, cancer recurrence, or noticeable increases in AST and ALT were excluded from this study to eliminate confounding factors that could arise in cases of cancer or acute infection. Moreover, there was no difference in the average AST and ALT values among groups of patients with chronic hepatitis, compensated liver cirrhosis, and decompensated liver despite clear differences in GDF-15 as well as strong correlations between the liver fibrosis and severity prediction models including CDS, CTP score, and MELD score (r=0.447, r=0.474, and r=0.415, respectively). In conclusion, increases in blood GDF-15 concentration were not due to hepatocellular damage from infection but appeared to be related to fibrosis level. The relationship between GDF-15 and fibrosis of various tissues and organs is increasingly being described. Overexpression of GDF-15 caused by chronic and repetitive injuries of tissues or dysfunction of regulation the expression of this cytokine is a major factor in the pathogenesis of organ fibrosis. [bib_ref] Current review on the role of transforming growth factor beta (TGF-beta) in..., Krzemień [/bib_ref] [bib_ref] Vascular endothelial growth factor (VEGF)-part 1: in physiology and pathophysiology, Kajdaniuk [/bib_ref] An increased GDF-15 expression was observed among others in patients with pulmonary, kidney, and liver fibrosis. [bib_ref] Role of transforming growth factor beta in human disease, Blobe [/bib_ref] In patients with chronic liver disease, the prolonged stimulation of hepatic stellate cells being the result of chronic damage to hepatocytes results in the release of profibrogenic abundant factors as GDF-15 and leads to the development of liver cirrhosis. GDF-15 leads to the extracellular matrix (ECM) accumulation in the mechanism: (1) directly increasing the synthesis of ECM components as procollagen 1a, (2) inhibition of tissue collagenases expression, (3) increasing synthesis of ECM-degrading enzyme inhibitors (as plasminogen activator inhibitor type 1, tissue inhibitors of metalloproteinases). [bib_ref] Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth..., Matsuzaki [/bib_ref] [bib_ref] Differential regulation of TGF-beta signal in hepatic stellate cells between acute and..., Tahashi [/bib_ref] [bib_ref] TGF-beta1 up-regulates the expression of PDGF-beta receptor mRNA and induces a delayed..., Shah [/bib_ref] Patients with chronic liver diseases would have deposited not only extracellular protein but collagen when liver fibrosis occurred; continuous deposits of such substances in the liver would result in structural changes of the liver tissues and induce functional liver impairment, which ultimately leads to portal hypertension and clinical complications. Therefore, understanding liver fibrosis severity is important in predicting the prognosis of patients with chronic liver diseases and choosing treatment and surveillance periods; it is also used to assess treatment reactions. Regarding studies of the relationships between GDF-15 and fibrosis, when Lambrecht et al. [bib_ref] Growth differentiation factor 15, a marker of lung involvement in systemic sclerosis,..., Lambrecht [/bib_ref] measured the serum GDF-15 concentration in 119 patients with systemic sclerosis, disease activity and organ involvement showed a strong relationship; in particular, more severe fibrosis in the liver showed a higher GDF-15 concentration. Lok et al. [bib_ref] Circulating growth differentiation factor-15 correlates with myocardial fibrosis in patients with non-ischaemic..., Lok [/bib_ref] identified a relationship between myocardial fibrosis and GDF-15 in nonischemic dilated cardiomyopathy. No studies have been performed on the correlation between GDF-15 and liver fibrosis in patients with chronic hepatitis or liver cirrhosis. This study confirmed that there was a strong relationship between them. Finally, the most critical result of this study was that GDF-15 showed extremely high sensitivity and specificity in distinguishing between chronic hepatitis and liver cirrhosis, and especially in distinguishing the patients with compensated and decompensated liver cirrhosis (at a cutoff of >869 ng/L; sensitivity, 97.9%; specificity, 94.1%). Of the previous prediction models, CDS is a prediction model for liver fibrosis that is composed of platelets, AST/ALT ratio, and PT INR. A score >8 may reduce the need for liver biopsy for the diagnosis of liver fibrosis, [bib_ref] Utility of a discriminant score for diagnosing advanced fibrosis or cirrhosis in..., Bonacini [/bib_ref] but it fails to predict chronic liver disease severity. Also, CTP and MELD scores are applied to the prediction models for liver disease severity. CTP scoring had the following weaknesses: two of its five factors, ascites and hepatic coma, are not objective; all factors were treated the same regardless of weighted values, the cutoff value was arbitrarily set without any statistical basis; and patients within the same class were not uniform, so they could have different prognoses. [bib_ref] Predicting surgical risk in patients with cirrhosis: from art to science, O&apos;leary [/bib_ref] Unlike CTP score, MELD score is composed only of objective factors and gives weighted values to factors according to its importance. It is also continuous without the use of arbitrary restrictions. The strength of the MELD score was that even an increase in a single score was proportional to the risk, but it was ambiguous for selecting among the different factors used in multivariable analysis for completing the model and it ignored the importance of ascites; unlike CTP score, which could easily be calculated and applied to clinical settings, its calculation was complicated. [bib_ref] Predicting surgical risk in patients with cirrhosis: from art to science, O&apos;leary [/bib_ref] Furthermore, most critically, it could not distinguish between or describe compensated and decompensated liver cirrhosis.In such cases, the use of GDF-15 is convenient for comparing CDS, CTP score, and MELD score and able to more accurately predict liver fibrosis and liver disease severity. In particular, GDF-15 is believed to be a biomarker that is capable of diagnosing compensated and decompensated liver cirrhosis, which is clinically critical. Limitations of this study were expressed as follows. Firstly, because a small number of patient group, it was not possible to analyze association between GDF-15 according to the cause of chronic liver disease. And secondly, liver biopsy was not performed to examine the association of GDF-15 and fibrosis. Despite having a number of limitations, our work generates important findings. In other words, this is the first study to investigate the effectiveness of GDP-15 in patients with chronic liver diseases, and our findings confirmed it to be a very valuable biomarker for predicting liver fibrosis and chronic liver disease severity. In particular, in the diagnosis of clinically important decompensated cirrhosis, it showed high diagnostic rate; when the cutoff valve of 869 ng/L was set, it had a sensitivity of 97.9% and specificity of 94.1%. ## Conflicts of interest No potential conflict of interest relevant to this article was reported. [fig] Figure 1: (A) Correlation between the Model for End-stage Liver Disease (MELD) score and growth differentiation factor 15 (GDF-15); (B) Correlation between the Child-Pugh score and GDF-15; (C) Correlation between the cirrhosis discriminant score (CDS) and GDF-15. When the significant serum markers from the analysis of the correlations between the factors that predicted fibrosis and liver disease severity and GDF-15 were ranked from the highest bivariate Spearman rank correlation coefficient to the lowest, the results were as follows: Child-Pugh score (r=0.474), CDS (r=0.447), and MELD score (r=0.415) (p<0.01). [/fig] [fig] Figure 2: Receiver operating characteristic (ROC) curve of the growth differentiation factor 15 (GDF-15) value in patients with chronic liver diseases (A), chronic hepatitis (B), compensated liver cirrhosis (C), and decompensated liver cirrhosis (D). The area under the curve (AUC) value of GDF-15 in the ROC curves for chronic liver disease, chronic hepatitis, compensated liver cirrhosis, and decompensated liver cirrhosis patients were 0. [/fig] [table] Table 1: Characteristics of Study Participants (n=246) [/table] [table] Table 2: Multivariate Analysis of Blood GDF-15 Concentration Using a Multivariate Logistic Regression Test [/table] [table] Table 3: Diagnostic Accuracy of GDF-15 for Chronic Liver Disease [/table]

Identification to species level of live single microalgal cells from plankton samples with matrix-free laser/desorption ionization mass spectrometry Introduction Marine planktonic communities are complex microbial consortia often dominated by microscopic algae. The taxonomic identification of individual phytoplankton cells usually relies on their morphology and demands expert knowledge. Recently, a live single-cell mass spectrometry (LSC-MS) pipeline was developed to generate metabolic profiles of microalgae. Objective Taxonomic identification of diverse microalgal single cells from collection strains and plankton samples based on the metabolic fingerprints analyzed with matrix-free laser desorption/ionization high-resolution mass spectrometry. Methods Matrix-free atmospheric pressure laser-desorption ionization mass spectrometry was performed to acquire singlecell mass spectra from collection strains and prior identified environmental isolates. The computational identification of microalgal species was performed by spectral pattern matching (SPM). Three similarity scores and a bootstrap-derived confidence score were evaluated in terms of their classification performance. The effects of high and low-mass resolutions on the classification success were evaluated. Results Several hundred single-cell mass spectra from nine genera and nine species of marine microalgae were obtained. SPM enabled the identification of single cells at the genus and species level with high accuracies. The receiver operating characteristic (ROC) curves indicated a good performance of the similarity measures but were outperformed by the bootstrapderived confidence scores. Conclusion This is the first study to solve taxonomic identification of microalgae based on the metabolic fingerprints of the individual cell using an SPM approach. # Introduction Phytoplankton inhabiting aquatic ecosystems worldwide is highly complex and can contain thousands of interacting species, which coexist and potentially compete for the same 1 3 28 Page 2 of 10 resources [bib_ref] Chemical ecology of marine plankton, Schwartz [/bib_ref]. The classical taxonomic identification of larger phototrophic microalgae over 20 µm is based on morphological features of the cells that are investigated by light or electron microscopy. The identification requires expert knowledge and often leads to misclassifications due to morphological similarities of different species. Automated approaches, such as PlanktoVision have been developed and use a neural network that analyzes light-microscope pictures to classify microalgae [bib_ref] Plank-toVision-An automated analysis system for the identification of phytoplankton, Schulze [/bib_ref]. More advanced workflows combine the algorithm-assisted picture analysis with fluorescence information, and yield taxonomic resolution on a single-cell level, even deriving the growth phase of the analyzed microalgae [bib_ref] Combining high-throughput imaging flow cytometry and deep learning for efficient species and..., Dunker [/bib_ref]. This approach has been applied for identification based on features from morphological criteria (e.g. ornamentation, contour, shape) or image characteristics (e.g. transparency, area) of living algal cells [bib_ref] Automated taxonomic classification of phytoplankton sampled with imaging-in-flow cytometry, Sosik [/bib_ref] [bib_ref] Automatic plankton image classification combining multiple view features via multiple kernel learning, Zheng [/bib_ref] or purified diatom cell walls [bib_ref] Automated diatom classification (part A): Handcrafted feature approaches, Bueno [/bib_ref]. Complementary molecular genetic methods determining the taxonomic identity of algae are available since the 1970s and include now the genome sequencing of single microbial cells [bib_ref] Single cell genomics: An individual look at microbes, Stepanauskas [/bib_ref]. Nevertheless, most of these existing methods require time-consuming sample preparation and do not give information on the physiological status of cells, nor on cellular metabolites. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms such as bacteria, fungi, and algae, based on mass spectra of protein extracts from groups of cells [bib_ref] Rapid characterization of microalgae and microalgae mixtures using matrix-assisted laser desorption ionization..., Barbano [/bib_ref] [bib_ref] New method for the identification of arbuscular mycorrhizal fungi by proteomic-based biotyping..., Crossay [/bib_ref] [bib_ref] MALDI-TOF MS based discrimination of coccoid green microalgae (Selenastraceae, Chlorophyta), Mello [/bib_ref] [bib_ref] MALDI-TOF MS profilingadvances in species identification of pests, parasites, and vectors, Murugaiyan [/bib_ref]. MALDI-TOF mass spectra analysis is established for the automated identification of bacteria by spectral pattern matching (SPM) and clustering algorithms, even when consortia of organisms were analyzed [bib_ref] Characterization of microbial mixtures by mass spectrometry, Sandrin [/bib_ref] [bib_ref] Direct MALDI-TOF MS identification of bacterial mixtures, Yang [/bib_ref]. However, a group of cells, even sampled from a single clonal culture, is far from being homogeneous. Cells of the same population with very different physiological status can coexist (e.g. mitotically dividing, encysting, sexually reproducing, actively growing, resisting against pathogen). Working at the single cell level is the only way to explore both the identity and the physiology of a specimen. Single-cell analysis has been introduced in the last decade to profile the cellular content, providing information on the genes, proteome, transcriptome or metabolome of the organism under study [bib_ref] Challenges and emerging directions in single-cell analysis, Yuan [/bib_ref]. Single-cell mass spectrometry of algal cells has already been developed with matrix-free laser desorption-ionization using the ionization-enhancing effect of diatom cell walls [bib_ref] Matrix-free single-cell LDI-MS investigations of the diatoms Coscinodiscus granii and Thalassiosira pseudonana, Jaschinski [/bib_ref]. Live single-cell mass spectrometry (LSC-MS) with laser-desorption ionization high-resolution mass spectrometry was developed to profile reliably the low-molecular-weight metabolites of living cells kept in their native environment prior to analysis. This allows the study of the cellular physiology in microalgae . High-resolution atmospheric pressure scanning microprobe laser desorption/ionization mass spectrometry with its high spatial resolution (10 µm) enables individual targeting of intact live microbial cells under ambient conditions. Coupling of the source to an Orbitrap mass spectrometer provides high-resolution mass spectra [bib_ref] Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging, Schober [/bib_ref]. The analysis of such high-throughput data is however challenging. To date, only a few bioinformatic classification tools are available for (MA)LDI derived workflows, including the Bruker Biotyper® [bib_ref] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial..., Bizzini [/bib_ref] , VITEK® MS [bib_ref] Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for..., Branda [/bib_ref] and the freeware Matlab based tool MicrobeMS. Recently, [bib_ref] Bacterial whole cell typing by mass spectra pattern matching with bootstrapping assessment, Yang [/bib_ref] introduced an optimized SPM pipeline, whereby the reliability of the prediction was boosted by confidence scores that were derived from the identification results of bootstrap spectra from the query spectrum [bib_ref] Bacterial whole cell typing by mass spectra pattern matching with bootstrapping assessment, Yang [/bib_ref]. This study aimed to demonstrate the utility of LSC-MS for the taxonomic discrimination of live single cells picked from natural samples. Therefore, we combined LSC-MS to generate single-cell mass spectra with the optimized SPM approach to enable microalgal cell identification, based solely on singlecell mass spectrometry profiles. # Materials and methods ## Sampling and identification of microalgae Algae strains were purchased from the Roscoff Culture Collection (RCC,and maintained under fluorescent lamps (irradiance 100 mE m −2 s −1 ) with a 14 h photoperiod coupled to a thermo-regulated cycle (16-12 °C day-night). Novel microalgal isolates were obtained during field sampling at the bloom season in the waters of Penzé Estuary (France, June 2018), Lesvos (Greece, May 2018), Helgoland (Germany, August 2016 and 2017) and Farsund (Norway, September 2017). The workflow from algal cell isolation to single-cell analysis is detailed in . The field samples were concentrated by filtration using nylon mesh (40, 70 and 100 µm pore size, Corning Life Sciences) and washed with sterile-filtered seawater. Single algal cells were picked by micromanipulation under a binocular stereomicroscope (binocular VisiScope®, VWR International GmbH, Pennsylvania, US). Cells were transferred into Petri dishes with 5 mL sterile natural seawater (ATI, Gebesee, Germany). Single cells were then either re-isolated by pipetting and/or purified by dilution. Almost all of the isolated cells divided and multiplied to give sufficient individuals for analysis within 15 days. Meanwhile, cultures were visualized with light microscopy, photographed, and their morphological characteristics were compared with descriptions from previous studies of phytoplankton blooms in these areas. The isolated algal cells were identified to the genus level using light microscopy. All algae from field samplings and culture collection were maintained under the same culture conditions and grown in Guillard's (F/2) enrichment medium (Sigma-Aldrich, Munich, Germany) prepared with natural 0.2 µm filtered and autoclaved seawater. Among the algal isolates, 15 were deposited in the RCC collection under strain numbers RCC6807 to RCC6821. Pictures of cells were taken with a 20 × /0.4 Ph2-Korr-Achroplan on an Axiovert 200 microscope (Zeiss, Jena, Germany). ## Lsc-ms analysis and pre-processing of single-cell spectra Single algal cells growing in the replete medium at the early growth stage were manually collected with a 20 µL pipette and deposited onto a GF/C glass fiber filter wetted with medium (Whatman, Maidstone, United Kingdom) according to [bib_ref] Live single-cell metabolomics with matrix-free laser/ desorption ionization mass spectrometry to address..., Baumeister [/bib_ref]. For chain-forming diatoms such as Chaetoceros spp. or Thalassiosira spp., only cells separated from chains were analyzed, since the spatial resolution of the laser does not allow an analysis of single cells in chains. The AP-SMALDI ion source (Trans-MIT, Gießen, Germany) was coupled to a Q Exactive™ Plus (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer to record high-resolution LSC-MS spectra. Individual cells were either analyzed in positive or negative polarity with 120 cycles per cell (1 min acquisition time). One cycle comprises 30 laser shots with a frequency of 60 Hz with an approximate energy of 1.5 μJ per shot. Mass spectra were recorded in the mass range from m/z 100 to 1000 with a resolving power of 280 000 (at m/z 200). This range was chosen to cover a sufficient amount of metabolites for fingerprinting but it could be extended if metabolites of interest fall outside of the range. Every single-cell mass spectrum represents an average of all scans from the oneminute data acquisition. MS raw files were converted with the Thermo File Converter from the Xcalibur suite 3.0.63 to the netCDF format and processed with the MALDIquant R package [bib_ref] MALDIquant: A versatile R package for the analysis of mass spectrometry data, Gibb [/bib_ref]. Sample spectra were de-noised (signal-to-noise ratio 5) and peaks co-occurring in medium blanks acquired from the sterile medium were removed. Processed spectra were conserved in the commaseparated values (CSV) file format. Upfront similarity scoring, spectra were normalized to the most abundant signal in the individual spectrum (base peak normalization). Integer mass spectra were generated from the high-resolution mass spectra by rounding the m/z values to integers and summing up the corresponding intensities of those signals that matched together after rounding. Datasheets with metadata were created (data files S11-S14), and a unique identifier (ID) was assigned to each LSC-MS profile. The metadata files contain information about the sampling site, date of isolation, date of LSC-MS analysis, growth medium used, and strain availability in a culture collection. The dataset structure and content are presented in . Spectra, R scripts, metadata files and result data files used in this study are available in the Pohnert-Lab GitHub repository (https :// githu b.com/Pohne rt-Lab/SC-MS-Ident ifica tion). Workflow of the spectral database generation and data analysis using SPM. a Microalgae were obtained from culture collection and field sampling during the bloom season. Microalgal cells from the field samplings were isolated and identified. Single algal cells were analyzed in their native environment with matrix-free laser desorption/ionization high-resolution mass spectrometry. After pre-processing, each single-cell spectrum received a unique identification num-ber and was recorded in the single-cell profile database. b Structure and content of the datasets analyzed with SPM: the collection strain dataset is a subset of the mixed dataset which includes single-cell spectra from field sampling algae. c Principle of the SPM, from the database (DB) containing N spectra, each spectrum (n) was once isolated and used as a query against the reduced DB ## Spectral similarity matching and statistical analysis Data analysis was conducted in R 3.4.2 (R Core Team 2017). Normalization, similarity scoring, and bootstrap assessment (n = 500) of top hits from spectral pattern matching (SPM) were performed based on a method established by [bib_ref] Bacterial whole cell typing by mass spectra pattern matching with bootstrapping assessment, Yang [/bib_ref]. Mass tolerance for matching of highresolution masses was set to ± 5 ppm, and for integer masses to ± 500 ppm. SPM of live single-cell mass spectra was performed with three similarity measures, the cosine correlation (Cos), the relative Euclidean distance similarity (Eu) and the intensity-weighted relative Euclidean distance similarity (iEu). Each spectrum of the in-house database was removed once from the database and used as the query . Only the match result with the highest score (top hit) was used for data evaluation. Sensitivities and error rates were calculated according to [bib_ref] Bacterial whole cell typing by mass spectra pattern matching with bootstrapping assessment, Yang [/bib_ref] , corresponding threshold scores were determined and the receiver operating characteristic (ROC) curves produced using the pROC package 1.10.0 [bib_ref] pROC: An open-source package for R and S+ to analyze and compare..., Robin [/bib_ref]. Confusion matrices were produced using the ModelMetrics package 1.1.0. A qualitative rating of the AUC values was established according to [bib_ref] Translational biomarker discovery in clinical metabolomics: An introductory tutorial, Xia [/bib_ref]. The plots displaying the number of peaks per genus or species (Figs. S2, S3) and the plots showing the frequencies of peaks per spectrum (bin size m/z 10) per genus or species (Figs. S4-S7) were produced with the ggplot2 package 2.2.1. Graphics were processed in Adobe Illustrator CS5. # Results and discussion ## Microalgae samplings and lsc-ms acquisition Microalgae belonging to the group of bloom-forming single-cell eukaryotes, coexisting in marine ecosystems, were selected for the study. Several diatom genera and one dinoflagellate genus were obtained from monoclonal public collection strains and from field samplings at many different locations in Europe. The established workflow, from algae isolation, maintenance in culture to single-cell analysis is depicted in . Single algal cells from the field samplings were purified by dilution, photographed under a light microscope , and identified based on morphological characteristics and literature. A high number of strains, mainly belonging to the genera Guinardia, Coscinodiscus, and Chaetoceros were recovered . The process of obtaining one mass spectrum of one cell, including sample preparation, data acquisition to computation can be performed within few minutes. An important advantage of the method introduced here, compared to competing techniques is the low effort in sample preparation, which involves only filtration. Also the possibility to rely on data bases for identification once expert knowledge has been put in to classify the species is superior compared to traditional light microscopy approaches. Highresolution single-cell mass spectra of intact live algal cells were acquired in negative and positive polarity . The overall data set, denoted as the "mixed dataset", contained 662 mass spectra acquired in positive (383 spectra) or negative polarity (279 spectra), obtained from 64 strains of 9 genera . A subset of the whole dataset, referred to as the "collection strain dataset", consisted of spectra obtained from 9 species from culture collections. The collection strain dataset contained 224 spectra, including 137 single-cell spectra acquired in positive polarity and 87 spectra in negative polarity . To first assess the SPM methodology a dataset that contains only spectra from cells unambiguously identified to the species level was used. Later the mixed species datasets with more genera or isolates from the field were analyzed. Most of the spectra were rich in peaks but the number of peaks per cell was dependent on the individual and varied according to genus and species (Figs. S2, S3). The total count varied in the range from less than ten to several thousand peaks per spectrum. The absolute count of peaks tended to be higher in spectra obtained in positive polarity, with Thalassiosira being an exception . The spectra were not further filtered, nor were peaks removed, as it is the practice in the generation of MALDI reference spectra of bacteria [bib_ref] Phylogenetic classification and identification of bacteria by mass spectrometry, Freiwald [/bib_ref]. Frequencies of m/z values per spectrum (bin size m/z 10, split by genus and species) showed a similar trimodaltype pattern (Figs. S4-S7), whereby the region in between m/z 100-330 usually contained most of the signals, followed by the range m/z 430-660 and few but pronounced signals were observed in the range of m/z 760-880. The following taxonomic identification approach relied entirely on the whole mass spectral fingerprint and therefore the underlying pattern produced by all detected signals. Nevertheless, the molecular classes that are principally addressed with this technique are noteworthy. It was shown that direct LDI-MS of intact microalgal cells addresses photosensitive molecules such as pigments (e.g. carotenes and chlorophyll), as well as lipids and even zwitterionic molecules such as DMSP [bib_ref] Multidimensional analysis of single algal cells by integrating microspectroscopy with mass spectrometry, Urban [/bib_ref] [bib_ref] Live single-cell metabolomics with matrix-free laser/ desorption ionization mass spectrometry to address..., Baumeister [/bib_ref]. ## Identification of microalgae based on spectral pattern matching Each single-cell mass spectrum was used as a query for the SPM and was therefore isolated from the database following the method from Yang et al. 2017. Identification success was evaluated independently for the respective polarity and spectral similarity measure. Results were visualized in confusion matrices [fig_ref] Figure 3: Confusion matrices of identification results of microalgae at the species level by... [/fig_ref] , S8-S10). A confusion matrix gives an overview of the classification success (hits) and misclassification of the queried spectra dependent on the used classifier [bib_ref] Combining high-throughput imaging flow cytometry and deep learning for efficient species and..., Dunker [/bib_ref]. The analysis of the collection strain dataset revealed convincing identification results, with overall accuracies in the range of 88.6% to 100% at the genus and 73.4% to 98.7% at the species level [fig_ref] Figure 3: Confusion matrices of identification results of microalgae at the species level by... [/fig_ref]. The similarity measures Eu and iEu performed slightly better than Cos, especially at the species level [fig_ref] Figure 3: Confusion matrices of identification results of microalgae at the species level by... [/fig_ref] , as did spectra obtained in negative polarity [fig_ref] Figure 3: Confusion matrices of identification results of microalgae at the species level by... [/fig_ref]. However, the direct comparison of both polarities is challenging, since each cell could only be analyzed by one polarity. Misclassifications at the species level often occurred in the way that species were misclassified as a species from the same genus, as indicated by the very high accuracies of up to 100% at the genus level. The mixed dataset which extended the collection strain dataset by cells from field sampling showed lower overall classification accuracies of 79.0% to 89.9% . Especially Pleurosigma and Rhizosolenia were wrongly assigned quite often to various different genera. The differences in accuracy between the similarity measures and also the ionization polarities were negligible and small. Nevertheless, the obtained accuracies are in the same range as of taxonomical experts [bib_ref] Do experts make mistakes? A comparison of human and machine identification of..., Culverhouse [/bib_ref] or machine learning approaches [bib_ref] Automatic plankton image classification combining multiple view features via multiple kernel learning, Zheng [/bib_ref] which assign the algal identity based on microscopy images. ## Statistical assessment of the spm-driven identification To evaluate the performance of the microalgal identification based on SPM, the receiver operating characteristic (ROC) curves were obtained for the three similarity measures and the bootstrap-derived confidence scores, further divided by dataset, genus or species level and polarity (Figs. 4, 5, S11, S12 a Underlying high-resolution single-cell spectra acquired in positive polarity. b Underlying high-resolution single-cell spectra acquired in negative polarity. Confidence intervals (95%) of overall accuracies are indicated above each plot sensitivity (true positive rate) and specificity (true negative rate) of one or more classifiers by constantly altering the decision threshold [bib_ref] Receiver-operating characteristic (ROC) plots: A fundamental evaluation tool in clinical medicine, Zweig [/bib_ref]. In this study, the classifiers are the three similarity measures and the corresponding bootstrap-derived confidence scores. The area under the ROC curve (AUC) is an established measure of performance of analyzed classifiers, whereby an area greater than that under the diagonal (AUC > 0.5) indicates a positive non-stochastical classification [bib_ref] The meaning and use of the area under a receiver operating characteristic..., Hanley [/bib_ref]. We first evaluated the classifier performance at the genus and species level based on the collection strain dataset . At the genus level, the best scores were obtained with Cos (AUC: 0.931) and Eu (AUC: 0.942) for single-cell spectra acquired in positive and negative polarity, respectively . Fair AUCs of 0.753 to 0.791 (positive polarity) and good to excellent AUCs of 0.850 to 0.970 (negative polarity) were reached at the species level . The scoring measures Eu and iEu surpassed Cos in all analyses, except for the spectra at the genus level in positive polarity . The classification performance dropped when the mixed dataset was used and poor to fair AUCs in the range of 0.680 to 0.789 were obtained [fig_ref] Figure 5: Receiver operating curves and corresponding areas under the curves [/fig_ref]. The bootstrap-dependent confidence scores improved the classification performance for most of the analyses, but especially for those that exhibited poor to fair AUCs when the similarity scores were used as the classifier [fig_ref] Figure 5: Receiver operating curves and corresponding areas under the curves [/fig_ref]. Furthermore, we determined threshold scores and the corresponding sensitivities at error rates below a fixed value . For example, error rates of less than 5%, sensitivities of up to 100% were achieved at the genus level using the collection strain dataset. The confidence scores yielded in general higher sensitivities than the similarity measures Cos, Eu, and iEu at the same error rate . This finding is in accordance with the initial study by classifying bacterial species by protein mass spectra [bib_ref] Bacterial whole cell typing by mass spectra pattern matching with bootstrapping assessment, Yang [/bib_ref]. Based on these results, it is recommended using the bootstrapping assessment for the classification of microalgae by single-cell mass spectrometry profiling and SPM. ## Assessment of the mass resolution on microalgal species identification In proteomics, metabolomics, and related fields, high resolution and accuracy of the analyzed masses is a desired feature [bib_ref] Categorizing cells on the basis of their chemical profiles: Progress in single-cell..., Comi [/bib_ref]. Many database-driven SPM methods still rely on unit mass resolution spectra [bib_ref] High-resolution filtering for improved small molecule identification via GC/MS, Kwiecien [/bib_ref] [bib_ref] An integrated method for spectrum extraction and compound identification from gas chromatography/mass..., Stein [/bib_ref]. SPM of bacterial spectra obtained with MALDI-TOF works with very high relative mass deviations (over 200 ppm) [bib_ref] Mass spectrometry tools for the classification and identification of bacteria, Sauer [/bib_ref]. To evaluate if the single-cell identification depends solely on high mass resolution data, the spectra were rounded to unit mass resolution, referred then as integer mass spectra, and analyzed with the SPM workflow. The number of peaks per genus and per species dropped substantially in some cases (Figs. S2, S3) and four spectra had to be removed from the database for Chaetoceros since only one peak remained in the respective spectra. However, the confusion matrices and ROC curves showed high similarities with those obtained from high-resolution spectra (Figs. S9-S12). Consequently, future acquisitions of mass spectra from microalgae could be performed at a lower resolution, which would allow the use of mass spectrometers with less resolving power, or, for Orbitrap measurements, to acquire more scans per time interval [bib_ref] Orbitrap mass spectrometry, Zubarev [/bib_ref] , hence increasing the sensitivity. Furthermore, SPM with integer mass spectra allows faster computation, since peak matching between reference and query spectrum is simplified as the mass deviation no longer has to be taken into account. However, it can be assumed that increasing the database with more algal genera and species will require more resolution to distinguish between taxonomic groups. In terms of mass spectra, high-resolution delivers greater space in the m/z domain, which would allow for more taxonomic resolution as long as the metabolite diversity correlates with the taxonomic diversity of the analyzed species. With a bigger single-cell profile database, the SPM algorithm would have to be simply optimized to avoid an increase in computation time. # Conclusion Here, we combined live-single cell mass spectrometry with an SPM approach in one workflow to reliably derive the taxonomic identity of a microalgal cell through its metabolic fingerprint. The analysis of single-cell spectra in both negative and positive polarity resulted in robust assignments of the taxonomic identity. Of the three tested similarity measures, Eu and iEu performed better in almost all Receiver operating characteristic curves and corresponding areas under the curves (AUC) for the identification of single microalgal cells for the collection strain dataset. Assessment of identification at either the genus (a-d) or species levels (e-h). The analysis was performed with bootstrap assessment (b, d, f, h) or without (a, c, e, g). High-resolution single-cell spectra recovered from the positive (a, b, e, f) or negative (c, d, g, h) polarity were independently analyzed. The AUC curves obtained for each classifier (Cos, Eu, iEu) analyzed are indicated in purple, green or orange color, respectively test situations. The bootstrap-derived confidence scores improved the classification, mainly when applied to the more diverse mixed dataset [fig_ref] Figure 5: Receiver operating curves and corresponding areas under the curves [/fig_ref]. The comparison of high-resolution spectra versus unit-mass resolution showed little gain in the success of the method at this early stage of the database development. The herein described method demands no phycological expert knowledge, once a single-cell profiling database is established, ideally as an open-source repository. Since only cultures in well-defined conditions have been investigated, future studies will implement various stress conditions, as those were shown to have a strong influence on the metabolic profile of a microalgal cell [bib_ref] Single-cell and bulk fluorescence excitation signatures of seven phytoplankton species during nitrogen..., Faulkner [/bib_ref] [bib_ref] Metabolic responses of eukaryotic microalgae to environmental stress limit the ability of..., Driver [/bib_ref]. The present approach that monitors the metabolome has the potential to generate data about the physiological state of the single cells and thus about the metabolic heterogeneity of a plankton population. The discrimination of nutrient-depleted or aged cells could already be explained with single-cell metabolic profiling [bib_ref] Single-cell mass spectrometry reveals the importance of genetic diversity and plasticity for..., Krismer [/bib_ref]. In future studies, a broader set of spectra that also takes into account the cells´ physiological condition could be implemented so that not only the cells identity but also its health status is revealed. Author contributions TB, MV, and GP conceived and designed the study. MV isolated and identified the samples from wild samplings, and maintained the algal collection. LG provided the coordinated Penzé sampling and helped with the identification. FK acquired the AP-LDI-HR-MS data. TB analyzed the data together with MV. TB and MV wrote the manuscript with contributions from GP, AS, FK, and LG. [fig] Figure 3: Confusion matrices of identification results of microalgae at the species level by Cos, Eu, and iEu, for the collection strain dataset. a Underlying high-resolution single-cell spectra acquired in positive polarity. b Underlying high-resolution single-cell spectra acquired in negative polarity. Confidence intervals (95%) of overall accuracies are indicated above each plot [/fig] [fig] 0 0, Figure 4: Cos -AUC: 0.856 Eu -AUC: 0.748 iEu -AUC: 0.861 Cos -AUC: 0.753 Eu -AUC: 0.784 iEu -AUC: 0.791 Cos -AUC: 0.893 Eu -AUC: 0.973 iEu -AUC: 0.962 Cos -AUC: 0.850 Eu -AUC: 0.970 iEu -AUC: Cos -AUC: 0.913 Eu -AUC: 0.850 iEu -AUC: 0.919 Cos -AUC: 0.931 Eu -AUC: 0.774 iEu -AUC: 0.920 Cos -AUC: 0.953 Eu -AUC: 1.000 iEu -AUC: 0.982 Cos -AUC: 0.847 Eu -AUC: 0.942 iEu -AUC: [/fig] [fig] Figure 5: Receiver operating curves and corresponding areas under the curves (AUC) for the identification of single microalgal cells at the genus level for the mixed dataset comprising collection strains and field isolates. The analysis was performed with bootstrap assessment (b, d) or without (a, c). High-resolution single-cell spectra recovered from the positive (a, b) or negative (c, d) polarity were independently analyzed. The AUC curves obtained for each classifier (Cos, Eu, iEu) analyzed are indicated in purple, [/fig]

Visual short-term memory for coherent motion in video game players: evidence from a memory-masking paradigm In this study, we investigated visual short-term memory for coherent motion in action video game players (AVGPs), non-action video game players (NAVGPs), and non-gamers (control group: CONs).Participants performed a visual memory-masking paradigm previously used with macaque monkeys and humans. In particular, we tested whether video game players form a more robust visual short-term memory trace for coherent moving stimuli during the encoding phase, and whether such memory traces are less affected by an intervening masking stimulus presented 0.2 s after the offset of the tobe-remembered sample. The results showed that task performance of all groups was affected by the masking stimulus, but video game players were affected to a lesser extent than controls. Modelling of performance values and reaction times revealed that video game players have a lower guessing rate than CONs, and higher drift rates than CONs, indicative of more efficient perceptual decisions. These results suggest that video game players exhibit a more robust VSTM trace for moving objects and this trace is less prone to external interference.www.nature.com/scientificreports www.nature.com/scientificreports/ better than non-gamers in a colour-wheel task aimed at measuring VSTM precision 29 , use a more efficient search strategy in a scene change detection task 30 , and perform better on an enumeration task than non-gamers 31 . All these results suggest enhanced VSTM for AVGPs compared to non-gamers. Additionally, Blacker and Curby 20 using a colour-change detection task showed better performance for AVGPs than NAVGPs, but the VSTM advantage did not depend on the processing speed of the to-be-remembered information. However, Appelbaum et al.32showed that AVGPs advantage in perceptual and cognitive tasks may depend on an enhanced sensitivity to visual stimuli, rather than higher capacity or longer retention of information in short-term memory, partially contradicting previous findings.In this study we further explored whether video game players exhibit better VSTM performance compared to non-gamers in a motion direction change discrimination task. Additionally, we also investigated whether video game players form a visual short-term memory representation that is more robust and less prone to external interference than in non-gamers. Data were also fitted with computational models of VSTM performance and reaction times, the results of which can inform us with respect to the underlying mechanisms in terms of precision, precision variability across trials, guessing rate, stimulus encoding efficiency and response execution.In particular, we investigated VSTM for moving objects using a memory-masking paradigm similar to that used by Pasternak and Zaksas 33 in macaque monkeys and by Pavan et al. 34 in humans. There is psychophysical and brain-imaging evidence that VSTM is selective for different attributes of the stimulus including contrast, colour, spatial frequency, orientation, speed and motion direction[35][36][37][38]investigated VSTM for global motion using a memory-masking paradigm 39 . These authors 34 presented global motion random dot kinematograms (RDKs) in two visual quadrants, whereas in the two remaining quadrants they presented random-motion RDKs. This pattern of stimulation was displayed in two distinct temporal intervals (i.e., sample and test intervals), separated in time by a 3.2 s delay interval. During the delay interval, directional masking RDKs were presented for 0.2 s in each of the four visual quadrants either at the beginning, in the middle or at the end of the delay interval. The task was to report which RDK in the test interval changed motion direction with respect to the sample interval (direction change discrimination task). The results showed that the mask mainly interfered with performance when displayed 200 ms after the offset of the sample interval, and when its direction, speed and spatial position matched that of the remembered sample (see Pasternak and Zaksas 33 for similar results on macaque monkeys). These results suggest that the short-term representation for global motion is selective for direction, speed and spatial position, being compromised by intervening directional stimuli presented immediately after the encoding phase 38 . Using a similar paradigm, we investigated whether action video game players encode and retain the direction of coherent and translationally moving stimuli more efficiently than non-gamers. Moreover, we also assessed whether in action video game players, the visual short-term memory trace for coherent motion is more robust and less prone to interference during the retention period. We also wanted to identify the importance of the type of video games engaged in by our participants. To this end we defined a further group of video gamers who engage in non-action video game playing. There is extensive behavioural evidence that playing action video games enhances a range of perceptual [bib_ref] Improved probabilistic inference as a general learning mechanism with action video games, Green [/bib_ref] [bib_ref] Selectively enhanced motion perception in core video gamers, Hutchinson [/bib_ref] [bib_ref] Action Video Games Improve Direction Discrimination of Parafoveal Translational Global Motion but..., Pavan [/bib_ref] [bib_ref] Intensive video gaming improves encoding speed to visual short-term memory in young..., Wilms [/bib_ref] and cognitive functions [bib_ref] Action video games and improved attentional control: Disentangling selection-and response-based processes, Chisholm [/bib_ref] [bib_ref] Playing an action video game reduces gender differences in spatial cognition, Feng [/bib_ref] [bib_ref] Functional Integration between Salience and Central Executive Networks: A Role for Action..., Gong [/bib_ref] [bib_ref] Learning, attentional control, and action video games, Green [/bib_ref] [bib_ref] Action video game modifies visual selective attention, Green [/bib_ref] [bib_ref] Action video game training reduces the Simon Effect, Hutchinson [/bib_ref]. In addition, it has been demonstrated that training on action video games improves reading abilities in children with developmental dyslexia [bib_ref] Report Action Video Games Make Dyslexic Children Read Better, Franceschini [/bib_ref] [bib_ref] Shall We Play a Game?": Improving Reading Through Action Video Games in..., Franceschini [/bib_ref] [bib_ref] Action video games improve reading abilities and visual-to-auditory attentional shifting in English-speaking..., Franceschini [/bib_ref] [bib_ref] Multiple Causal Links between Magnocellular-Dorsal Pathway Deficit and Developmental Dyslexia, Gori [/bib_ref] , although this conclusion has been recently disputed [bib_ref] Neither action nor phonological video games make dyslexic children read better, Łuniewska [/bib_ref]. These beneficial effects on cognitive functions may depend on the fact that action video game players have to track, store in visual short-term memory and react to multiple auditory and visual stimuli, which are changing continuously over space and time [bib_ref] Action Video Games Improve Direction Discrimination of Parafoveal Translational Global Motion but..., Pavan [/bib_ref] [bib_ref] Perceptual learning during action video game playing, Green [/bib_ref]. This massive information processing involves a high degree of perceptual, attentional and motor load (see for a review on the role of action video games in psychological research and Bediou et al. [bib_ref] Meta-analysis of action video game impact on perceptual, attentional, and cognitive skills, Bediou [/bib_ref] for a meta-analysis on the effects of action video game playing on perceptual and cognitive abilities). In the present study, we investigated visual short-term memory (VSTM) for parafoveal moving objects in action video game players (AVGPs), non-action video game players (NAVGPs), and non-gaming controls (CONs). The ability to store and maintain task-relevant visual information is crucial to deal with a changing external environment by controlling gaze during visual search [bib_ref] Understanding the Function of Visual Short-Term Memory: Transsaccadic Memory, Object Correspondence, and..., Hollingworth [/bib_ref] , for learning and problem solving 20 , for reducing distraction by maintaining the prioritization of relevant visual information [bib_ref] The role of working memory in visual selective attention, De Fockert [/bib_ref]. In general, VSTM reflects the ability of the brain to briefly retain information that can be used to drive learning and/or guide on-going actions. Given its implication in maintaining relevant information from the visual environment, it is important to assess whether it is possible to enhance VSTM with specific training regimes. Such an enhancement is also important because of working-memory limitations in terms of capacity and resources [bib_ref] Failure of self-consistency in the discrete resource model of visual working memory, Bays [/bib_ref] [bib_ref] Visual working memory capacity: From psychophysics and neurobiology to individual differences, Luck [/bib_ref] [bib_ref] Discrete fixed-resolution representations in visual working memory, Zhang [/bib_ref]. Video game training could provide a cost-effective and reliable tool to improve VSTM, which would be useful for example to counteract normal visual working memory decline in aging [bib_ref] Age-related changes in working memory and the ability to ignore distraction, Mcnab [/bib_ref] [bib_ref] Age-related decline of precision and binding in visual working memory, Peich [/bib_ref]. Previous studies have shown that AVGPs exhibit superior performance in a change-detection task when compared to persons who do not play video games [bib_ref] The effects of video game playing on attention, memory, and executive control, Boot [/bib_ref]. Furthermore, AVGPs perform Based on earlier studies [bib_ref] Effects of action video game training on visual working memory, Blacker [/bib_ref] we predict that action video game players can encode visual motion information more efficiently than their non-playing counterparts and that their visual short-term memory trace is more resistant to interference immediately after the encoding phase. To test these hypotheses, we used a memory masking discrimination task [bib_ref] Stimulus specificity and temporal dynamics of working memory for visual motion, Pasternak [/bib_ref] [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref]. In the memory interval we presented four RDKs (one in each visual quadrant), two RDKs were composed of random motion, whereas the other two patches were coherent RDKs moving in different directions. After a retention interval of 3.2 s, we presented a test interval, identical to the sample interval with the exception that one of the coherently moving RDK patches changed direction. The task of the participants was to discriminate the location of the moving patch with a different motion direction with respect to the sample. On some of the trials, an intervening masking stimulus was presented 0.2 s after the offset of the sample stimuli. The RDKs composing the masking stimuli were all composed of 100% coherent moving dots with different directions. The temporal parameters of the experiment were chosen on the basis of our previous work on visual short-term memory, in which we found that memory masking is more effective when the mask (1) is presented 0.2 s after the offset of the sample stimuli, (2) it contains directional motion rather than random motion, and (3) its direction of motion and speed match that of the target sample [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref]. Masking RDKs contained directional motion since we showed that masking RDKs with random motion did not affect performance when using a similar memory-masking task [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref]. It is important to note that, prior to the main experiment, action video game players (AVGPs), non-action video game players (NAVGPs) and non-gamers (CONs) were trained on a direction discrimination task to match their performance. In this fashion, we could be sure that the three groups did not differ with respect to the visual-processing requirements of the tasks. To anticipate the main findings, the results showed similar VSTM performance in direction change discrimination in all three groups. However, video game players overall outperformed controls showing more accurate VSTM across all the masking conditions. Compared to controls, video game players exhibited less interference in the masking conditions. # Methods ## Participants. A group of fifteen participants with no previous experience in video game playing (i.e., non-gaming controls: CONs), a group of twelve action video game players (AVGPs), and a group of ten non-action video game players (NAVGPs) took part voluntarily in the experiment. Subjects were matched on age and socio-economic status. The AVGP group was composed of one female and eleven males, the NAVGP group was composed by six females and four males, whereas the CON group was composed by ten females and six males. In order to be considered an action video game player, the participant needed to have played a minimum of 3 to 4 days a week in the past 6 months and for a minimum of 2 hours a day. On average, AVGPs played action video games 4.75 days a week (SEM = 0.41) and for 5.1 hours a day (SEM = 0.45), whereas NAVGPs played non-action video games (including strategy and role-play games) 4.2 days a week (SEM = 0.63) and for 2.7 hours a day (SEM = 0.49). Independent-sample t-tests revealed a significant difference between the two video-game groups in terms of gaming hours a day (t 20 = 3.708, p = 0.001) but not gaming days (t 20 = 0.76, p = 0.457). Action video games played by our observers were, for example, Call of Duty, Battlefield, Fallout, Far Cry, whereas non-action video games were for example, Sims, Titan Quest, Path of Exile, Risen. There were no obvious effect on the VSTM performance based on the type of action video games played. For the control group, non-gamers had played no video games in the past 6 months. To allocate participants into control and VGP groups, we used the questionnaire adapted from Rosser et al. [bib_ref] The impact of video games on training surgeons in the 21st century, Rosser [/bib_ref]. The questionnaire is useful to determine the genres of the games played and make sure that VGPs played either action or non-action video games for a minimum of 2 hours a day and had not recently played any games from other genres. This is to make sure that video game players were specifically experienced in either action or non-action video games. All participants had normal or corrected to normal visual acuity and sat in a dark room at a distance of 57 cm from the screen. Viewing was binocular. Ethical considerations and informed consent. Methods were carried out in accordance with the World Medical Association Declaration of Helsinki 42 . This study was approved by the Ethics Committee of the University of Lincoln (Proposal number: PSY1718534) and the University of Regensburg (Proposal Number: 13-101-0029). Written informed consent was obtained from each participant prior to the enrolment in the study. At the University of Lincoln participants received university credits for their participation, whereas at the University of Regensburg participants were paid for their time. Apparatus. Data from ten AVGPs and ten CONs were gathered from the University of Lincoln using a 24-inch Dell P2414H monitor with a refresh rate of 60 Hz and a resolution of 1920 × 1080 pixels. Each pixel subtended 1.66 arcmin. Data from two AVGPs, six CONs and a new sample of ten NAVGPs were collected at the University of Regensburg using a 24-inch Dell S2417DG monitor with a refresh rate of 120 Hz and a resolution of 2560 × 1440 pixels. Each pixel subtended 1.26 arcmin. Though at the University of Regensburg data were collected using a different setup, spatial and temporal parameters were adjusted to determine the same stimulus size and dots' drifting speed. Stimuli were generated with Matlab Psychtoolbox Stimuli. Stimuli were random dot kinematograms (RDKs) consisting of 100 white dots (diameter: 0.15 deg) presented within a circular aperture with a diameter of approximately 8 deg (density: 2 dots/deg 2 ). All the dots moved along translational trajectories with 100% coherence. The dots' Weber contrast was set at 0.99 and moved on a grey background (luminance: 38.2 cd/m 2 ) at a speed of 13.3 deg/s. Dots had a limited lifetime; after 83 ms, each dot vanished and was replaced by a new dot at a different randomly selected position within the circular window. Dots also appeared asynchronously on the display [bib_ref] Conditions for motion flow in dynamic visual noise, Morgan [/bib_ref] [bib_ref] A selective impairment of motion perception following lesions of the middle temporal..., Newsome [/bib_ref]. In addition, moving dots that travelled outside the circular window were also replaced by a new dot at a different randomly location within the circular window, thus always maintaining the same density. The duration of the motion sequence was 0.2 s. Procedure. The procedure used in the experiment was similar to that reported in Pavan et al. [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref] and consisted of two phases: (i) in a control experiment, VGPs and CONs were trained in a motion-direction discrimination task in order to match their performance. This phase of the experiment consisted of a single presentation interval (duration 0.2 s) in which an RDK was displayed in each visual quadrant. One RDK was composed by coherently moving dots (100% coherence), whereas the other three RDKs were random-motion RKDs in which dots were randomly placed within the circular window at every frame (i.e., random position 47 ). Participants had to discriminate the motion direction of the coherent RDK which could move in one of the eight cardinal directions (8AFC). Each block consisted of 40 trials, and participants performed as many blocks as needed to get an accuracy equal or higher than 0.95, which was achieved in all cases. (ii) Main VSTM experiment. The sample interval (0.2 s) was composed by two directional RDKs (100% coherence) and two noisy RDKs. Observers were required to remember the location of the two coherent RDKs and their motion directions. A masking interval (duration 0.2 s) was then presented 0.2 s after the offset of the sample stimulus, i.e., 0.2 s after the onset of the retention interval . The masking interval was composed by four coherently moving patches (100% coherence) with the same speed as the RDKs presented in the sample interval. On each trial, one of the two coherent RDKs that made up the sample was selected as target RDK. In the masking interval, the RDK occupying the same location of the target RDK could drift in the same or different direction with respect to the target RDK (the minimum difference in motion direction between sample and masking was 45 deg). All the other RDKs in the masking interval drifted in different directions than the sample RDKs, but always in one of the eight cardinal directions. After the masking interval there was a delay interval of 3 s in which only the fixation point was presented. It should be noted that the masking interval was presented for 0.2 s during the retention interval, therefore the total duration of the retention interval was 3.2 s (see . After the delay interval we presented a test interval of 0.2 s. In the test interval, the spatial location of the 100% coherent RDKs and the noise RDKs was the same as in the sample interval. However, the RDK occupying the spatial location of the target RDK had a different motion direction, whereas the other RDK had the same motion direction to that of the sample interval. The observers' task was to judge in which quadrant the direction of the test RDK differed from that of the sample RDK. Coherently moving RDKs could be presented in the upper left and upper right quadrants, upper left and lower left quadrants, lower left and lower right quadrants, upper right and lower right quadrants. , for example, represents a trial in which the test RDK presented in the upper right visual quadrant differed from the direction of the sample RDK. Observers were also instructed to ignore the masking interval and only attend to the sample and test RDKs. They responded by pressing one of 4 buttons to indicate the quadrant in which the sample and test RDKs had different directions. Observers performed a total of 288 trials split in 12 blocks (i.e., 24 trials per block). There were 32 trials with no masking. Each of the eight directions of the target sample was repeated 32 times. Moreover, in the masking trials, www.nature.com/scientificreports www.nature.com/scientificreports/ the direction of the masking RDK presented in the same location to that of the target sample could drift in one of the eight cardinal directions. For example, if the target sample drifted at 90°, the masking patch presented in the same location to that of the target could drift either at 0°, 45°, 90° (sample-mask direction match), 135°, 180°, 225°, 270°, or 315°. # Results Accuracy. The results of the training in motion direction discrimination showed that AVGPs, NAVGPs and CONs needed respectively 2.25 sessions (SEM: 0.58), 2.2 sessions (SEM: 0.2), and 4.87 sessions (SEM: 1.16) to get the desired level of accuracy (>0.95 correct performance rate). A Shapiro-Wilk normality test showed that number of sessions were not normally distributed for the three groups (p < 0.05). Additionally, a mean-based Levene test showed that variances were not homogeneous (p = 0.002). Therefore, to analyse whether there was a significant difference on the number of training sessions between the three groups, we used an independent-sample Kruskal-Wallis test. The Kruskal-Wallis test did reveal a barely significant effect of group (χ 2 = 6.127, df = 2, p = 0.047). Bonferroni-Holm corrected Mann-Whitney post-hoc comparisons did not report a significant difference between AVGPs and NAVGPs (p = 0.254), between AVGPs and CONs (p = 0.123), and between NAVGPs and CONs (p = 0.16). However, a non-parametric trend analysis (Jonckheere-Terpstra) showed a significant trend for increasing number of training sessions for the order AVGPs, NAVGPs and CONs (one-sided p = 0.0052), suggesting a significant increment of the number of training sessions across the so-ordered groups. We also compared the performance of the three groups in motion-direction discrimination in the last training session. A one-way ANOVA indicated no significant difference in motion direction discrimination between the three groups post training (F 2,34 = 0.153, p = 0.859). Additionally, Bonferroni corrected one-sample t-tests showed that accuracies of AVGPs, NAVGPs and CONs were significantly higher than 0.95 (AVGPs: mean = 0.987, SEM = 0.0049, t 11 = 7.71, p = 0.0001; NAVGPs: mean = 0.99, SEM = 0.01, t 9 = 4.00, p = 0.003; CONs: mean = 0.985, SEM = 0.00476, t 14 = 7.36, p = 0.0001), suggesting equally high performance in all three groups. Additionally, we tested whether the absolute direction of the target had any effect on performance. A mixed ANOVA on the last training session including as a between-subjects factor the group (i.e., AVGPs, NAVGPs, and CONs) and as a within-subjects factor the target direction (i.e., eight cardinal directions) did not reveal any significant main effect (group: F 2,34 = 0.153, p = 0.859, η p 2 = 0.009; target direction: F 7,238 = 0.811, p = 0.58, η p 2 = 0.023) or interaction (F 14,238 = 0.7, p = 0.775, η p 2 = 0.039), suggesting that at the end of training performance was constant over all target directions. We next determined the effect of memory masking on the retention of direction-specific motion information. [fig_ref] Figure 2: Results of the memory-masking experiment [/fig_ref] shows the proportion of correct responses as a function of the direction difference between sample and mask in the memory-masking paradigm used in the main experiment. A mixed ANOVA including as a between-subjects factor the group (i.e., AVGPs, NAVGPs, CONs) and as a within-subjects factor the masking condition (including the no-mask condition and the five direction differences between sample and mask), revealed a significant effect of the group (F 2,34 = 10.1, p < 0.001, η p 2 = 0.372), and a significant effect of the masking condition (F 5,170 = 22.69, p < 0.001, η p 2 = 0.40). The interaction was not significant (F 10,170 = 1.51, p = 0.139, η p 2 = 0.082), suggesting that the broad tuning of masking was similar across groups. A one-way ANOVA between the accuracy of AVGPs, NAVGPs and CONs in the no-masking condition revealed a significant effect of the group (F 2,34 = 5.75, p = 0.007). Post-hoc comparisons using a false discovery rate (FDR) at 0.05 48 revealed only a significant difference between CONs and NAVGPs in accuracy for the no-masking . Representation of the procedure used in the memory-masking experiment. The black square frame in the sample interval (not presented during the experiment) indicates the target RDK. In the masking interval, the RDK occupying the same spatial location to that of the target RDK has the same motion direction to that of the target RDK. In the test interval, the same RDK changes direction with respect to the target RDK. The task of the observers was to report which 100% coherently moving patch in the test interval changed direction with respect to the sample interval. They were instructed to ignore the randomly moving RDKs. www.nature.com/scientificreports www.nature.com/scientificreports/ condition (adjusted-p = 0.006). The one-way ANOVA did not reveal any significant difference between AVGPs and NAVGPs (adjusted-p = 0.12) and between AVGPs and CONs (adjusted-p = 0.12). In order to further assess the effect of the masking stimulus we conducted a separate repeated measures ANOVA for each group. For the AVGPs group the ANOVA revealed a significant effect of the masking condition (F 5,55 = 5.67, p = 0.0001, η p 2 = 0.34). Post-hoc comparisons using FDR at 0.05 revealed that all the target-sample-mask direction differences were significantly different from the no-mask condition (p < 0.05). A one-way repeated-measures ANOVA performed only on the five target-sample-mask direction differences revealed no significant effect (F 4,44 = 0.618, p = 0.65, η p 2 = 0.053). Additionally, a one-way repeated-measures ANOVA with polynomial trend analysis did not discover any significant trend (linear, quadratic, cubic, quartic, all p > 0.05), suggesting negligible tuning of masking. For the NAVGPs group the repeated measures ANOVA revealed a significant effect of the masking (F 5,45 = 6.38, p = 0.0001, η p 2 = 0.42). Post-hoc comparisons using FDR at 0.05 revealed that all the target-sample-mask direction differences were significantly different from the no-mask condition (p < 0.05). A one-way repeated-measures ANOVA performed on the five target-sample-mask direction differences revealed no significant effect (F 4,36 = 1.49, p = 0.225, η p 2 = 0.142). The trend analysis did not report any significant trend (all p > 0.05). Similarly, for the CONs group the ANOVA revealed a significant effect of the masking (F 5,70 = 13.97, p < 0.001, η p 2 = 0.5). Post-hoc comparisons using FDR at 0.05, revealed that all the target sample-mask direction differences were significantly different from the no-mask condition (p < 0.001). A one-way repeated-measures ANOVA performed on the five target-sample-mask direction differences did not reveal any significant effect (F 4,56 = 1.135, p = 0.349, η p 2 = 0.075). Additionally, the trend analysis did not report any significant trend (all p > 0.05), again suggesting negligible tuning of masking. Therefore, we found that the masking stimulus affected task performance regardless of its motion direction relative to that of the sample. However, there is little or no tuning of masking. [fig_ref] Figure 2: Results of the memory-masking experiment [/fig_ref] shows the performance of AVGPs, NAVGPs and CONs in the no-mask and mask conditions, after pooling over all the sample-mask direction differences. On average, the intervening masking reduced performance by 8.31%, 9.7% and 16.85% in AVGPs, NAVGPs and CONs, respectively. A mixed ANOVA revealed a significant effect of the group (F 2,34 = 8.93, p < 0.001, η p 2 = 0.344), a significant effect of the masking condition (i.e., no-masking vs. masking) (F 1,34 = 93.88, p < 0.001, η p 2 = 0.734) and a significant interaction between masking condition and group (F 2,34 = 4.07, p = 0.026, η p 2 = 0.193). For the group, post-hoc comparisons using FDR at 0.05 reported a significant difference between AVGPs and CONs (adjusted-p = 0.018), between NAVGPs and CONs (adjusted-p = 0.0003), but not a significant difference between AVGPs and NAVGPs (adjusted-p = 0.136). For the condition x group interaction, post-hoc comparisons reported a significant difference between the no-masking condition and the mask condition for all the three groups (adjusted-p < 0.001). Additionally, for the no-masking condition we found a significant difference in performance only between NAVGPs and CONs (adjusted-p = 0.006), however, for the masking condition post-hoc comparisons revealed a significant difference between AVGPs and CONs (adjusted-p = 0.0045), between NAVGPs and CONs (adjusted-p = 0.0003), but no difference between AVGPs and NAVGPs (adjusted-p = 0.27), suggesting very similar performance in the no-masking condition for the video-gaming groups. Additionally, both AVGPs and NAVGPs seem to have similar tolerance to the intervening masking stimulus, affecting their motion discrimination performance to a lesser degree than in CONs. In [fig_ref] Figure 2: Results of the memory-masking experiment [/fig_ref] it is evident that AVGPs and NAVGPs have higher accuracies than CONs, but all three groups show better performance in the no-mask condition (i.e., all data points fall above the equal-performance diagonal with www.nature.com/scientificreports www.nature.com/scientificreports/ one exception). This pattern of results was consistent across nine of the twelve AVGPs and for all the NAVGPs and CONs. Modelling visual short-term memory. In order to assess whether components of visual short-term memory differed across the three groups, accuracy data were fitted with a prominent model of visual short-term memory; the variable precision (VP) model [bib_ref] Variability in the quality of visual working memory, Fougnie [/bib_ref] [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] [bib_ref] Factorial comparison of working memory models, Van Den Berg [/bib_ref]. Previous studies showed that visual short-term memory precision is continuous and variable across memory items and trials [bib_ref] Variability in the quality of visual working memory, Fougnie [/bib_ref] [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] [bib_ref] Factorial comparison of working memory models, Van Den Berg [/bib_ref] [bib_ref] The precision of visual working memory is set by allocation of a..., Bays [/bib_ref] , rather than continuous but equally distributed across items to be remembered 53 , or variable but deployed over a discrete number of memory slots [bib_ref] Discrete fixed-resolution representations in visual working memory, Zhang [/bib_ref]. Based on Zhang and Luck 25 , below the slot limit, memory items can be stored in more than one slot and averaged, thus producing high precision and high-resolution memory traces. However, beyond the slot limit, no information is stored resulting in an increase of guess rate (i.e., random responses). Therefore, the slot + averaging model of Zhang and Luck [bib_ref] Discrete fixed-resolution representations in visual working memory, Zhang [/bib_ref] would predict that the errors in memory at recall emerge as a result of guessing and noise with which the item is stored. The VP model does not assume any memory slot limit, but resources are variable across trials and scale with the number of items to be remembered. Following the rationale of van den Berg et al. [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] , an observer should simultaneously memorize both the location and the motion direction of the two coherent RDKs. Each RDK is encoded with a certain precision. The VP model assumes that the observer's internal measurement of a stimulus is noisy, and it follows a von Mises (circular normal) distribution, in which a high precision produces a narrower von Mises distribution. In the VP model, memory precision is variable across trials and the model assumes that it is drawn, independently across trials, from a gamma distribution. The model also assumes that mean precision (i.e., mean of the gamma distribution with a certain scale parameter), depends on set size in a power-law fashion (see [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] ,b in van der Berg et al. [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] for a schematic representation of the model), however, in our experiment we tested only one set size (i.e., two memory items). The VP model is characterized by three parameters: guess rate (g), mode SD and SD variability. The guess rate (g) expresses the probability with which the observer did not remember which of the two coherently moving patches in the test interval was drifting in a different direction with respect to the sample, and consequently guessed randomly. Mode SD represents the precision of the remembered items; high values in mode SD indicate a less precise memory representation. SD variability indicates trial-to-trial variation in memory precision; high values of SD variability indicate high trial-to-trial variability. We choose to fit the VP model instead of other available VSTM models also because of recent brain imaging evidence that shows how the superior intraparietal sulcus (sIPS) may be involved in the modulation of variability of memory precision [bib_ref] Superior Intraparietal Sulcus Controls the Variability of Visual Working Memory Precision, Weber [/bib_ref]. We fitted a two-alternative forced-choice (2AFC) version of the VP model using the Matlab MemToolbox 55 (http://visionlab.github.io/MemToolbox/) in order to assess whether there are differences in the guessing rate (g), mode precision (mode SD) and SD variability (SD var) between the three groups. For each sample-mask direction difference we calculated the direction difference between the target sample and the relative test patch (i.e., the test patch occupying the same spatial location of the target sample) and we assigned the value 1 if the observer responded correctly or 0 otherwise. Based on Suchow et al. [bib_ref] Modeling visual working memory with the MemToolbox, Suchow [/bib_ref] , the model input was an array of target sample-test direction differences and an array of observer's responses. The output of the model was an estimate of parameters g, mode SD and SD var. The MemToolbox uses Bayesian inference to derive a probability distribution over parameter values. This probability distribution describes the reasonableness of parameters after considering the observed data in light of a prior distribution (see Suchow et al. [bib_ref] Modeling visual working memory with the MemToolbox, Suchow [/bib_ref] for a detailed description of model fitting and parameters estimation). The VP model was fitted to the entire set of each observer's data. Since in the previous analysis we did not find any significant difference between the different masking directions used, the model was fitted just to the no-masking condition and to the masking condition, composed by all the sample-mask direction differences pooled. [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] shows the parameters estimate of the variable precision model. For guessing rate (g) [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] , a mixed ANOVA revealed a significant effect of the group (F 2,34 = 9.734, p < 0.001, η p 2 = 0.364), a significant effect of the masking condition (F 1,34 = 94.716, p < 0.001, η p 2 = 0.735), but www.nature.com/scientificreports www.nature.com/scientificreports/ no significant interaction between group and masking condition (F 2,34 = 1.465, p = 0.245, η p 2 = 0.0793). For the group, post-hoc comparisons using FDR at 0.05 revealed a significant difference between AVGPs and CONs (adjusted-p = 0.006), between NAVGPs and CONs (adjusted-p = 0.0001), but no significant difference between AVGPs and NAVGPs (adjusted-p = 0.255), suggesting an overall higher guessing rate in CONs. A higher guessing rate was also evident in the masking condition compared to the no-masking condition (mean difference: 0.219, SE: 0.0223). ## Results of the variable precision model. For mode precision (Mode SD) [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] , a mixed ANOVA did not reveal any significant main effect or interaction (group: F 2,34 = 0.386, p = 0.682, η p 2 = 0.022; masking condition: F 1,34 = 0.542, p = 0.467, η p 2 = 0.0157; interaction group x masking condition: F 2,34 = 1.517, p = 0.234, η p 2 = 0.0819). Additionally, from [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] , there seems to be a linear trend for the no-masking condition, with mode SD linearly increasing for the three groups. A Jonckheere-Terpstra trend analysis did not reveal a significant trend for the order AVGPs, NAVGPs and CONs (p = 0.161). For trial-to-trial precision variability (SD var) [fig_ref] Figure 3: Parameters estimate of the variable precision model [/fig_ref] , a mixed ANOVA revealed no significant effect of the group (F 2,34 = 2.675, p = 0.083, η p 2 = 0.136) and masking condition (F 1,34 = 0.001, p = 0.971, η p 2 = 0.0001. However, the interaction masking condition x group was significant (F 2,34 = 4.675, p = 0.016, η p 2 = 0.216). For the interaction, post-hoc comparisons using FDR at 0.05 revealed a significant difference between AVGPs and CONs for the no-masking condition (adjusted-p = 0.021), but not a significant difference between AVGPs and NAVGPs (adjusted-p = 0.229) and between NAVGPs and CONs (adjusted-p = 0.237). Additionally, post-hoc comparisons also revealed a nearly significant difference between no-masking and masking conditions only for CONs (adjusted-p = 0.051). In sum, fitting the accuracy data with the VP model 50 revealed that the three groups do not differ in terms of precision of the memory representation (i.e., Mode SD). However, CONs exhibited a significantly higher guessing rate than video gamers and also higher trial-to-trial precision variability (SD var) than AVGPs. Additionally, a nearly significant difference between no-masking and masking conditions for SD var was found for CONs. Reaction Times. [fig_ref] Figure 4: Reaction times in the visual short-term memory experiment [/fig_ref] shows the log 10 reaction times (RTs) for correct responses only. Before transforming RTs in log 10 , for each observer and condition, outlier RTs were filtered out following the procedure of Lachaud and Renaud 56 . Specifically, we calculated the median of the RTs, and trials in which RTs were ±3 MAD (median absolute deviation) or more from the median were removed from the analysis. A mixed ANOVA on log 10 (RT s ) revealed a significant effect of the group (F 2,34 = 3.587, p = 0.039, η p 2 = 0.174), a significant effect of direction difference between sample and mask (F 3.376,114.78 = 4.24, p = 0.005, η p 2 = 0.111), but no interaction between sample-mask difference and group (F 10,170 = 0.422, p = 0.934, η p 2 = 0.024). When the sphericity assumption was violated, degrees of freedom were corrected using the Greenhouse-Geisser correction. For the group, post-hoc comparisons using FDR at 0.05 reported a significant difference between AVGPs and NAVGPs (adjusted-p = 0.039), but no difference between CONs and AVGPs (adjusted-p = 0.135) and between CONs and NAVGPs (adjusted-p = 0.276). With respect to the effect of direction difference between sample and mask, post-hoc comparisons using FDR at 0.05, revealed that reaction times in the no-masking condition were systematically greater than the reaction times of all the other sample-mask direction differences (all adjusted-p values < 0.01). Additionally, there were no significant differences in reaction times between the other sample-mask direction differences (all adjusted-p values > 0.05). An additional analysis of variance for repeated measures with trend analysis on sample-mask direction differences revealed significant linear (F 1,34 = 8.217, p = 0.007, partial-η 2 = 0.195) and cubic (F 1,34 = 12.74, p = 0.001, partial-η 2 = 0.273) trends. Higher-order trends were not significant (Quadratic, F 1,34 = 2.09, Additionally, a one-way ANOVA conducted between log 10 (RT s ) of AVGPs, NAVGPs and CONs in the no-masking condition, revealed a significant effect of the group (F 2,34 = 3.757, p = 0.034). Post-hoc comparisons using FDR at 0.05, reported a significant difference between AVGPs and NAVGPs (adjusted-p = 0.03), but no difference between AVGPs and CONs (adjusted-p = 0.151) and between NAVGPs and CONs (adjusted-p = 0.22). On average, CONs exhibit reaction times that fell in between those of AVGPs and NAVGPs. Additionally, response times of NAVGPs were longer than reaction times of AVGPs, but NAVGPs showed similar performance values to those of AVGPs. In order to better understand this speed-accuracy trade-off between the two video game groups, reaction times data were fitted with the standard diffusion decision model [bib_ref] A theory of memory retrieval, Ratcliff [/bib_ref] [bib_ref] Modeling Response Times for Two-Choice Decisions, Ratcliff [/bib_ref] [bib_ref] Estimating parameters of the diffusion model: Approaches to dealing with contaminant reaction..., Ratcliff [/bib_ref]. # Diffusion model analysis. For a combined analysis of response times and accuracy rates, we fitted the standard diffusion decision model [bib_ref] A theory of memory retrieval, Ratcliff [/bib_ref] [bib_ref] Modeling Response Times for Two-Choice Decisions, Ratcliff [/bib_ref] [bib_ref] Estimating parameters of the diffusion model: Approaches to dealing with contaminant reaction..., Ratcliff [/bib_ref] to the data of all participants. The diffusion model is a model for perceptual decisions with two alternatives [bib_ref] The diffusion decision model: Theory and data for two-choice decision tasks, Ratcliff [/bib_ref]. It is based on the assumption that decision times can be described by a Wiener diffusion process subject to two response criteria, representing evidence thresholds for each of the two alternatives. Due to random perturbations, a response criterion is reached sometimes sooner, sometimes later, and can also reach the criterion associated with an incorrect response [fig_ref] Figure 5: The diffusion model of response times [/fig_ref]. Hence, the diffusion model allows for a complete analysis of RTs, including response accuracy and the distributions of both correct and incorrect responses. We fitted the standard diffusion model with trial-to-trial variation in drift rates, starting point, and non-decision times to the RT distributions (i.e., the 10%, 30%, 50%, 70%, and 90% quantiles) of trials without masking and trials with masking between the sample and the test intervals. Since we did not find a significant effect of the sample-mask direction difference with respect to accuracies, RT data were pooled across all masking conditions. The standard version of the diffusion model has seven free parameters: in addition to the barrier separation (a), drift rate (v), starting point (z), and non-decision time (T er ), the model includes trial-by-trial variation in drift rate (η), starting point (s z ), and non-decision time (s t ). When fitting this model, we allowed for separate drift rates for decisions in trials with and without masking. A difference in drift rates -if present -would reflect an effect of the masking, reflecting how detrimental the mask had been to the memory representation of the sample stimulus. It turned out that fixing the starting point to a/2 (i.e., unbiased decision making) did not affect the model fits much, so we fixed this parameter and set its variation to zero. The whole model had six free parameters to explain RT distributions for correct and error RTs in two conditions (i.e., four distributions). Predictions of the diffusion model were obtained using an efficient evaluation of the first-passage distribution with variable drift [bib_ref] The first-passage time distribution for the diffusion model with variable drift, Blurton [/bib_ref] and numerical integration to account for variable non-decision time [bib_ref] The efficient computation of the cumulative distribution and probability density functions in..., Tuerlinckx [/bib_ref]. Parameter estimates were obtained by quantile maximum likelihood estimation [bib_ref] Quantile maximum likelihood estimation of response time distributions, Heathcote [/bib_ref] using the aforementioned quantiles. The resulting estimates of the model were tested for group differences by means of a one-factorial ANOVA. Since this involved six statistical tests, one for each parameter, the results were corrected for multiple comparisons using FDR correction [bib_ref] Controlling the false discovery rate: a practical and powerful approach to multiple..., Benjamini [/bib_ref]. Only parameters that exhibited significant group differences after FDR correction at 0.05 were subsequently tested in single group comparisons by means of (two-tailed) t-tests. Wiener process with drift µ, starting at z, to one of two evidence thresholds, placed at a and 0. Due to random noise, the first-passage occurs at different times and at different criteria. The corresponding density functions of first passage is plotted at either criterion. In addition to the decision latency, there is a non-decision latency T er . In the full model, drift, starting point, and non-decision time are assumed to vary from trial-to-trial. The drift is assumed to be normally distributed with mean v and variance η 2 . Starting point and non-decision time are assumed to be uniformly distributed with distribution width s z and s t , respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ Results. The parameter estimates of the model were in the usual range for a perceptual decision task and the overall model fit was acceptable [fig_ref] Figure 6: Empirical [/fig_ref]. The fit to RTs for the RT distributions in the masking condition was very good [fig_ref] Figure 6: Empirical [/fig_ref] , whereas in the no-masking condition the fit was slightly worse [fig_ref] Figure 6: Empirical [/fig_ref]. This lack of fit was detected during model selection, and it was found that an additional free parameter for the no-masking condition (i.e., a separate response criterion or non-decision time latency) increased the quality of the fit. The quantitative increment of the goodness-of-fit of the model was marginal, however, since the no-masking condition contained distinctly fewer trials than the masking condition. Therefore, we did not include a separate non-decision time latency for those trials. All diffusion model parameters except the drift rate are the same between conditions. Fixing the drift rate to be equal would lead to identical model predictions and, hence, considerably worse model fits [fig_ref] Figure 6: Empirical [/fig_ref]. In statistical terms, the drift rates between the two conditions are significantly different for AVGPs (X 2 = 23.16, df = 12, p = 0.03) and CONs (X 2 = 60.78, df = 15, p < 0.001), but not for NAVGPs (X 2 = 16.05, df = 10, p = 0.10). We obtained a significant group difference for the drift rate in masking trials (F 2,34 = 9.40, adjusted-p = 0.008). There were also significant differences between the drift rate in no-masking trials and non-decision time latency, but these differences were no longer significant after FDR correction. Most importantly, we found no evidence for different response criteria (a) across the three groups (F 2,35 = 2.20, adjusted-p = 0. 46), which speaks against a speed-accuracy trade-off as an explanation for the observed group differences in accuracy and RT. The drift rate in masking trials was significantly higher for AVGPs (v mask = 0.16) than for CONs (v mask = 0.1, t 25 = 2.54, p = 0.018) and significantly higher for NAVGPs (v mask = 0.23) than for CONs (t 23 = 3.93, p = 0.001). There was also a significant difference between AVGPs and NAVGPs (t 20 = -2.1, p = 0.049). These results speak against a speed-accuracy trade-off explanation for the differences across groups. Instead, the differences seem to reflect the quality of the VSTM representation: in all three groups, the mask after the sample stimulus negatively affected the representation in VSTM, and this masking effect was most pronounced in the CONs. In this group, the VSTM representation was particularly susceptible to the mask. But even in the conditions without the mask, VSTM representation at the end of the 3.2 s period was better in video game players than non-video game players. The fact that AVGPs had the fastest responses is partly due to a lower non-decision time latency. While this effect is not significant after FDR correction, non-decision latency was on average 120 ms to 180 ms lower in the AVGP group. The reason for this lower latency is difficult to determine; non-decision processes comprise processes such as encoding of the test stimulus, retrieval of the sample stimulus from VSTM, www.nature.com/scientificreports www.nature.com/scientificreports/ and the motor response, amongst others. Any of these processes could have contributed to a faster non-decision latency. A problem with applying the standard diffusion model in this situation is that is does not account for guessing due to a total loss of the VSTM representation like the variable precision model. To overcome this shortcoming, we fitted a diffusion model that takes into account that the decision depends on an intact representation in VSTM. If this representation is lost, the drift necessarily must be (close to) zero. We accounted for this by fitting a mixture diffusion model in which some trials are modelled by the standard model as described in the previous section, but other trials are described by a diffusion model with zero drift. With zero drift, the unbiased decision model can predict "guessing", since reaching either criterion happens with probability ½. The probability of losing the VSTM representation must be estimated from data; this adds two free parameters to the model because this probability is presumably different between masking and no-masking conditions. Two more parameters (response criterion a and non-decision time T er ) for the guessing trials were needed to obtain an acceptable fit. On the other hand, the drift rate was no longer different between mask and no-masking conditions, so we constrained them to be equal. The guessing diffusion model had 9 free parameters. The fit of the diffusion model that accounts for loss of the representation in VSTM by assuming a zero drift decision is comparable to the standard diffusion model and the estimated parameters closely reflect those of the standard model. However, the drift rates are no longer significantly different among groups (F 2,34 = 0.84, adjusted-p = 0.99). Instead, the probability of losing the VSTM representation in the masking condition is significantly different across the three groups (F 2,34 = 10.5, adjusted-p = 0.007). Post-hoc comparisons using FDR at 0.05 showed that AVGPs (t 25 = -3.73, p = 0.001) and the NAVGPs (t 23 = -3.25, p = 0.004) guessed significantly less than CONs. In other words, CONs had a significantly higher amount of trials in which the VSTM representation was lost. The two video gamer groups did not differ significantly (t 20 = 0.50, p = 0.62). We found no such effect in the no-masking condition and none of the other parameters differed significantly across the groups. This includes the drift rate, suggesting that, if the representation in VSTM was intact, the quality of information available for the decision was about the same in all three groups, irrespective of gaming experience. This essentially supports conclusions drawn from the variable precision model (see above), however, using the diffusion model, response times are taken into account as well. Learning Curves. Previous studies [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref] have argued that the generalized benefits seen in action video game players might be because AVGPs have an enhanced ability with respect to 'learning to learn' . That is, when faced with a novel task, AVGPs start out not different from CONs, but they are able to learn the relevant statistics of the task at a faster rate, leading to a more rapid improvement in performance, as a function of exposure to the task. However, in our study we did not find differences in performance values between AVGPs and NAVGPs across all the masking conditions tested, suggesting that, with respect to this type of memory task, non-action video game playing is associated with approximately the same benefits as is action video game playing. In the current study, observers performed a total of 288 trials split in 12 blocks. In order to examine whether video game players were better than non-video game players from the outset or whether all video game players started at the same level, with the difference first emerging over the course of exposure to the task, as would be predicted by the learning to learn hypothesis 64 , the 12 blocks were grouped in bins of two blocks, leading to 6 bins in total. We will refer to the bins as learning sessions. Binning was necessary because in each block the number of conditions differed, therefore we had to pool at least two blocks in order to estimate how performance varied with exposure to the task. To increase statistical power of this comparison, data from the masking conditions were pooled, since we did not find a significant difference between performance values of the different masking directions. We also estimated learning curves for the no-masking condition. [fig_ref] Figure 7: Learning curves for video game players and non-video game players with proportion... [/fig_ref] shows performance values as a function of the learning session. In order to test whether there were differences between learning rates of the three groups and whether video game players were better than non-video game players from the outset of the task, learning sessions were fitted with an elaborated power function of the form: [formula] = + − − − f x ax c x ( ) (1 ) (1) b b [/formula] The function has been recast from the elaborated power function of Bejjanki et al. [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref]. Parameter a indicates the accuracy in the first learning session, b is the learning rate (smaller values of b indicate slow improvements of the performance across learning sessions), and c indicates the asymptotic accuracy after an arbitrarily large number of learning sessions. Since parameters a and c indicate accuracies, they were constrained to vary between 0 and 1, whereas parameter b was constrained to assume only positive values in order to achieve strictly increasing learning curves. The fitting procedure and model selection used was similar to that reported in Bejjanki et al. [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref] For the three groups, the fully saturated model consisted of nine parameters . The maximally restricted model, i.e., the model that postulated no change between the groups, had only three parameters , and assumed that the three parameters were the same across the groups (see . Between the fully saturated model and the maximally restricted model, a lattice of models with different numbers of parameters were fitted. The best-fitting model selection was based on the following F-test: [formula] F RSS RSS df df RSS df ( ) /( ) /(2)1 2 1 2 2 2 = − − [/formula] www.nature.com/scientificreports www.nature.com/scientificreports/ where RSS 1 and df 1 are the residual sum of squares and the degrees of freedom of the simpler model, whereas RSS 2 and df 2 are the residual sum of squares and the degrees of freedom of the more complex model. The associated probability (p) can be computed as: [formula] = − p fcdf F df df df ( , , )(3) 1 2 2 [/formula] where fcdf is the F cumulative distribution function [bib_ref] Sex Alloc. within broods intrabrood sharing-out hypothesis, Burnham [/bib_ref]. If the resulting p-value was higher than the significance level (0.05), then the simpler model was likely to be correct. On the other hand, if the p-value was lower than the significance level, then the model with more parameters was likely to be correct. It should also be noted that the F-test can be used only with nested models (i.e., model A is nested in model B if parameters in model A are a subset of the parameters in model B). Comparisons between models with the same number of parameters (i.e., the same degrees of freedom) were performed using both the Akaike Information Criterion (AIC) test and the Bayesian Information Criterion (BIC). In this case the model with lower AIC and BIC is likely to be correct. [fig_ref] Table 3: Lattice of models fitted to the learning sessions of the three groups... [/fig_ref] reports the lattice of models fitted to the performance values. All the possible pairs of models were compared without repetitions. For the no-masking condition [fig_ref] Figure 7: Learning curves for video game players and non-video game players with proportion... [/fig_ref] the best fitting model corresponded to the Restricted Model 4, i.e., the model with five parameters in which only the parameter a was varied across the three groups. As stated above a difference in parameter a indicates different accuracies between groups in the first learning session. The Restricted Model 4 had a global R 2 = 0.851. The a parameter was 0.79 (SE: 0.02) for the AVGP group, 0.86 (SE: 0.02) for the NAVG group and 0.74 (SE: 0.02) for the CON group. The Restricted Model 4 was significantly better than the fully saturated model (i.e., the model with 9 parameters) (F 4,9 = 1.17, p = 0.39), better than the maximally An additional analysis was performed in order to test whether parameter a differed between video gamers and between gamers and controls. In order to do this, the Restricted Model 4 was fitted separately to each dataset, but parameters b and c were fixed at 0.41 and 0.99, respectively. These values were taken from the previous global fit in which the same model (Restricted Model 4) was fitted to the three groups. Therefore, for the purpose of this analysis only the parameter a was free to vary and was constrained to assume values between 0 and 1. When comparing the a parameter between AVGPs and NAVGPs the analysis reported that parameter a was different for each dataset (AIC same: −75.24, AIC different: −81.55). The same difference was found between AVGPs and CONs (AIC same: −82.13, AIC different: −83.57), and between NAVGPs and CONs (AIC same: −67.99, AIC different: −82.38). These results suggest that in the no-masking condition, the learning rate is similar across the three groups, and the three groups have the same asymptotic accuracy after an arbitrarily large number of learning sessions. However, video gamers performed better at the beginning of the experiment relative to non-gamers. Additionally, our analysis also pointed out that in the no-masking condition NAVGPs exhibited significantly higher initial accuracy rates than AVGPs. For the masking condition [fig_ref] Figure 7: Learning curves for video game players and non-video game players with proportion... [/fig_ref] the best fitting model resulted to be Restricted Model 6, i.e., the model with five parameters in which only the c parameter was varied across groups. As stated above the c parameter indicates the asymptotic accuracy after an arbitrarily large number of learning sessions. The Restricted Model 6 had a global R 2 = 0.946. The c parameter for the AVGP group was 0.91 (SE: 0.06), for the NAVG group was 0.97 (SE: 0.07) and for the CON group was 0.75 (SE: 0.03). The Restricted Model 6 and was significantly better than the fully saturated model (i.e., the model with 9 parameters) (F 4,9 = 1.87, p = 0.2), better than the maximally restricted model ( ## Model name Model function ## Number of parameters Fully Saturated www.nature.com/scientificreports www.nature.com/scientificreports/ An additional analysis was performed in order to test whether the asymptotic accuracy (i.e., parameter c) differed between video gamers and between gamers and controls. In order to do this, the Restricted Model 6 was fitted separately to each dataset, but parameters a and b were fixed at 0.63 and 0.85, respectively. These values were taken from the previous global fit in which the same model (Restricted Model 6) was fitted to the three groups. Therefore, for the purpose of this analysis only the parameter c was free to vary. When comparing the learning rate between AVGPs and NAVGPs the analysis reported that the asymptotic accuracy was different for each dataset (AIC same: −82.22, AIC different: −86.50). The same difference was found between AVGPs and CONs (AIC same: −64.70, AIC different: −82. [bib_ref] The capacity of visual working memory for features and conjunctions, Luck [/bib_ref] , and between NAVGPs and CONs (AIC same: −58.27, AIC different: −83.72). These results suggest that in the masking condition, the three groups had similar performance at the beginning of the experiment and also exhibited similar learning rates, but the asymptotic accuracy differed between the three groups, with video gamers exhibiting higher accuracy than controls after several learning sessions. Additionally, it seems that NAVGPs exhibited significantly higher asymptotic accuracy than both AVGPs and CONs. [formula] f1(x) = a1x −b1 + c1(1 − x −b1 ) 9 f2(x) = a2x −b2 + c2(1 − x −b2 ) f3(x) = a3x −b3 + c3(1 − x −b3 ) Restricted 1 f1(x) = ax −b1 + c1(1 − x −b1 ) 7 f2(x) = ax −b2 + c2(1 − x −b2 ) f3(x) = ax −b3 + c3(1 − x −b3 ) Restricted 2 f1(x) = a1x −b + c1(1 − x −b ) 7 f2(x) = a2x −b + c2(1 − x −b ) f3(x) = a3x −b + c3(1 − x −b ) Restricted 3 f1(x) = a1x −b1 + c(1 − x −b1 ) 7 f2(x) = a2x −b2 + c(1 − x −b2 ) f3(x) = a3x −b3 + c(1 − x −b3 ) Restricted 4 f1(x) = a1x −b + c(1 − x −b ) 5 f2(x) = a2x −b + c(1 − x −b ) f3(x) = a3x −b + c(1 − x −b ) Restricted 5 f1(x) = a1x −b1 + c(1 − x −b1 ) 5 f2(x)=ax −b2 + c(1 − x −b2 ) f3(x) = a3x −b3 + c(1 − x −b3 ) Restricted 6 f1(x) = ax −b + c1(1 − x −b ) 5 f2(x) = ax −b + c2(1 − x −b ) f3(x) = ax −b1 + c3(1 − x −b ) Maximally Restricted f(x) = ax −b + c(1 − x −b ) 3 [/formula] # Discussion In this study we investigated visual short-term memory (VSTM) in action video game players (AVGPs), non-action video game players (NAVGPs), and in non-video game playing controls (CONs). In particular, we used a visual masking paradigm previously used with macaque monkeys and humans. Participants were presented with a sample memory interval composed of two coherently moving RDKs and two randomly moving RDKs, each occupying a visual quadrant. Participants had to memorize both the spatial location and direction of the two coherently moving RDKs. After an interval of 3.2 s, a test display was presented and participants had to report which of the two coherently moving RDKs changed direction with respect to the sample display. In some of the trials, 0.2 s after the offset of the sample display, a masking stimulus was presented. The masking stimulus was composed by four coherently moving RKDs, and participants were instructed to ignore the intervening masking stimulus. We manipulated the motion direction of the masking RDK corresponding to the sample RDK that changed direction in the test interval (i.e., the target RDK). We measured both accuracy and response times. The results showed that the intervening masking display presented shortly after the offset of the sample display interferes with performance showing a decrement in accuracy in mask compared to no-mask conditions. Though memory masking interfered with the performance of the three groups, the effects of masking were more pronounced for the non-video game playing controls (see [fig_ref] Figure 2: Results of the memory-masking experiment [/fig_ref]. It should be noted that this result does not depend on group differences in their ability to discriminate the direction of the RDKs, since after initial training their performance was similar. However, the performance of the three groups significantly differed in the no-masking condition [fig_ref] Figure 2: Results of the memory-masking experiment [/fig_ref]. The results showed a significant difference between CONs and NAVGPs in accuracy for the no-masking condition, but no significant difference between AVGPs and NAVGPs nor between AVGPs and CONs. Overall, in the no-masking condition, video game players show a better performance than controls right at the outset of the experiment, as demonstrated by our analysis on learning sessions. Additionally, in the no-masking condition, we found that the learning rate and the asymptotic accuracy did not significantly differ across the three groups, indicating that the three groups differed in performance values at the outset of the experiment, subsequently converging with similar speed. On the other hand, for the masking condition, the three groups exhibited the same accuracy at the outset of the experiment and also the same learning rate, but the asymptotic accuracy differed amongst the groups. That is, video gamers could achieve better performance after a number of training sessions. These results are only partially in agreement with those reported by Bejjanki et al. [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref]. The authors claimed that AVGPs do not enter the perceptual task with improved perceptual templates, rather they found that the performance of AVGPs was indistinguishable from that of non-gaming controls at the beginning of the task. We confirmed these results only for the masking condition in which the best fitting model assumed the same starting accuracy. However, this does not hold for the no-masking condition in which the best fitting model assumed different starting accuracies, showing initially superior VSTM for video gamers than controls. Additionally, we could not confirm that video gamers learned more rapidly the statistics as they progressed on the task. This is because the best fitting models for the no-masking and masking conditions did not assume different learning rates across groups. On the other hand, in the masking condition, video game players outperformed controls showing significantly higher asymptotic accuracy after an arbitrarily large number of learning sessions, thus showing less interferences to intervening masking stimuli as they progressed on the task. The reason behind the discrepancy in the present results and those of Bejjanki et al. [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref] might depend on the task used. In the case of Bejjanki et al. [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref] , participants had to discriminate the orientation of a Gabor patch under different levels of external noise. It might be possible that for this type of low-level perceptual task, both video game players and non-gaming controls need time to learn the relevant statistics of the task to form a proper template of the target stimuli. The authors [bib_ref] Action video game play facilitates the development of better perceptual templates, Bejjanki [/bib_ref] found that AVGPs are faster in learning statistics related to that perceptual task, as suggested by their perceptual training results. In our case, at least for the no-masking condition, it is possible that both action and non-action video game players had already an enhanced VSTM leading in self-selection and engagement in video game playing, though this advantage is lost when introducing the masking RDKs after the sample. In the no-masking condition it should also be noted that even though video game players were better than controls right at the outset of the experiment, they showed no faster improvement as the task progressed. Additionally, when correct and error responses were fitted with the variable precision model [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] , the results revealed a difference in terms of guessing rate between groups, with CONs guessing more than video-game players, and exhibiting slightly higher trial-to-trail variability in memory precision than AVGPs, but not with respect to NAVGPs. The VSTM modelling results then suggest that the overall VSTM precision is similar across the three groups, but CONs made more random responses than video-game players. www.nature.com/scientificreports www.nature.com/scientificreports/ In an analysis of the reaction times, we did not find any evidence for faster responses (i.e., lower reaction times) in AVGPs compared to CONs. This result is consistent with our previous work 3 in which we did not find differences in terms of reaction times between AVGPs and CONs. However, our NAVGPs showed a different pattern of results. Though VSTM performance was very similar across AVGPs and NAVGPs, the reaction times of NAVGPs were much greater than those of AVGPs. On the other hand, we found intermediate response times for CONs. In order to better investigate this possible speed-accuracy trade-off between AVGPs and NAVGPs, reaction times data were analysed with the standard diffusion decision model [bib_ref] A theory of memory retrieval, Ratcliff [/bib_ref] [bib_ref] Modeling Response Times for Two-Choice Decisions, Ratcliff [/bib_ref] [bib_ref] Estimating parameters of the diffusion model: Approaches to dealing with contaminant reaction..., Ratcliff [/bib_ref] [bib_ref] The diffusion decision model: Theory and data for two-choice decision tasks, Ratcliff [/bib_ref]. According to the analysis of the diffusion model, the aforementioned RT differences between the three groups are mostly attributable to the latency of non-decision processes and do not reflect a speed-accuracy trade-off. This is further supported by the finding that the response criteria are approximately the same across the three groups. The non-gamers set the lowest (i.e., most liberal) criterion, on average, but not significantly lower than the video gamers. The same holds for the estimated non-decision time in the three groups who show the same ordering as the mean reaction times, but these differences were not statistically significant. The results of the diffusion model fits further suggest that persons who regularly play video games were less susceptible to interference by the mask. Indicative of the speed of information accumulation, the diffusion drift rates were higher for the video-gaming groups compared to the non-gamers. This trend was observed in both masking and no-masking conditions, whereas the effect was significant only in the masking condition. It seems unlikely that this difference is attributable to the test stimulus. Rather, this finding suggests that video gamers were more efficient in holding the sample stimulus in visual working memory until the test stimulus was presented. A similar conclusion can be drawn from the diffusion model that includes guessing due to loss of the VSTM representation. According to this model, the two groups of video gamers had to rely less on guessing because they more often had the information from VSTM available for the decision. In sum, the results of the diffusion model support the notion that video game players were more efficient in shielding the visual working memory trace of the sample stimulus from interference of the mask. Non-gaming controls, on the other hand, exhibited an overall poorer performance and higher guessing rates, as estimated both with the variable precision model [bib_ref] Variability in the quality of visual working memory, Fougnie [/bib_ref] [bib_ref] Variability in encoding precision accounts for visual short-term memory limitations, Van Den Berg [/bib_ref] [bib_ref] Factorial comparison of working memory models, Van Den Berg [/bib_ref] and the diffusion model [bib_ref] A theory of memory retrieval, Ratcliff [/bib_ref] [bib_ref] Modeling Response Times for Two-Choice Decisions, Ratcliff [/bib_ref] [bib_ref] Estimating parameters of the diffusion model: Approaches to dealing with contaminant reaction..., Ratcliff [/bib_ref] [bib_ref] The diffusion decision model: Theory and data for two-choice decision tasks, Ratcliff [/bib_ref]. These results are in line with those reported by Green et al. [bib_ref] Improved probabilistic inference as a general learning mechanism with action video games, Green [/bib_ref] , in which they found that AVGPs exhibit a higher rate of information processing and that motor processing was similar between AVGPs and non-gaming controls. It is difficult to assess whether the deterioration of performance in the mask condition is better described by a total loss of VSTM information in some trials or by a degradation of VSTM information. The variable precision model and the diffusion model modified to account for guesses are in support of the former interpretation, whereas the standard diffusion model analysis supports the latter. Another result that is worth mentioning is that in our previous study [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref] we found that masking interfered more when its direction matched that of the target sample. However, in the present study there is no difference in performance as a function of the sample-masking direction difference. The reason is not clear, but it might depend on our use of 100% coherent motion in the sample and mask displays, whereas in our earlier study [bib_ref] Visual short-term memory for global motion revealed by directional and speed-tuned masking, Pavan [/bib_ref] we used globally moving RDKs with a signal-to-noise ratio producing approximately 84% correct performance in direction discrimination. Physiological and psychophysical evidence indicates that in motion area MT the directional tuning of neurones is largely invariant with motion coherence and contrast [bib_ref] Tuning bandwidths for near-threshold stimuli in area MT, Britten [/bib_ref] [bib_ref] The invariance of directional tuning with contrast and coherence, Fine [/bib_ref]. In conclusion, we showed that both action and non-action video game playing is associated with an improvement in VSTM for moving objects leading to a visual memory representation that is more robust to external interference. Video game players also exhibit a lower guess rate than non-gaming controls, although memory precision and across trial precision variability were relatively similar across the three groups. Computational modelling of reaction times and accuracies pointed out additional advantages of AVGPs over both NAVGPs and CONs. AVGPs exhibit more efficient information processing and faster response execution than NAVGPs and CONs. Owing to the lack of random group assignment we cannot make any definitive statements concerning the cause and effect of the observed group differences. Further studies should determine the effects of repeated video game playing on VSTM task behavior in a longitudinal study approach with random assignment to the video gaming and control groups. Why it is important to examine VSTM in video game players? As mentioned above, VSTM is involved in a number of important cognitive functions. Video game training and in particular training with action video games may represent a cost-effective and non-invasive training tool to improve VSTM in vulnerable populations. Action video game training can be used for example to counteract the normal VSTM decay in aging [bib_ref] Age-related changes in working memory and the ability to ignore distraction, Mcnab [/bib_ref] [bib_ref] Age-related decline of precision and binding in visual working memory, Peich [/bib_ref] , and even for rehabilitation purposes in patients with mild cognitive impairment 68 , patients with medial temporal lobe damage [bib_ref] Visual short-term memory for high resolution associations is impaired in patients with..., Koen [/bib_ref] , mild Alzheimer diseaseand Parkinson disease [bib_ref] Visual short-term memory deficits associated with GBA mutation and Parkinson's disease, Zokaei [/bib_ref]. Beneficial effects of action video game training on VSTM could also be used to improve visually guided behaviour, with important implications in everyday tasks, driving or sport activities [bib_ref] Understanding the Function of Visual Short-Term Memory: Transsaccadic Memory, Object Correspondence, and..., Hollingworth [/bib_ref]. ## Data availability The analysed datasets are available from the corresponding author upon request. [fig] Figure 2: Results of the memory-masking experiment. (a) Proportion of correct responses as a function of the sample-mask direction difference (deg) (AVGPs: n = 12, NAVGPs: n = 10, CONs: n = 15). Confidence regions (denoted by colour shading) represent standard error of the mean (SEM). (b) Proportion of correct responses for the no-mask and mask conditions. Data were pooled across all sample-mask motion direction differences. Error bars ± SEM. (c) Data points represent accuracies (proportion correct) for individual members of the three groups. Mask data points are the mean performance levels for each participant, averaged over each of the five sample-mask direction differences. The black continuous line represents the equal-performance line for mask and no-mask conditions. [/fig] [fig] Figure 3: Parameters estimate of the variable precision model. (a) g (random guessing) for AVGPs, NAVGPs and CONs. (b) Mode SD (Precision) for the three groups. (c) SD var (Trial-to-trial precision variability) for the three groups. Error bars ± SEM. [/fig] [fig] Figure 4: Reaction times in the visual short-term memory experiment. Filtered log 10 reaction times (ms) of correct responses as a function of the direction difference between sample and mask. Results of the no-mask condition are plotted along the leftmost position of the abscissa. The colours signify the results from the three groups tested. Error bars ± SEM.www.nature.com/scientificreports www.nature.com/scientificreports/ p = 0.157, partial-η 2 = 0.058; quartic, F 1,34 = 1.192, p = 0.283, partial-η 2 = 0.034; quintic, F 1,34 = 0.124, p = 0.727, partial-η 2 = 0.004). [/fig] [fig] Figure 5: The diffusion model of response times. Decision latency is modelled as the first-passage time of a [/fig] [fig] Figure 6: Empirical (10%, 30%, 50%/median, 70%, and 90% quantiles plus overall response probability, circles) and predicted (unmarked points connected with lines) cumulative distribution function of correct (solid circles and lines) and error responses (open circles and dashed lines). (a) Data and model predictions for the nomasking condition. (b) Data and model predictions for the (pooled) masking condition. (2019) 9:6027 | https://doi.org/10.1038/s41598-019-42593-0 [/fig] [fig] Figure 7: Learning curves for video game players and non-video game players with proportion of correct response as a function of the learning session. (a) No-masking condition. (b) Masking condition. Each learning session corresponds to a bin in which data from two consecutive experimental blocks were pooled. The curves represent an elaborated power function fitted to the performance values (see text for more details). Error bars ± SEM. [/fig] [fig] 2: Mean parameter estimates (±SE) and tests of the VSTM/diffusion model fit. Note-Parameters are evidence threshold (a), (mean) drift rates (v), drift rate standard deviation (η), (mean) non-decision time (T er ), and variation of non-decision time (s t ). The guessing parameter (p g ) refers to the percentage of trials in which the content in VSTM was lost. Parameters in parentheses are fixed. Starting point was fixed (z = a/2, s z = 0).F-and p-values refer to results of group comparisons (ANOVA), the adjusted p-values (p adj ) were obtained after FDR correction. (2019) 9:6027 | https://doi.org/10.13 = 15.2, p < 0.001), and better than the model in which only parameter b (i.e., learning rate) varied across groups (i.e., Restricted model 5), with lower AIC and BIC (AIC Restricted model 4 = −117.43, AIC The Restricted Model 5 = −116.43; BIC Restricted Model 4 = −119.73, BIC Restricted Model 5 = −118.72). The Restricted Model 4 was also better than the model in which only the parameter c varied across groups (i.e., Restricted Model 6; AIC Restricted Model 6 = −113.35; BIC Restricted Model 6 = −115.64). The Restricted Model 4 was also better than all the models with 7 parameters (Restricted 4 vs. Restricted 1: F 2,11 = 0.62, p = 0.56; Restricted 4 vs. Restricted Model 2: F 2,11 = 2.8, p = 0.1; Restricted Model 4 vs. Restricted Model 3: F 2,11 = 2.87, p = 0.1). [/fig] [table] Table 3: Lattice of models fitted to the learning sessions of the three groups and for no-masking and masking conditions. f1(x) indicates the functions fitted to the AVGPs group, f2(x) indicates the functions fitted to the NAVGPs group and f3(x) the functions fitted to the CONs group. [/table]

Energy Saving in Data Processing and Communication Systems The power management of ICT systems, that is, data processing (Dp) and telecommunication (Tlc) systems, is becoming a relevant problem in economical terms. Dp systems totalize millions of servers and associated subsystems (processors, monitors, storage devices, etc.) all over the world that need to be electrically powered. Dp systems are also used in the government of Tlc systems, which, besides requiring Dp electrical power, also require Tlc-specific power, both for mobile networks (with their cell-phone towers and associated subsystems: base stations, subscriber stations, switching nodes, etc.) and for wired networks (with their routers, gateways, switches, etc.). ICT research is thus expected to investigate into methods to reduce Dp-and Tlc-specific power consumption. However, saving power may turn into waste of performance, in other words, into waste of ICT quality of service (QoS). This paper investigates the Dp and Tlc power management policies that look at compromises between power saving and QoS. # Introduction The growth in ICT energy consumption is driven by the growth of demand for greater data processing (Dp) and larger access to telecommunications (Tlc), within almost every organization. This growth has a number of important implications, including(i) increased energy costs for business and government, (ii) increased emissions, including greenhouse gases, from electricity generation, (iii) increased strain on the existing power grid to meet the increased electricity demand, (iv) increased capital costs for expansion of data center capacity and construction of new data centers, (v) increased capital costs for expansion of wired and wireless access to communications. Making 100 the total electrical power consumption for the ICT, around 14% is taken by the mobile Tlc, 74% by the wired Tlc, and the remaining 12% by the Dp technology. Dp, however, is only apparently the less powered sector, since Tlc is itself is a Dp consumer, and so any effort to reduce the Dp power consumption may produce cascade effects that also reduce the Tlc one. Studying ways to save Dp power is thus central to any study for ICT power control and optimization. In the US, power absorbed by data centers is estimated in more than 100 billion kW, for an expenditure of $ 8 billion a year that corresponds to the expenditure in electricity of about 17 million homes [bib_ref] Open problems in power management of data centers, Harchol-Balter [/bib_ref]. This same US-local data-center problem becomes a global one when seeing power consumption by web companies, say Google, Yahoo, and so forth. The number of Google servers will reach an estimated 2,376,640 units by the end of 2013. Assuming a busy server absorbs around 240 W of power, Google will need about 600 MW of electrical power by the end of year 2013. In Tlc systems, about 90% of Tlc-specific power consumption is concentrated in the routers. The links only absorb 10%. Current routers consume between 0.01 and 0.1 W/Mbps [bib_ref] Power consumption in telecommunication networks: overview and reduction strategies, Vereecken [/bib_ref]. IT research is thus expected to investigate methods to reduce power absorbed by Dp and Tlc systems. To do that, one may decide to adopt policies to periodically switch off Dp servers or Tlc routers when they are in an idle state. Such policies, however, are to be sufficiently intelligent not to degrade the system quality of service (QoS). Indeed, returning an off server or an off router to its on state requires spending a nonnegligible amount of setup time that makes the server or router slower to respond to customer requests. This may turn into low-quality services such as low response to web queries and unsatisfactory VoIP communications and streaming of data. Any research in power management should thus look at compromises between power saving and QoS. In this paper, Section 2 studies Dp power management policies and Section 3 studies Tlc power management policies. ## Power management in dp systems Data centers have become common and essential to the functioning of business, communications, academic, and governmental systems. During the past years, increasing demand for Dp services has led to significant growth in the number of data centers, along with an estimated doubling in the energy used by servers and the power and cooling infrastructure that supports them. [fig_ref] Figure 1: Data Ccnter energy consumption sources[25] [/fig_ref] illustrates the way energy is spent in data centers. Heating and services for ventilation and air conditioning/backup (HVAC/UPS) absorb around 40% of electrical energy and Dp services the remaining 60%. The latter is in turn divided between AC/DC losses (25%), DC/DC losses (10%), fans, drives, PCI, and so forth, and memory consumptions (for a total 35%) and the remaining 30% is consumption in server processors. In other words, the processors consumption totalizes 0.30 × 0.60, that is, 20% of total data center consumption. Such an amount, even though apparently negligible with respect to the total, is the main cause of the remaining 80%. Thus, any effort to reduce the processors 20% may produce cascade effects that also reduce the remaining 80%. [fig_ref] Figure 2: Cascade effect of energy saving in data centers [/fig_ref] shows that 1 W savings at servers component level (processor, memory, hard disk, etc.) create a reduction in data center energy consumption of approximately 2.84 W. For this reason, any research in ICT power saving should concentrate on policy to reduce Dp consumption at server components level. Data centers can be seen as composed of a number of servers that can be organized into single farms or multifarms. In the following, Section 2.1 sees power saving policies in the single-farm case and Section 2.2 at the multifarm one. ## Energy saving in single-farm data centers. Most of power absorbed by the servers of a farm is wasted, since servers are busy (i.e., making processing work) only 20% to 30% of the time, on average. So, energy saving requires the adoption of management policies to avoid powering the servers when they are not processing. In other words, policies decide in which state (idle or off) to keep the servers when not busy. Two types of server management policies will be considered: static and dynamic policies. ## Energy saving with static policies. One may assume that a busy server in the on state absorbs around 240 W ( ON ), an idle server about 160 W ( IDLE ), and an off server 0 W ( OFF ). So why not keep in the idle state or in the off state the servers when not busy? Just since switching a server from off to on consumes a time overhead. Thus, a power-saving policy. may result in a time-wasting problem. As a consequence, the servers may lose performance (e.g., increased response time to the incoming jobs, lower throughput of communication packets, etc.) and its service may become unacceptable to customers. To turn on an off server, we must first put the server in setup mode. During the setup period, the server cannot process jobs. The time spent in setup is called setup time. In [bib_ref] Server farms with setup costs, Gandhi [/bib_ref] the authors consider server farms with a setup cost. Setup costs always take the form of a time delay, and sometimes there is also a power penalty, since during that entire period the server consumes the same power as being in the on state. In [bib_ref] Server farms with setup costs, Gandhi [/bib_ref] three different policies are studied to manage server farms: On/Idle policy, On/Off policy, and On/Off/Stag policy. Under the On/Idle policy, servers are never turned off. All servers are either on or idle and remain in the idle mode when there are no jobs to serve. Assume that the farm consists of servers; if an arrival finds a server idle, it starts serving on the idle server. Arrival that finds all servers on busy, joins a central queue from which the servers pick jobs when they become idle. The On/Off policy consists instead of immediately turning off the servers when not in use. As said above, however, there is a setup cost (in terms of time delay and of additional power penalty) for turning on an off server. Finally, the On/Off/Stag policy is the same as the On/Off one, except that at most 1 server can be in setup at any point of time. This policy is known as the "staggered boot up" policy in data centers or "staggered spin up" in disk farms [bib_ref] Server farms with setup costs, Gandhi [/bib_ref] [bib_ref] Pergamum: replacing tape with energy efficient, reliable, diskbased archival storage, Storer [/bib_ref].compares the On/Off and On/Idle policies for an example case. The On/Idle policy proves to be better in terms of response time, because the incoming jobs do not suffer from setup time delays but involves a larger amount of power waste with respect to the On/Off policy, because of the amount of power an idle server absorbs. [fig_ref] Figure 4: Effects of server management policies and setup time on response time and... [/fig_ref] compares the three server management policies in a farm consisting of = 10 servers, when the average setup time changes from 1 to 100 sec and the average processing load (i.e., average job arrival rate) from 1 to 7 job/sec. The mean job size (service time) is assumed to be 1 sec. Comparison is on the basis of the resulting mean response time [ ] to the incoming jobs and the average power consumption [ ]. In the On/Idle case, when is low, there is no waiting and thus the mean response time [ ] is of about the mean job service time (1 sec) and increases for increasing . A similar trend can be observed for the On/Off/Stag policy, since [formula] [ ] ON/OFF/STAG = [ ] ON/IDLE + [setup time] ,(1) [/formula] as shown in [bib_ref] Server farms with setup costs, Gandhi [/bib_ref]. For the On/Off policy, instead, the response time curve follows a bathtub behavior. When the load is low, the mean response time is high, since almost every arrival finds servers in the off state, and thus every job incurs the setup time. For medium loads, servers are less frequently switched to the off condition and thus jobs are more likely served by available servers in the on state and do not incur in setup times. For high loads, finally, the mean response time increases due to large queueing in the system. For [fig_ref] Table 1: Synthetic view of the On/Off, On/Idle and On/Off/Stag power optimization policies [/fig_ref] gives a synthetic comparison of the three considered policies in terms of response time and power consumption. (b) Experimental results with same parameters of (a) except for SPTF or SJF queueing discipline. In conclusion, any reduction in power consumption is paid by an increase in response times. So, why not adopt queueing disciplines that minimize average response times? The SPTF (shortest processing time first)or SJF (shortest job first)queueing discipline is known to perform better than the common FIFO (first in first out). Its use can then reduce the amount to pay in terms of response time to obtain a given power saving.(that illustrates the FIFO queueing case) shows that to reduce the power consumption from 780 to 320 W we have to pay an increase from 11 to 39 sec in average response time.illustrates that if the SPTF discipline is used instead; the debt to pay in response time is much smaller (from 39 to 27 sec) as proved in our simulations studies. ## Seeking the optimal ( , ) strategy for the single-farm Data Centers. Under the assumption of Poisson arrivals, exponential service times, and deterministic setup times, authors in [bib_ref] Optimality analysis of energy-performance trade-off for server farm management, Gandhi [/bib_ref] prove that the optimal, or nearly optimal, combination of ( , ), with being one policy from the set {On/Off, On/Idle} and one queueing discipline from the set {FIFO, LIFO, RAND}, means minimizing a new metric called ERP (energy-response time product) and is defined as [formula] ERP ( , ) = [ ] ( , ) × [ ] ( , ) .(2) [/formula] Minimizing ERP( , ) can be seen as maximizing the performance per Watt, with performance defined as the inverse of the mean response time [bib_ref] Optimality analysis of energy-performance trade-off for server farm management, Gandhi [/bib_ref]. In other words, according to their results, there is no need to consider other policies than the On/Off and the On/Idle policies. They, however, only study the effects of moving from one policy to another, without paying attention to the effects of also moving from a = FIFO discipline to another timeindependent discipline. Under the FIFO assumption, however, they find that the On/Idle policy is typically superior to the remaining two in terms of ERP ( , ). Our aim is to extend such results by studying the effects of the queueing discipline , both on the ERP( , ) index and on the [ ]( , ) and [ ]( , ) indices separately. More precisely, the following four ( , ) strategies are investigated in the paper: Similarly, two largely different farm data center loads, low ( ≤ 0.5) and high ( → 1), will be used to stress the effect of the queueing disciplines. [formula] (1) (On/Idle, FIFO),(2) [/formula] The following farm data center characteristics are assumed: server mean setup time [setup] = 1 sec (or 100 sec), server ON = 240 W, server SETUP = 240 W, server IDLE = 150 W, server OFF = 0 W, mean job service time = 1 sec, and = 30 servers. [fig_ref] Table 2: Server farm results for low setuptime [/fig_ref] shows that the optimal ERP( , ) is obtained for the (On/Off, FIFO) strategy and for the (On/Off, SPTF) strategy when is low, while it is obtained for the (On/Idle, SPTF) strategy only when is high. For the high setup time case ( [setup] = 100 sec), the reader is sent to [bib_ref] Power management of server farms, Iazeolla [/bib_ref]. In summary, making predictions of the Dp power management policies that optimizes (ii) the Dp performance (minimum response time) or (iii) the Dp performance-per-Watt (minimum response time-per-Watt) is a nontrivial task. The most significant policies are first to be drawn from the universe of all possible policies. Then, for each such policy, the effects of time-dependent and timeindependent queueing disciplines are to be studied. On the other hand, once the modeling work has been done, the work the server-farm manager has to perform to direct his Dp is greatly simplified, since the universe of all possible ( , ) strategies he needs to choose from is drastically reduced to very small set of most significant strategies. ## Energy saving with dynamic policies. In any practical situation, the load changes over time, according to a given pattern ( ). One should then find policies that adapt themselves to changing load patterns. This is not the case of the policies introduced in Section 2. For this reason, two adaptive versions of the On/Off and On/Idle policies are known in the literature, respectively, called DelayedOff and LookAhead, which dynamically adapt themselves to changing loads [bib_ref] Optimality analysis of energy-performance trade-off for server farm management, Gandhi [/bib_ref]. The DelayedOff policy is an improvement of the On/Off. According to DelayedOff, when a server goes idle, rather than turning off immediately, it sets a timer of duration wait and sits in the idle state for wait seconds. If a request arrives at the server during these wait seconds, the server goes back to the busy state (with zero setup cost); otherwise, the server is turned off. The LookAhead policy is an improvement of the On/Idle. Under such a policy, the system fixes an optimally chosen number * of servers maintained in the on or idle states. According to the standard On/Idle, if an arrival finds a server idle it starts serving on the idle server. Arrivals that find all * server on busy, join a central queue from which servers pick jobs when they become idle. The optimal wait and the optimal * of the two policies, respectively, are chosen to minimize the ERP index. As said above, minimizing ERP can be seen as maximizing the performance per Watt, with performance defined as the inverse of the mean response time [bib_ref] Optimality analysis of energy-performance trade-off for server farm management, Gandhi [/bib_ref]. In the LookAhead policy, * changes as a function of time. Indeed, the policy calculates * ( ) for each time t basing on the forecast of the load ( ) at time . [fig_ref] Figure 5: Effects of dynamic policies with respect to the static On/Off[11] [/fig_ref] illustrates the autoscaling capabilities of the LookAhead and DelayedOff policies [bib_ref] Optimality analysis of energy-performance trade-off for server farm management, Gandhi [/bib_ref] , with respect to the conventional On/Off, for Poisson arrivals with ( ) changing sinusoidally with time (period = 6 hrs). The illustration shows how, with the two dynamic policies, number busy+idle ( ) and number ( ) almost completely follow the behavior of the demand pattern ( ), while in the On/Off case such numbers are somewhat dispersed; in other words, some servers remain in the idle state whereas they should be busy and vice versa, with the consequence of waste of power and worsened response time. The two dynamic policies above simply try to optimize the [ ] by [ ] product. In many practical situations, instead, the objective is to meet a given average response time, according to requirements dictated by specific service level agreements (SLAs). In this case, specific dynamic policies have been introduced, which try to respect the [ ] requirement while minimizing the average power consumed by the servers ( avg ) and the average number of used servers ( avg ). Such policies are known as the AutoScale policy [bib_ref] Autoscale: dynamic, robust capacity management for multi-tier data centers, Gandhi [/bib_ref] , the AlwaysOn policy [bib_ref] Energy-aware server provisioning and load dispatching for connection-intensive internet services, Chen [/bib_ref] , the Reactive policy [bib_ref] Dynamic provisioning of multi-tier Internet applications, Urgaonkar [/bib_ref] , and the Predictive MWA policy [bib_ref] Statistical machine learning makes automatic control practical for internet datacenters, Bodìk [/bib_ref] [bib_ref] Policies for dynamic clock scheduling, Grunwald [/bib_ref] [bib_ref] The Simulation and evaluation of dynamic voltage scaling algorithms, Pering [/bib_ref] [bib_ref] Server workload analysis for power minimization using consolidation, Verma [/bib_ref]. The latter will not be dealt with here, and we will only treat the AutoScale policy, which is an evolution of the remaining three. The AutoScale policy generalizes the use of the wait time already seen for the DelayedOff policy. Differently from this latter, however, is that in the AutoScale case each server decides autonomously when to turn off, setting a timer of duration wait and sitting in the idle state for wait sec. As with the DelayedOff, however, if a request arrives at the server during these wait sec, then the server goes back to the busy state (with zero setup cost). Otherwise, the server is turned off. The AutoScale and the three remaining policies have been evaluated in [bib_ref] Autoscale: dynamic, robust capacity management for multi-tier data centers, Gandhi [/bib_ref] according to a specific load pattern ( ) varying over time between 0 and 800 req/s (see . Such a pattern, known as dual phase pattern is used to represent the diurnal nature of typical data center traffic, where the request rate is low at the night time and high at day time. illustrates the performance of the AutoScale policy, when the time requirement to meet is a 95-percentile response time goal of 400 to 500 ms (denoted T 95 ). In the illustration, the red lines denote the number busy+idle+setup ( ) of busy+idle+setup servers and the blue lines the number busy+idle ( ) of busy+idle servers at time . The ideal line represents the number of servers that should be on at any given time to fully satisfy the demand ( ). The illustration shows how, with the AutoScale policy, there is no dispersion in the available servers and the number busy+idle+setup ( ) and number busy+idle ( ) almost totally follow the demand pattern ( ), and a T 95 = 491 ms goal is achieved, with avg = 1,297 W and avg = 7.2 servers. In the mentioned similar policies (AlwaysOn, Reactive, and Predictive MWA), instead, the T 95 requirement can be seen to be met only at the expense of server dispersion and/or at the expense of avg and avg [bib_ref] Autoscale: dynamic, robust capacity management for multi-tier data centers, Gandhi [/bib_ref]. Indeed, in the AlwaysOn case the T 95 requirement is met (T 95 = 291 ms) but at the expense of a large dispersion in the available servers and large power consumption ( avg = 2,322 W and avg . In the Reactive case, instead, a low dispersion of servers is achieved, with low power and low number of servers ( avg = 1,281 W, avg = 6.2), but the time requirement is absolutely out of range (T 95 = 11,003 ms). A better time performance (T 95 = 7,740 ms) is found in the Predictive MWA with similarly low dispersion of servers and similarly low power and number of servers ( avg = 1,276 W, avg = 6.3). ## Energy saving in multifarm data centers. Energy saving in multifarms is based on so-called self-organization and selfdifferentiation algorithms, whose goal is to transfer the load from a server to a less loaded one, to maximize the power efficiency of the whole data center. These algorithms are widely adopted in the autonomic computing field. The term autonomic indicates systems able to self-manage, self-configure, self-protect, and self-repair; thus, systems have no need of external action to be managed [bib_ref] A bio-inspired algorithm for energy optimization in a selforganizing data center, Barbagallo [/bib_ref]. [fig_ref] Figure 8: Example of a multifarm data center [/fig_ref] illustrates the typical multifarm architecture that consists of a series of server farms (1 through ) each farm controlled by a so-called autonomic component (AC), with the ACs interacting through an overlay network (ON). Each farm serves a number of clients (1 trough ). The ON is a self-organized network, in other words a network which is created, maintained, and optimized through self-organization algorithms which cluster the ACs according to their properties or type [bib_ref] Self-organized server farms for energy savings, Martínez [/bib_ref]. The ACs, in turn, execute a particular kind of selforganization algorithm called self-differentiation algorithm, which takes decentralized decisions on the state and configuration of the ACs. The AC aims to put state the servers in idle and transfer the load on the other servers to limit performance degradation. Three types of self-differentiation algorithms are known: Stand-by, Load Distribution, and Wake-up algorithms whose details can be found in [bib_ref] Self-organized server farms for energy savings, Martínez [/bib_ref]. The algorithms were evaluated by means of simulations of a use-case in which server farms are in charge to serve requests issued by a set of clients. Each client performs several requests, before terminating the connection. The percentage of energy that can be saved in a day goes from about 7% to about 12%, with a debt to pay in terms of response time from about 9 units of time (when the power saving is 7%) to about 11 units of time (when the power saving is 12%). ## Energy management in tlc systems Tlc systems may consist of wired or wireless access networks or of a combination thereof. In addition to the basic Dp infrastructure, Tlc systems also include Tlc-specific subsystems: cell-phone, towers with associated base stations, subscriber stations, switching nodes, and so forth, for the wireless part, and communication processors, routers, gateways, switches, and so forth, for the wired part. Power management in Tlc systems, thus, includes not only power optimization of their Dp infrastructure, but also power optimization of Tlc-specific subsystems. In this section we will only deal with Tlc-specific subsystems, since the power optimization of Dp infrastructure is dealt with is already seen in Section 2. [fig_ref] Figure 9: Typical Tlc network architecture[4] [/fig_ref] describes a typical Tlc architecture, which combines wired and wireless communication networks. In the wired part, three main types of connections are found: (1) the twisted pair copper cable connection based on the DSL (digital subscriber line) technology; (2) the coax cable connection based on the DOCS (data over cable service) technology; and (3) the optical fiber connection based on the GPON (gigabit passive optical network) technology, used when higher bit rates are required. The illustration also shows the DSLAM (DSL access multiplexer) nodes, the OLT (optical line termination) nodes, and the FTTB (fiber to the building) nodes. In order to interconnect different user areas, a core network is used, that consists of a number of core nodes that are interconnected through wavelength-division multiplexed (WDM) optical fiber links, usually in a mesh or ring topology. In the wireless part of the network we find base stations (BS) to which the user's devices are connected by means of radio signals. Each BS is further connected to the core network through a so-called backhaul network. Different technologies can be found, from WiMAX (worldwide interoperability for microwave access) [bib_ref] A distributed approach to wireless system simulation, Iazeolla [/bib_ref] , to HSPA (high speed packet access), and to the most recent LTE (long term evolution). In such a system, about 90% of Tlc-specific power consumption is concentrated in the routers (with 75% the line cards, 10% the power supply and fans, and 10% the switch fabric) [bib_ref] Power control and optimization in ICT, Iazeolla [/bib_ref]. Current routers consume between 0.01 and 0.1 W/Mbps. One can calculate that at ADSL access rates (8 Mbps) the power absorbed per subscriber is of about 0.24 W/subs, while at 100 Mbps becomes of about 3 W/subs [bib_ref] Power consumption in telecommunication networks: overview and reduction strategies, Vereecken [/bib_ref]. Currently, Tlc networks are designed to handle the peak loads. Designing adaptable networks, where one can switch off line cards when the demand is lower and can lead to lower power consuming networks. In core networks this can be achieved by use of dynamic topology optimization algorithms: from all possible topologies that satisfy the required traffic demand, the topologies with lower overall power consumption are chosen. By such algorithms, reductions of power consumption for more than 50% during off-peak hours can be achieved [bib_ref] Power reduction techniques in multilayer traffic engineering, Puype [/bib_ref]. Base stations (BS) with differentiated cell sizes are the key in wireless networks optimization if the so-called hybrid hierarchical BS deployment is used. ## 10 The Scientific World Journal A low layer access network is first created, providing a low bit rate (but large cell sizes) to the users. In the higher layers, BS with higher bit rates (but smaller cell sizes) is utilized to provide high bandwidth connections when required. The advantage is that the higher layers can be switched to the idle and only switched on with high traffic demand. Tlc power optimization also tries to minimize the power consumption of the home gateways. These are individual devices that only need to be on when the user is active. At other times, they could be switched off. In reality this is rarely operated, but legislations concerning standby power consumption standards of 0.5 W are emerging [bib_ref] Power consumption in telecommunication networks: overview and reduction strategies, Vereecken [/bib_ref]. # Conclusions The power management of ICT systems, that is, data processing (Dp) and telecommunication (Tlc) systems, is a complex issue with implications in economical terms. The paper has illustrated methods to optimize Dp power consumption by use of power management policies (static and dynamic policies) that yield electrical power saving while maintaining the system QoS at acceptable levels. The paper has also illustrated methods to optimize Tlc power consumption by use of power management policies to be adopted in wired and wireless Tlc systems. This achieves electrical power saving without compromising the service quality. [fig] Figure 1: Data Ccnter energy consumption sources[25]. [/fig] [fig] Figure 2: Cascade effect of energy saving in data centers [/fig] [fig] Figure 3: (a) Experimental results with 4 servers, utilization = 30% average setup time = 200 sec; average job size (service time) = 7 sec[2]. [/fig] [fig] Figure 4: Effects of server management policies and setup time on response time and power consumption[2]. [/fig] [fig] Figure 5: Effects of dynamic policies with respect to the static On/Off[11]. [/fig] [fig] Figure 6 2, Figure 7: Dual ms, P avg = 1, 297 W, N avg = 7.Effects of dynamic AutoScale policy[13]. [/fig] [fig] Figure 8: Example of a multifarm data center. [/fig] [fig] Figure 9: Typical Tlc network architecture[4]. [/fig] [table] Table 1: Synthetic view of the On/Off, On/Idle and On/Off/Stag power optimization policies. [/table] [table] Table 2: Server farm results for low setuptime ( [setup] = 1 sec). [/table]

MicroRNA-mediated Regulation of the Development and Functions of Follicular Helper T cells The germinal center reaction is a key event of humoral immunity, providing long-lived immunological memory. Follicular helper T (T FH ) cells are a specialized subset of CD4 + T cells located in the follicles, which help B cells and thus control the germinal center reaction. T FH cell development is achieved by multi-step processes of interactions with dendritic cells and B cells along with the coordination of various transcription factors. Since the T helper cell fate decision program is determined by subtle changes in regulatory molecules, fine tuning of these dynamic interactions is crucial for the generation functional T FH cells. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulatory molecules for gene expression, which consequently modulate diverse biological functions. In the last decade, the miRNA-mediated regulation network for the germinal center reaction has been extensively explored in T cells and B cells, resulting in the identification of several key miRNA species and their target genes. Here, we review the current knowledge of the miRNAmediated control of the germinal center reaction, focusing on the aspect of T cell regulation in particular. In addition, we highlight the most important issues related to defining the functional target genes of the relevant miRNAs. We believe that the studies that uncover the miRNA-mediated regulatory axis of T FH cell generation and functions by defining their functional target genes might provide additional opportunities to understand germinal center reactions. # Introduction Humoral immunity plays an important role in host defense by conferring the sophisticated antibodies required to fight against foreign targets. The production of antibodies largely depends on the generation of plasma cells that are differentiated from naïve B cells. Upon antigenic stimulation, naïve B cells begin to proliferate and undergo a cognate interaction with primed CD4 + T cells at the T-B cell border in the secondary lymphoid tissues. Some of the primed B cells are then further differentiated into extrafollicular plasmablasts. Although these plasmablasts are short-lived, they are responsible for the first wave of antigen-specific antibody production, and mainly secrete IgM-type low-affinity antibodies; these instantly secreted antibodies are very important in providing the first line of defense against invading pathogens [bib_ref] IgM in microbial infections: taken for granted?, Racine [/bib_ref]. Subsequently, these low-affinity antibodies are gradually replaced with classswitched high-affinity antibodies produced by the experienced plasma cells of the germinal center to ultimately fortify host immunity. The germinal center is a transient structure formed in the follicles of the secondary lymphoid tissues during infection or vaccination. A small number of specific antigen-primed B cells migrate toward the B cell follicle area from the T-B cell border, proliferate massively, and begin to form the germinal center. During the proliferation stage, the germinal center B cells undergo a class-switch and somatic hypermutation, and selected germinal center B cells are further differentiated into class-switched memory B cells or plasma cells. These reactions waned as the pathogens or antigens are cleared from the system, and the germinal centers also eventually disappear. The size, numbers, and duration of germinal centers must be tightly regulated for maintaining immune homeostasis [bib_ref] Dysregulation of germinal centres in autoimmune disease, Vinuesa [/bib_ref] ; indeed, dysregulation of the germinal center reaction can result in many immunological disorders, including autoimmunity, immunodeficiency, and lymphoma . A small CD4 + T cell population has been observed in the germinal centers, and these cells are believed to play important roles in antibody production in humans and mice (5,6) because they control the Ig class-switch, survival, selection, and differentiation of germinal center B cells via cytokine secretion and ligand-mediated interactions [bib_ref] T follicular helper cell differentiation, function, and roles in disease, Crotty [/bib_ref]. Recently, this specialized subset of CD4 + T cells, termed follicular helper T (T FH ) cells, has been proposed as a key regulator of the magnitude of the germinal center reaction [bib_ref] Control of TFH cell numbers: why and how?, Pratama [/bib_ref]. For instance, strong functional activity and/or increased numbers of T FH cells are strongly linked with autoimmune diseases such as systemic lupus erythematosus [bib_ref] T follicular helper (Tfh) cells in lupus: activation and involvement in SLE..., Blanco [/bib_ref]. By contrast, low numbers of T FH cells cause impaired humoral immunity that eventually leads to immunodeficiency [bib_ref] Pathophysiology of T follicular helper cells in humans and mice, Ueno [/bib_ref]. Therefore, a key to understanding humoral immunity regulation mechanisms might lie in elucidating the detailed mechanisms underlying the development of T FH cells. The correct positioning of T FH cells in the B cell area is one of the most important steps in generating functional T FH cells in the germinal centers. Therefore, it is not surprising that the earliest sign of the T FH cell fate decision is upregulation of C-X-C chemokine receptor (CXCR) 5 [bib_ref] ICOS receptor instructs T follicular helper cell versus effector cell differentiation via..., Choi [/bib_ref] , which is positively controlled by the transcriptional factor achaete scutelike 2 (Ascl2) [bib_ref] Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development, Liu [/bib_ref]. Interestingly, the CXCR5 + antigen-primed T cells are repositioned at the T-B cell border by EBI2 and its ligand 7α,25-dihydroxycholesterol, which mediates chemotactic activity [bib_ref] EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells, Li [/bib_ref]. These pre-T FH cells then interact with CD25 + dendritic cells to confer inducible costimulator ligand (ICOSL)-inducible costimulator (ICOS)-mediated signals, which are responsible for the expression of B cell CLL/lymphoma (Bcl) 6 and sustained CXCR5 expression on pre-T FH cells [bib_ref] EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells, Li [/bib_ref]. In addition, the CD25 + dendritic cells result in an IL-2-deprived environment that is favorable for T FH cell development, because IL-2-mediated signal transducer and activator of transcription (STAT) 5 activation can suppress T FH cell development [bib_ref] EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells, Li [/bib_ref] [bib_ref] STAT5 is a potent negative regulator of TFH cell differentiation, Johnston [/bib_ref] [bib_ref] STAT5 protein negatively regulates T follicular helper (Tfh) cell generation and function, Nurieva [/bib_ref]. Finally, the pre-T FH cells move across the T-B border and reside in B cell follicles where they fully differentiate into T FH cells. Surprisingly, crossing of the border does not rely on CXCR5; instead, the interaction of ICOS-ICOSL with bystander B cells is the crucial event for this step [bib_ref] Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility, Xu [/bib_ref]. In addition to the migratory control of T FH cell differentiation, networks of various transcriptions factors are also critical for the development and functions of T FH cells. The transcription factors Bcl6, interferon-regulatory factor (IRF) 4, B cell-activating transcription factor (BATF), c-Maf, Ascl2, and STAT3 have been revealed as positive drivers, while B lymphocyte induced maturation protein 1 (Blimp-1), forkhead box protein O (FoxO) 1, forkhead box protein P (FoxP) 1, Fosl2, Krüppel-like factor (KLF) 2, and STAT5 are known as repressors of the development and functions of T FH cells [bib_ref] Transcriptional regulation of follicular T-helper (Tfh) cells, Liu [/bib_ref]. Because of the fundamental impact of the amount of lineage-specific transcription factors for the commitment of the fate and plasticity of CD4 + T helper (Th) cells, even subtle changes of the expression of transcription factors and other regulatory genes have large impacts on T cell fates and functions [bib_ref] Transcriptional regulation of follicular T-helper (Tfh) cells, Liu [/bib_ref] [bib_ref] Dynamic balance between master transcription factors determines the fates and functions of..., Fang [/bib_ref]. Therefore, exploration of the mechanisms that underlie the delicate control of the expression of these genes might shed new light on understanding the T cell differentiation program and related immune modulation. MicroRNAs (miRNAs), which are endogenously expressed small (up to 22 nucleotides) single-stranded RNA species, have emerged as important post-transcriptional gene regulatory factors in various types of cells and species [bib_ref] Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells, Chekulaeva [/bib_ref]. MiRNAs integrated in an RNA-induced silencing complex recognize their target mRNAs through partial sequence complementarity and then bind to the targets to suppress their translation or result in their decay; perfect base pairing to the target sequence mediates the degradation of target mRNAs. Although the miRNA-mediated suppression of gene expression is not substantial in terms of magnitude, recent research has revealed an association between the dysregulation of miRNAs and many pathological conditions, including immunological disorders [bib_ref] MicroRNA therapeutics: towards a new era for the management of cancer and..., Rupaimoole [/bib_ref]. In the past few years, extensive studies have been conducted to identify roles of miRNAs in the regulation of the germinal center reaction. Since the miRNAs directly interact with several target mRNAs, revealing the miRNA species that regulate the germinal center reaction and their functional target genes might shed light on the underlying molecular mechanisms. In this review, we discuss the state of the current knowledge on the miRNA-mediated regulation of T FH cell differentiation for the germinal center reaction, and highlight the current topics of debate in understanding of the molecular mechanisms of miRNA-mediated gene regulation. ## Role of mirnas in t fh cell differentiation Among the thousands of miRNAs that have been identified in many species to date, only a few hundreds of these miRNA species are expressed in any given cell type [bib_ref] Regulation of microRNA expression and abundance during lymphopoiesis, Kuchen [/bib_ref] [bib_ref] A mammalian microRNA expression atlas based on small RNA library sequencing, Landgraf [/bib_ref]. CD4 + T cells are not an exception to this general pattern, as the expression profiles of miRNAs in CD4 + T cells are dramatically changed during T cell activation and effector T cell differentiation [bib_ref] T cell activation induces proteasomal degradation of Argonaute and rapid remodeling of..., Bronevetsky [/bib_ref]. For instance, the expression levels of many miRNA species are downregulated upon T cell activation. Only a handful of miRNAs, including miR-19b, miR-106b, miR-155, and miR-146a, are expressed at higher levels in activated T cells than in naïve CD4 + T cells [bib_ref] T cell activation induces proteasomal degradation of Argonaute and rapid remodeling of..., Bronevetsky [/bib_ref]. A comprehensive study on the roles of miRNAs in T cells was performed using mice deficient in Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), which are key enzymes of miRNA biogenesis. Dicer1 or Dgcr8-deficient T cells showed severe defects in activation, proliferation and survival; surprisingly, however, the production of functional cytokines of Th1-like interferon-gamma was not impaired in Dicer-deficient CD4 + T cells [bib_ref] Aberrant T cell differentiation in the absence of Dicer, Muljo [/bib_ref] [bib_ref] MicroRNA-29 regulates T-box transcription factors and interferon-γ production in helper T cells, Steiner [/bib_ref]. Moreover, Dgcr8-deficient CD4 + T cells are preferentially differentiated into Th1 and Th2 cells, but show impaired generation of T FH cells [bib_ref] The microRNA cluster miR-17-92 promotes TFH cell differentiation and represses subset-inappropriate gene..., Baumjohann [/bib_ref]. These results strongly suggest the specific regulatory roles of miRNAs in Th differentiation together with their general roles in T cell proliferation and survival. These findings have prompted researchers to explore the more detailed molecular mechanisms of individual miRNA species on T FH cell differentiation. To date, the potential roles of miR-17-92, miR-155, and miR-146a on T FH cell differentiation and functions have been most extensively studied. Interestingly, these miRNAs show different expression patterns with respect to T FH differentiation. Although all of these miRNAs are induced upon T cell receptor stimulation, miR-155 and miR-146a sustain high expression levels during the further progression of T FH differentiation, whereas induced miR-17-92 species waned out at this stage (24, [bib_ref] MicroRNAs of the miR-17-92 family are critical regulators of T(FH) differentiation, Kang [/bib_ref] [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref]. Six different miRNAs, miR-17, miR-18a, miR-19a, miR-20a, miR19b, and miR-92a, are encoded by a single miR-17-92 transcript, and are expressed and processed together as a cluster. Initially, miR-17-92 was identified as a negative regulator of T FH cell function through CXCR5 suppression [bib_ref] The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment, Yu [/bib_ref]. However, more careful studies with loss-or gain-of-function approaches revealed that miR-17-92 expression in CD4 + T cells is essential for a functional germinal center reaction. Upon antigen priming in T cells, miR-17-92 induces the expression of the target gene Rora, which in turn suppresses the expression of inappropriate genes for T FH cells such Ccr6, Il1r1, Ilr2, and Il22 that are positively regulated by Roar [bib_ref] The microRNA cluster miR-17-92 promotes TFH cell differentiation and represses subset-inappropriate gene..., Baumjohann [/bib_ref]. In addition, miR-17-92 allows for antigen-primed CD4 + T cells to migrate further into the B cell follicles and become functional T FH cells through regulating the strength of ICOS-mediated phosphoinositide 3-kinase (PI3K) activity by targeting the negative regulators of PI3K signaling pathway Pten and Phlpp2 [bib_ref] MicroRNAs of the miR-17-92 family are critical regulators of T(FH) differentiation, Kang [/bib_ref]. Interestingly, fully differentiated T FH cells retain miR-17-92 expression at low levels, which suggests that tight regulation of the miR-17-92 expression level is an important regulatory mode for ensuring an appropriate germinal center reaction. Indeed, overexpression of the miR-17-92 cluster in CD4 + T cells leads to the generation of an excess number of T FH -like cells and activated B cells, ultimately leading to lymphoproliferative disease and death [bib_ref] MicroRNAs of the miR-17-92 family are critical regulators of T(FH) differentiation, Kang [/bib_ref]. In contrast to miR-17-92, induced miR-155 and miR-146a expression upon T cell receptormediated stimuli was found to be sustained at high levels on fully differentiated T FH cells. The B cell integration cluster (bic) gene encodes miR-155, and bic-null mice show impaired T cell-dependent antibody production resulting in failure to protect against virulent Salmonella typhimurium infection, suggesting an important role of miR-155 in humoral immunity [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. miR-155-deficient T cells are activated normally but are prone to become IL-4-producing Th2 cells via the de-repression of c-Maf in vitro, which is reminiscent of the phenomenon by which miR-17-92 deficiency relieves Rora expression and causes the induction of non-T FH cell-related genes [bib_ref] The microRNA cluster miR-17-92 promotes TFH cell differentiation and represses subset-inappropriate gene..., Baumjohann [/bib_ref] [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref]. Indeed, recent studies revealed a miR-155-mediated specific role for functional T FH cell generation via targeting Peli1 and Fosl2 in CD4 + T cells [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref] [bib_ref] miR-155 promotes T follicular helper cell accumulation during chronic, low-grade inflammation, Hu [/bib_ref]. Peli1 is an important regulator of c-Rel protein, a member of the NF-κB family, by means of the ubiquitination in T cells, thus protecting against T cell intrinsic autoimmunity in mice [bib_ref] The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity, Chang [/bib_ref]. In line with previous results, miR-155 deficiency was shown to give rise to a low level of c-Rel expression due to the de-repression of Peli1 during T FH cell development. Interestingly, the low level of c-Rel expression does not affect T FH cell lineage commitment but rather leads to depletion of T FH cells in the draining lymph node, mainly due to the impaired proliferation of pre-T FH cells during development [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref]. Fosl2 binds to Jun and compete with BATF-containing activating (AP-1) complexes for DNA binding on AP-1-IRF composite elements (AICEs), which is necessary for T FH cell generation with IRF4 recruitment. Therefore, the miR-155-mediated repression of Fosl2 is important for determining T FH cell fate commitment [bib_ref] miR-155 promotes T follicular helper cell accumulation during chronic, low-grade inflammation, Hu [/bib_ref]. Taken together, these results suggest that miR-155 acts as a driver of T FH cell fate commitment and as an inhibitor of Th2 cell differentiation by regulating several genes concurrently. miR-146a shows a similar expression pattern to miR-155 during T FH cell development, which indicates that miR-146a might also play important roles in T FH cell generation and functions. However, the ablation of miR-146a results in the accumulation of both T FH cells and germinal center B cells with increased expression of ICOS on T cells, which represents a restrictive role of this miRNA on T FH cell functions (24). Interestingly, the T FH cell-driven regulation of the germinal center reaction might occur through a regulatory interaction between miR-155 and miR-146a in T cells. In 7-10-month-old miR-146a-deficient mice, T cell-driven spontaneous germinal centers are formed followed by autoantibody production in the serum, and miR-155 knockout almost completely restored this aberrant activity of miR-146a-deficient T cells to that of wild-type T cells [bib_ref] miR-155 promotes T follicular helper cell accumulation during chronic, low-grade inflammation, Hu [/bib_ref]. These findings indicate the opposing roles of miR-146a and miR-155 as the brake and accelerator pedals for the function of T FH cells, respectively. IL-2 mediated STAT5 signaling attenuates T FH cell fate commitment at early stages of the differentiation program. One study demonstrated that miR-182 is induced by IL-2 and regulates the late phase of expansion by the post-transcriptional regulation of FoxO1 [bib_ref] The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of..., Stittrich [/bib_ref]. Inactivation of FoxO1 through the ICOS-mediated signaling pathway was also shown to be an important aspect in regulation of T FH cell differentiation at late stages [bib_ref] ICOS coreceptor signaling inactivates the transcription factor FOXO1 to promote Tfh cell..., Stone [/bib_ref] , indicating a potent role of miR-182 in T FH cell generation. However, genetic approaches of miR-182 deficiency in the context of the miR-183/96/182 cluster have revealed a dispensable role of miR-182 in generating functional T FH cells [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref]. ## Role of mirnas in regulatory t fh cells FoxP3 + regulatory T cell (Treg)-mediated immune modulation is a widely accepted concept. Initially, it was considered that homogenous FoxP3 + T cells suppress several distinct subsets of effector T cells [bib_ref] Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control..., Sakaguchi [/bib_ref]. However, recent growing evidence has shifted this concept toward a mechanism by which FoxP3 + Tregs employ distinct regulatory pathways to restrict the responses of different subsets of the effector T cells [bib_ref] Stability and function of regulatory T cells expressing the transcription factor T-bet, Levine [/bib_ref] [bib_ref] Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage..., Yang [/bib_ref]. The function of T FH cells is also controlled by a specialized subset of Bcl6 + FoxP3 + T cells located in the germinal centers, named follicular regulatory T (T FR ) cells [bib_ref] Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions, Chung [/bib_ref] [bib_ref] Foxp3+ follicular regulatory T cells control the germinal center response, Linterman [/bib_ref]. Moreover, T FR cells have been shown to suppress the numbers of T FH cells and germinal center B cells and consequently the amount of antibody produced. T FR cells are distinguished by T FH cells according to the expression of IL-10, glucocorticoid-induced tumor necrosis factor family receptor (GITR), and cytotoxic T lymphocyte antigen (CTLA)-4. More importantly, T FR cells share features in common with T FH cells, including high expression of CXCR5, programmed cell death protein (PD)-1, and ICOS in addition to Bcl6. Beyond the location and expression profiles of T FH cell signature molecules, the differentiation pathway of T FR cells has also been co-opted from that of T FH cells [bib_ref] T-follicular helper cell differentiation and the co-option of this pathway by non-helper..., Linterman [/bib_ref]. Therefore, it is highly possible that several of the miRNAs described above would control the germinal center reaction through regulation of T FR cell generation and function. Indeed, the miR-17-92 dosage is positively correlated with the number of antigen-specific T FR cells, but not with that of polyclonal Tregs in the peripheral lymph nodes [bib_ref] The microRNA cluster miR-17-92 promotes TFH cell differentiation and represses subset-inappropriate gene..., Baumjohann [/bib_ref]. In the absence of miR-146a in T cells, the numbers of both T FR and T FH cells are increased compared to those of wild-type T cells (24). However, miR-146a −/− T FR cells may lose their suppressive capacity due to the overexpression of STAT1, which can partially account for the spontaneous development of germinal centers over time, even in the presence of a high number of T FR cells [bib_ref] Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses, Lu [/bib_ref]. FoxP3 is responsible for the elevated expression of miR-155 in Tregs, indicating that miR-155 exerts certain important roles in FoxP3 + Tregs [bib_ref] Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory..., Kohlhaas [/bib_ref] [bib_ref] Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1..., Lu [/bib_ref]. Indeed, miR-155 has been shown to be responsible for maintaining the Treg pool in competitive settings via suppressing suppressor of cytokine signaling (Socs) 1 [bib_ref] Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1..., Lu [/bib_ref]. Downregulation of Socs1 expression confers Tregs with survival and proliferation ability because of the enhanced STAT5 activity through IL-2. IL-2-mediated STAT5 activation is a well-known inhibitory axis for T FH cell generation [bib_ref] STAT5 is a potent negative regulator of TFH cell differentiation, Johnston [/bib_ref] [bib_ref] STAT5 protein negatively regulates T follicular helper (Tfh) cell generation and function, Nurieva [/bib_ref]. Therefore, it is possible that miR-155 may suppress the generation of T FR cells from Tregs to consequently enable adequate germinal center reactions. However, there has been no detailed study on the roles of miR-155 in T FR cells; thus, it would be interesting to address this issue in the future. miR-10a is induced by transforming growth factor-beta and retinoic acid in induced Tregs, and is known to prevent the conversion of Tregs to T FH cells in Peyer's patches [bib_ref] TGF-β and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and..., Takahashi [/bib_ref]. Since miR-10a is also expressed in naturally occurring Tregs [bib_ref] MicroRNA 10a marks regulatory T cells, Jeker [/bib_ref] and directly targets Bcl6 and its co-repressor, Ncor2 [bib_ref] TGF-β and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and..., Takahashi [/bib_ref] , it is plausible that miR-10a restrains the conversion of naturally occurring Tregs to Bcl6 + T FR cells. In that regard, T FR cell generation might be accompanied by a decrease in the miR-10a expression level, representing another possible regulatory axis for the germinal center reaction. Although there is no direct evidence to date, it is plausible that such regulation would occur during T FR cell generation, because T FH cells reduce the copy number of miR-10a during T FH cell differentiation [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref]. However, it is still worthwhile to explore the role of miR-10a in humoral immunity using T cell-specific loss-of-function or gain-of-function approaches. Overall, it is clear that several miRNA species regulate T FR cell generation and function. However, this subject has received relatively less attention than the regulation of T FH cells, mainly because of the prevailing concept that regulatory mechanisms for T FR cells might be common with those of T FH cells. Nevertheless, the ontology of T FR cells is different from that of T FH cells (conversion from Tregs vs. differentiation from naïve T cells) and their functions are also in opposition. Therefore, there might be a specific regulatory axis for T FR cell generation and function, which is also fine-tuned by certain miRNAs. Defining these mechanisms would be an interesting and important task toward gaining a comprehensive understating of regulation of the germinal center reaction. ## Perspectives Over the past few years, extensive studies have uncovered the miRNA-mediated regulatory mechanisms of the germinal center reaction at both the cellular and molecular levels [bib_ref] MicroRNA regulation of the germinal center response, Baumjohann [/bib_ref]. These studies mainly focused on the development of T FH cells from naïve CD4 + T cells and their functions. Interestingly, most of the miRNAs identified to date, including the miR-17-92 cluster and miR-155, seem to share similar regulatory features by which they suppress non-T FH cell differentiation while concurrently facilitating the T FH cell development program [fig_ref] Figure 1: miRNA regulation of T FH cell differentiation [/fig_ref]. This regulatory mode is fundamentally possible because even a single miRNA species is able to bind to numerous different genes and repress their expression simultaneously. Discovery of this interesting mode of regulation also raises the question as to whether or not miR-17-92 and miR-155 are specific miRNAs for T FH cell development and functions; however, this question remains to be clearly resolved. Moreover, several studies have demonstrated that miR-17-92 and miR-155 also regulate the Th1 responses against lymphocytic choriomeningitis virus infection and the generation of many other function T cell subsets [bib_ref] Cutting edge: mir-17-92 is required for both CD4 Th1 and T follicular..., Wu [/bib_ref] [bib_ref] Molecular dissection of the miR-17-92 cluster's critical dual roles in promoting Th1..., Jiang [/bib_ref] [bib_ref] miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding..., Escobar [/bib_ref] [bib_ref] MicroRNA-155 confers encephalogenic potential to Th17 cells by promoting effector gene expression, Hu [/bib_ref] , raising the general question as to how a single miRNA species is able to control so many different aspects of immune responses. different experimental settings with miR-155 knockout mice [bib_ref] A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper..., Liu [/bib_ref] [bib_ref] Requirement of bic/microRNA-155 for normal immune function, Rodriguez [/bib_ref] [bib_ref] miR-155 promotes T follicular helper cell accumulation during chronic, low-grade inflammation, Hu [/bib_ref] [bib_ref] Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1..., Lu [/bib_ref] [bib_ref] miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding..., Escobar [/bib_ref] [bib_ref] Stage-specific regulation of natural killer cell homeostasis and response against viral infection..., Zawislak [/bib_ref]. These same studies indicated that Jarid2, Socs1, Fosl2, Peli1, Il23r, and c-Maf were functional targets, which are responsible for each of the observed phenotypes in the miR-155 knockout mice. Surprisingly, the Socs1 3′-UTR mutant mice phenocopied only a few phenotypes among those observed in the miR-155 knockout mice, such as NK cell expansion upon viral infection and enhanced Treg fitness in competitive settings [bib_ref] A single miRNA-mRNA interaction affects the immune response in a context-and cell-type-specific..., Lu [/bib_ref]. Considering all of these findings, Lu et al. [bib_ref] A single miRNA-mRNA interaction affects the immune response in a context-and cell-type-specific..., Lu [/bib_ref] proposed that a single miRNA is capable of regulating several distinct immune responses by selecting a functional target in a cell type-and context-specific manner. This insightful conclusion can explain the variation observed within and between studies in 1 or 2 functional target genes even when using the same miR-155 knockout mice. However, this idea further brings forth a new and important question to resolve: how do miRNAs select the right target for the cell type and context? Many research groups are currently pursuing answers to this abstruse question, including Xiao and colleagues [bib_ref] Understanding microRNA-mediated gene regulatory networks through mathematical modelling, Lai [/bib_ref] [bib_ref] MicroRNA mechanisms of action: what have we learned from mice?, Jin [/bib_ref]. Recently, this group executed an elegant systemic transcriptome and translatome analysis of miR-17-92-deficient or overexpressed primary B cells [bib_ref] Differential sensitivity of target genes to translational repression by miR-17-92, Jin [/bib_ref]. Although the conclusions of this study were not able to provide clear answers to this question, they nevertheless provided important insights on this issue. First, there are more putative miR-17-92-binding sites in the pool of mRNAs than the total copy number of miR-17-92 species at the single-cell level, indicating that the presence of conserved miRNA-binding sites in the 3′-UTR of mRNA is not a sufficient condition to be repressed by any given miRNA. This implies the existence of certain mechanisms by which miRNAs select their targets. Second, the authors found that the validated target genes of miR-17-92 show distinct repression sensitivity to miR-17-92 in primary B cells. In other words, certain miR-17-92 target genes are repressed only after miR-17-92 are up-regulated in the B cells; however, those genes are not necessarily de-repressed when miR-17-92 are down-regulated. In contrast, another group of genes are responsive only when miR-17-92 are down-regulated; these 2 groups of genes rarely overlap with each other, indicating that functional target genes of miR-17-92 are probably selected on the basis of miRNA levels in B cells. Therefore, miRNAs might be able to select their appropriate target based on cell type and the context in accordance with the expression level of a given miRNA. Third, the secondary structure of mRNA was found to have a greater impact on its possibility of being selected as a target than other cis element criteria such as the length of the 5′-UTR, 3′-UTR, location, and numbers of miRNA-binding sites. Taken all together, these findings provide mechanistic insights into how the same miRNA species is able to choose different target genes in a cell type-and context-dependent manner. The last few years has seen an explosion in research focused on defining the roles of miRNAs in the initiation and maintenance of the germinal center reaction. However, recent findings of the presence of a memory type of T FH cells implies the existence of another level of regulation of the germinal center reaction by memory T FH cells [bib_ref] Memory T follicular helper CD4 T cells, Hale [/bib_ref]. Indeed, memory T FH cells rapidly respond to the second challenged pathogen and acquire T FH effector functions. This recall response of memory T FH cells is considered as a key event in long-lasting protective humoral immunity. Thus, revealing the mechanisms of the generation and maintenance of memory T FH cells might be crucial in the development of effective vaccines. Since CXCR5 + T FH -like cells are also characterized by plasticity with respect to their cell fate commitment upon stimulation, the roles of the memory type of T FH cells in various aspects of immunology are actively being explored [bib_ref] Do memory CD4 T cells keep their cell-type programming: plasticity versus fate..., Crotty [/bib_ref]. Therefore, defining the specific miRNA species and deciphering the molecular mechanisms of the miRNA-mediated regulation of memory T FH cell generation and functions will be of great interest, but remain to be elucidated. [fig] Figure 1: miRNA regulation of T FH cell differentiation. During the priming of naïve CD4 + T cells, miR-17-92, miR-155, and miR-146a are induced upon T cell receptor mediated stimulation. miR-17-92 and miR-155 positively regulate T FH cell differentiation via repression of negative regulators of T FH cells, Rora, Phlpp2, Pten, c-Maf, Peli1, and Fosl2. However, miR-146a tones down T FH cell functions by suppressing ICOS expression on the surface of effector T FH cells. [/fig]

Active Components of Traditional Chinese Medicinal Material for Multiple Myeloma: Current Evidence and Future Directions Multiple myeloma (MM) is a hematological malignancy characterized by clonal expansion of plasma cells in bone marrow, leading to the overproduction of monoclonal immunoglobulins. The clinical manifestations resulting from monoclonal proteins and malignant cells include signs of end-organ damage, such as hypercalcemia, renal failure, anemia, and bone lesions. Despite improvement in the survival of MM patients with use of myeloma-targeted and immunomodulatory therapies, MM remains an incurable disease. Moreover, patients with relapsed or refractory MM show poor survival outcomes. In recent years, there has been a growing interest in the use of traditional Chinese medicinal materials (TCMMs) for management of a wide spectrum of diseases. The bioactive ingredients derived from TCMMs hold great potential for the development of anticancer drugs. Here we summarize the evidence of the pharmacological effects of the active components in TCMMs on MM, including curcumin, resveratrol, baicalein, berberine, bufalin, cinobufagin, gambogic acid, ginsenoside, icariin, daidzin, formononetin, polysaccharides extracts from Hedyotis difus, and scutellarein. Available evidence indicates that the anti-MM effects of these bioactive ingredients are mediated via regulation of proliferation, apoptosis, autophagy, cell cycle, osteogenic differentiation, and drug resistance. In the future, the underlying mechanisms of the anti-MM effects of these components should be further investigated. Large-scale and well-designed clinical trials are also required to validate the efficacy of these bioactive constituents for MM. # Introduction Multiple myeloma (MM) is the second most commonly diagnosed hematologic malignancy, accounting for nearly 10% of all hematological malignancies [bib_ref] Multiple Myeloma, Kumar [/bib_ref]. Environmental and occupational exposure [bib_ref] Occupational Exposure and Multiple Myeloma Risk: An Updated Review of Meta-Analyses, Georgakopoulou [/bib_ref] , genetic factors, and epigenetic alterations , race [bib_ref] Dissecting Racial Disparities in Multiple Myeloma, Marinac [/bib_ref] have been implicated in the causation of MM [bib_ref] Multiple Myeloma: 2020 Update on Diagnosis, Risk-Stratification and Management, Rajkumar [/bib_ref]. The disease is characterized by progressive monoclonal proliferation in the bone marrow, which leads to the overproduction of nonfunctional intact immunoglobulins (also known as M protein or monoclonal protein) [bib_ref] Biology and Therapy of Multiple Myeloma, Joshua [/bib_ref] or immunoglobulin chains in 15-20% of patients [bib_ref] Serum Free Light-Chain Measurements for Identifying and Monitoring Patients with Nonsecretory Multiple..., Drayson [/bib_ref]. Accumulation of these immunoglobulins and interaction of the aberrant monoclonal plasma cells with other cells in the bone marrow results in various serious complications including hypercalcemia, renal failure, anemia and bone lesions which are collectively referred to as CRAB features [bib_ref] International Myeloma Working Group Updated Criteria for the Diagnosis of Multiple Myeloma, Rajkumar [/bib_ref] [bib_ref] Multiple Myeloma: 2020 Update on Diagnosis, Risk-Stratification and Management, Rajkumar [/bib_ref]. Pathologically, MM is preceded by a premalignant phase termed monoclonal gammopathy of undetermined significance (MGUS), which is typically characterized by lower concentration of M proteins than that in MM [bib_ref] Shaping the Treatment Paradigm Based on the Current Understanding of the Pathobiology..., Ninkovic [/bib_ref]. MGUS is often asymptomatic, with or without an identified intervening stage, referred to as smoldering multiple myeloma (SMM). Most MGUS cases are clinically stable with a 1% annual risk of progression to MM [bib_ref] Long-Term Follow-Up of Monoclonal Gammopathy of Undetermined Significance, Kyle [/bib_ref]. Currently, there is a paucity of drugs that are able to recondition MM. Some new drugs, such as proteasome inhibitors bortezomib and carfilzomib as well as the immunomodulatory drugs (thalidomide, lenalidomide, and pomalidomide) have helped improve the treatment landscape for MM patients. However, the majority of patients ultimately develop recurrence. Therefore, development of novel therapeutic approaches for MM is a key imperative. Natural products often constitute the basis for the identification of effective drugs. Many widely used drugs are of natural origin [bib_ref] Natural Products as Sources of New Drugs over the Nearly Four Decades..., Newman [/bib_ref]. In other words, refinement, processing, and standardization of the active ingredients in natural products is still a common approach for the development of novel therapeutics. A recent study systematically reviewed anti-myeloma plant natural products including alkaloids, phenolics and terpenes. In addition, Traditional Chinese medicinal materials (TCMMs), as natural products, are also an attractive source of novel drugs for MM treatment. In recent years, a considerable body of scientific evidence has underlined the promising potential of bioactive ingredients derived from TCMMs for treatment of MM. To the best of our knowledge, there is no systematic summary of the anti-MM effects and clinical applications of these commonly utilized bioactive components extracted from TCMMs that are different from the natural products reviewed by Jöhrer et al. [bib_ref] Multiple Myeloma Inhibitory Activity of Plant Natural Products, Jöhrer [/bib_ref]. The objective of this review was to systematically summarize the available evidence of the anti-MM effects of the bioactive components isolated from TCMMs and to identify future research priorities for the development of novel agents for MM. ## Active components derived from tcmms for multiple myeloma curcumin Curcumin, a polyphenol extracted from Curcuma longa (also known as turmeric) is widely used in medicine and as a dietary constituent worldwide. It has gained increasing attention for its greater bioactivity compared to the other bioactive compounds isolated from turmeric. Curcumin belongs to a chemical class of polyphenols with a chemical formula of C 21 H 20 O 6 [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref]. Curcumin exhibits a wide spectrum of pharmacological effects including anti-microbial, anti-inflammatory, anti-oxidant, anticancer, anti-viral, and neuroprotective effects. The mechanisms of action of curcumin against various cancers and other disorders have been intensively reviewed, such as breast cancer [bib_ref] Anti-metastasis Activity of Curcumin against Breast Cancer via the Inhibition of Stem..., Hu [/bib_ref] , colorectal cancer [bib_ref] Curcumin and Colorectal Cancer: From Basic to Clinical Evidences, Pricci [/bib_ref] , lung cancer (Wan Mohd Tajuddin et al., 2019), cerebral ischemia [bib_ref] Neuroprotective Effects of Curcumin in Cerebral Ischemia: Cellular and Molecular Mechanisms, Subedi [/bib_ref] , neurodegenerative diseases [bib_ref] Curcumin and Neurodegenerative Diseases, Monroy [/bib_ref] , and diabetes [bib_ref] Curcumin and Type 2 Diabetes Mellitus: Prevention and Treatment, Pivari [/bib_ref]. An increasing body of evidence from clinical and experimental studies has shown the great potential of curcumin as anti-MM therapy. In a study by curcumin treatment (at a dose of 4 g daily) for 3 months led to a decrease in paraprotein load and bone resorption in patients with MGUS. However, only patients with a paraprotein of >20 g/ L responded to curcumin [bib_ref] The Potential Role of Curcumin in Patients with Monoclonal Gammopathy of Undefined..., Golombick [/bib_ref]. Subsequently, results from a randomized, double-blind placebo-controlled trial suggested that curcumin treatment (4 and 8 g daily) may potentially slow down the disease progression in patients with smoldering MM . In a recent clinical trial, patients with MM who received adjuvant curcumin treatment (at a dose of 3-4 g daily), as a replacement of dexamethasone due to intolerance to dexamethasone, showed decreased paraprotein load and plasmacytosis by 38 and 59%, respectively, when administered in combination with other anti-myeloma therapies. These findings indicated that curcumin may slow disease progression without inducing the adverse effects associated with steroid use [bib_ref] Use of Curcumin in Multiple Myeloma Patients Intolerant of Steroid Therapy, Ramakrishna [/bib_ref]. Besides, curcumin was shown to inhibit STAT3 phosphorylation in U266 MM cells in a dose-and time-dependent manner. Curcumininduced inhibition of STAT3 phosphorylation not only suppressed the growth of myeloma cells, but also sensitized MM cells to dexamethasone [bib_ref] Curcumin (Diferuloylmethane) Inhibits Constitutive and IL-6-inducible STAT3 Phosphorylation in Human Multiple Myeloma..., Bharti [/bib_ref]. A curcumin analog, FLLL332, was also found to specifically inhibit STAT3 phosphorylation and DNA binding activity, which resulted in inhibition of downstream target genes involved in cell proliferation including cyclin D1, Bcl-2, survivin, thus inducing apoptosis in MM cells [bib_ref] A Novel Small Molecule Inhibits STAT3 Phosphorylation and DNA Binding Activity and..., Lin [/bib_ref]. Another water-soluble curcumin analog, curcumin #12, was found to increase the sensitivity of MM cells to proteasome inhibitor bortezomib, thus inducing a considerable increase in caspase activity [bib_ref] Sensitizing Human Multiple Myeloma Cells to the Proteasome Inhibitor Bortezomib by Novel..., Mujtaba [/bib_ref]. Curcumin was also shown to reverse the resistance to melphalan chemotherapy in vitro possibly via inactivation of the Fanconi anemia/BRCA pathway [bib_ref] Reversal of Multidrug Resistance by Curcumin through FA/BRCA Pathway in Multiple Myeloma..., Xiao [/bib_ref]. [bib_ref] Curcumin Ameliorates the In Vitro Efficacy of Carfilzomib in Human Multiple Myeloma..., Allegra [/bib_ref] reported that curcumin inhibited NF-κB pathway, upregulated expression levels of p53 and p21 that were implicated in the regulation of apoptotic pathways and cell cycle in U266 cells. A combination of curcumin and carfilzomib exerted stronger proapoptotic effects than curcumin or carfilzomib alone. Consistently, in several studies, curcumin was found to ameliorate chemoresistance and to sensitize MM cells to bortezomib via inhibiting Notch1 pathwayand NF-κB pathway , leading to the downregulation of cyclin D1, survivin, Bcl-xL, cIAP-1, XIAP, Bcl-2, TRAF1, and VEGF, which are associated with proliferation, apoptosis, and angiogenesis . Anti-angiogenesis effects of curcumin were also confirmed by [bib_ref] Inhibitory Effect of Curcumin on Angiogenesis Induced by Brain Derived Neurotrophic Factor..., Wang [/bib_ref]. They observed that curcumin inhibited angiogenesis via interrupting the interaction between MM cells and endothelial cells by decreasing TrkB expression in endothelial cells and suppressing BDNF production in MM cells. In addition, curcumin-induced epigenetic regulation on MM has also been investigated. [bib_ref] Curcumininduced Promoter Hypermethylation of the Mammalian Target of Rapamycin Gene in Multiple..., Chen [/bib_ref] found that curcumin induced Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 13 | Article 818179 3 apoptosis and suppressed the proliferation of MM cells via hypermethylating the promoter of mTOR in CpG sites, which in turn lead to an upregulation of mTOR signaling involved in the regulation of apoptosis and autophagy in MM. To summarize, the available evidence indicates multiple mechanisms of the anti-MM effects of curcumin, including induction of apoptosis, and inhibition of angiogenesis and proliferation . ## Resveratrol Resveratrol (3,4 ′,5-trihydroxy-trans-stilbene, [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref] was first isolated from the roots of white hellebore in 1940s, and since then it has been extracted from a variety of other plant species. It was also abundantly found in the active ingredients isolated from several Chinese herbal medicines, such as Polygonum cuspidatum , Semen cassia (Jue Ming Zi), and Veratrum nigrum . Resveratrol has a broad spectrum of beneficial health effects including anti-aging, antioxidant, anti-inflammatory, neuroprotective, cardioprotective, anti-microbial, and immune-regulatory effects. Studies have shown that resveratrol may also exhibit significant anti-cancer activity against a wide range of solid tumors and hematological malignancies. Recently, the anti-MM effects of resveratrol have been gaining growing attention. In a study, resveratrol was found to suppress angiogenesis in RPMI 8226 cells by inhibiting the expressions of VEGF, bFGF, MMP-2, and MMP-9, which was consistent with the results of a previous study. In the study by Jin et al. [bib_ref] Combining the Mammalian Target of Rapamycin Inhibitor, Rapamycin, with Resveratrol Has a..., Jin [/bib_ref] , combination therapy with resveratrol and rapamycin exerted stronger effects in inhibiting proliferation and inducing apoptosis of MM cells, indicating that resveratrol may have an inhibitory effect on mTOR signaling. In another study, resveratrol was found to induce apoptosis of MM cells via mitochondrial apoptotic pathway and the recruitment of Fas/ CD95 death receptor, and downstream signaling molecules into lipid rafts [bib_ref] Involvement of Mitochondria and Recruitment of Fas/CD95 Signaling in Lipid Rafts in..., Reis-Sobreiro [/bib_ref]. Wang et al. [bib_ref] Resveratrol Triggers the Pro-apoptotic Endoplasmic Reticulum Stress Response and Represses Pro-survival XBP1..., Wang [/bib_ref] demonstrated that resveratrol can induce endoplasmic reticulum stress response via activating IRE1α/XBP1 pathway, leading to a pro-apoptotic effects on MM cells. Additionally, it also suppressed pro-survival XBP1 signaling. Besides, inhibition of NEAT-1-mediated Wnt/β-catenin pathway [bib_ref] Resveratrol Inhibits Proliferation, Migration and Invasion of Multiple Myeloma Cells via NEAT1-Mediated..., Geng [/bib_ref] , IL-6/STAT3 pathway [bib_ref] Resveratrol Inhibits Proliferation, Induces Apoptosis, and Overcomes Chemoresistance through Down-Regulation of STAT3..., Bhardwaj [/bib_ref] , NF-κB signaling [bib_ref] Resveratrol Downregulates the Constitutional Activation of Nuclear Factor-kappaB in Multiple Myeloma Cells,..., Sun [/bib_ref] [bib_ref] Resveratrol Inhibits Proliferation, Induces Apoptosis, and Overcomes Chemoresistance through Down-Regulation of STAT3..., Bhardwaj [/bib_ref] , cyclin D1 and Bcl-xL [bib_ref] Resveratrol Downregulates the Constitutional Activation of Nuclear Factor-kappaB in Multiple Myeloma Cells,..., Sun [/bib_ref] [bib_ref] Resveratrol Inhibits Proliferation, Induces Apoptosis, and Overcomes Chemoresistance through Down-Regulation of STAT3..., Bhardwaj [/bib_ref] , ERK1/2 and JNK pathways [bib_ref] Pterostilbene Inhibits Human Multiple Myeloma Cells via ERK1/2 and JNK Pathway In..., Xie [/bib_ref] may also be involved in the resveratrol-induced suppression of cell proliferation and invasion, cell-cycle arrest, and apoptosis induction in MM. Other than the pro-apoptosis effects, resveratrol also showed pro-autophagic activities. [bib_ref] Resveratrol Induces AMPK and mTOR Signaling Inhibition-Mediated Autophagy and Apoptosis in Multiple..., Ma [/bib_ref] found that resveratrol caused dose-dependent upregulation of the levels of LC3 and Beclin1 in several MM cell lines; further investigation demonstrated that resveratrol-induced autophagic flux was mediated by increasing the phosphorylation of AMPK at Thr172 site and decreasing the phosphorylation of mTOR (Ser2448), p70S6K(Thr389), and 4EBP1(Thr37/46). In addition, resveratrol not only sensitized the proteasome inhibitor carfilzomib-induced apoptosis via increasing the production of reactive oxygen species (ROS) in multiple kinds of MM cell lines, but also induced autophagy when administered in combination with low-dose carfilzomib, as evidenced by increased levels of LC3-II and p62/SQSTM1 . Increased osteoclast formation and bone resorption and absence of bone formation are the serious consequences of MM [bib_ref] Pathogenesis of Bone Disease in Multiple Myeloma: from Bench to Bedside, Terpos [/bib_ref]. In a study, resveratrol treatment was shown to promote osteogenic differentiation of bone marrow mesenchymal stem cells from patients with multiple myeloma via activating the SIRT1/RUNX2 pathway. Moreover, resveratrol inhibited osteoclastogenesis via receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation, which was associated with downregulation of RANK expression and decrease in NFATc1 stimulation and NF-κB nuclear translocation; these findings suggested that resveratrol may be an efficient osteoclast inhibitor in MM. Besides, resveratrol also induced the expressions of osteoblast markers osteocalcin and osteopontin in human bone marrow mesenchymal stem cells and sensitized their response to FIGURE 3 | Action pathways involved in the anti-MM effects of curcumin. represents inhibitory effects, represents stimulative effects. Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 13 | Article 818179 1,25(OH) 2 vitamin D 3 [bib_ref] Resveratrol Inhibits Myeloma Cell Growth, Prevents Osteoclast Formation, and Promotes Osteoblast Differentiation, Boissy [/bib_ref]. Collectively, these studies demonstrate that the anti-tumor effects of resveratrol in MM cell lines include inhibition of cell proliferation, upregulation of apoptosis and autophagy, induction of oxidative stress, cell cycle arrest, and suppression of osteoclastogenesis [fig_ref] FIGURE 4 |: Action pathways involved in the anti-MM effects of resveratrol [/fig_ref]. However, results from a phase-two study of resveratrol plus bortezomib for patients with relapsed and or refractory MM indicated minimal efficacy of resveratrol treatment; in addition, resveratrol caused severe adverse events such as nausea, vomiting, and even unexpected renal failure [bib_ref] A Phase 2 Study of SRT501 (Resveratrol) with Bortezomib for Patients with..., Popat [/bib_ref]. Till date, there have been few clinical trials of resveratrol therapy in MM patients. Whether resveratrol is a potential therapeutic intervention for MM patients or not warrants further investigation. ## Baicalein Baicalein (4H-1-benzopyran-4-one,5,6,7-trihy-droxy-2-phenyl-C 15 H 10 O 5, [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref] is a major bioactive flavone derived from the roots of Scutellaria baicalensis georgi (Hunag Qin). Studies have demonstrated the anticancer effects of baicalein in the context of various cancers, including breast cancer [bib_ref] Baicalein Induces Apoptosis and Autophagy of Breast Cancer Cells via Inhibiting PI3K/AKT..., Yan [/bib_ref] , lung cancer [bib_ref] Baicalein Inhibits Non-small-cell Lung Cancer Invasion and Metastasis by Reducing Ezrin Tension..., Zhang [/bib_ref] , colorectal cancer [bib_ref] Inhibition of Autophagy Amplifies Baicalein-Induced Apoptosis in Human Colorectal Cancer, Phan [/bib_ref] , and hepatocellular carcinoma [bib_ref] Baicalein: A Review of its Anti-cancer Effects and Mechanisms in Hepatocellular Carcinoma, Bie [/bib_ref]. Baicalein was shown to induce DNA damage without causing serious chromosomal instabilities or mutagenesis that may lead to severe side effects during chemotherapy [bib_ref] High-throughput Genotoxicity Assay Identifies Antioxidants as Inducers of DNA Damage Response and..., Fox [/bib_ref]. Recent studies have demonstrated the anti-MM effects of baicalein mediated via inhibition of proliferation and migration, and induction of apoptosis. In the study by, baicalein suppressed the growth and promoted apoptosis of myeloma U266 cells via downregulating IKZF1 and IKZF3 (two essential lymphoid transcription factors in MM). Similarly, baicalein not only inhibited myeloma cell proliferation and induced apoptosis through downregulating IL-6, but also abrogated IL-6-mediated signaling cascades including JAK, STAT3, MAPK, and Akt pathways associated with the proliferation and survival of MM cells [bib_ref] Inhibitory Effect of Baicalein on IL-6-mediated Signaling Cascades in Human Myeloma Cells, Liu [/bib_ref]. In addition, baicalein inhibited the proliferation and migration of RPMI 8226 and U266 MM cell lines via inhibition of Wnt/β-catenin pathway, c-myc, cyclin D1 and integrin β7, which are involved in cell proliferation. [bib_ref] PPARbeta-mediated Growth Suppression of Baicalein and Dexamethasone in Human Myeloma Cells, Otsuyama [/bib_ref] found that combination of baicalein and dexamethasone can suppress the growth of U266 MM cells via activating PPARβ and glucocorticoid receptors, thus inhibiting the transcriptional activity of NF-κB followed by decreased levels of IL-6 and IKBα. Besides, baicalein can increase the sensitivity of MM cells to immunomodulatory drugs (IMiDs) by upregulating the CRBN which is also the target protein of IMiDs. Side population cells show proliferation and differentiation potential similar to cancer stem cells. [bib_ref] Baicalein Decreases Side Population Proportion via Inhibition of ABCG2 in Multiple Myeloma..., Gu [/bib_ref] reported that baicalein decreased the proportion of side population cells via inhibition of ATP-binding cassette, subfamily G, isoform two protein (ABCG2) which is responsible for drug resistance in human MM cell line RPMI 8226. These findings suggest that baicalein may potentially target cancer stem cells of MM, indicating its potential use for the treatment of MM [fig_ref] FIGURE 5 |: Action pathways involved in the anti-MM effects of baicalein [/fig_ref]. ## Berberine Berberine [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref] is the main active component of Optidis rhizome . Several studies have demonstrated the anti-inflammatory, anti-oxidative, anti-microbial, anti-tumor, and neuroprotective effects of berberine. Recent experimental studies have also suggested the potential benefits of berberine in the treatment of MM . In the study by berberine administered in combination with bortezomib significantly increased the expressions of casepase-3, casepase-8, and casepase -9 in U266 cell line, and showed stronger proapoptotic effect than berberine or bortezomib alone. In a study by Qing et al. [bib_ref] Berberine Induces Apoptosis in Human Multiple Myeloma Cell Line U266 through Hypomethylation..., Qing [/bib_ref] , berberine Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 13 | Article 818179 decreased the p53 DNA CpG methylation level via suppressing DNA methyltransferases DNMT1 and DNMT3B, and affected mRNA levels of several primary apoptosis-related proteins, thus inducing apoptosis and cell cycle arrest in U266 MM cells. Berberine can also suppress MM cells via possibly downregulating miRNA clusters including miR-99a~125b, miR-17-92 and miR-106-25, which possibly affected MAPK, ErbB, and TP53 signaling pathways [bib_ref] Systematic Analysis of Berberine-Induced Signaling Pathway between miRNA Clusters and mRNAs and..., Feng [/bib_ref]. Further studies validated that miR-106b/25 cluster encoding miR-106b, miR-93, and miR-25 [bib_ref] Integrative Analysis of Signaling Pathways and Diseases Associated with the miR-106b/25 Cluster..., Gu [/bib_ref] , and miR-19a/92a cluster [bib_ref] Signal Pathways, Diseases, and Functions Associated with the miR-19a/92a Cluster and the..., Yin [/bib_ref] were involved in berberine-induced inhibition of MM cells. [bib_ref] Integrative Analysis of Differential miRNA and Functional Study of miR-21 by Seed-Targeting..., Luo [/bib_ref] found that berberine treatment suppressed MM cell growth via inhibiting IL6/STAT3/miR-21 pathway, which led to increased expression of PDCD4 and inhibition of p53 signaling. Similarly, berberine inhibited NF-κB translocation via Set9-mediated lysine methylation, resulting in a decrease in miR-21 level followed by decrease in Bcl-2 level, thereby triggering ROS generation and apoptosis in U266 MM cells [bib_ref] Set9, NF-Κb, and microRNA-21 Mediate Berberine-Induced Apoptosis of Human Multiple Myeloma Cells, Hu [/bib_ref]. Lastly, [bib_ref] Identification of Berberine as a Novel Drug for the Treatment of Multiple..., Gu [/bib_ref] also demonstrated that berberine treatment inhibited the growth of several MM cell lines via targeting UHRF1 (ubiquitin-like with PHD and RING Finger domains 1). Further molecular docking and surface plasmon resonance analysis confirmed UHRF1 as a berberine-binding protein and revealed that berberine binded UHRF1 in the tandem tudor domain and plant homeodomain. ## Chansu Chansu, also known as toad venom, is an expensive TCMM extracted from the skin or parotid venom glands of Bufo gargarizans Cantor . Bufadienolides (including bufalin, cinobufagin, and resibufogenin) are considered as the main bioactive ingredients of Chansu. Among these, bufalin and cinobufagin are the main agents contributing to the antitumor effects [bib_ref] Spatial Lipidomics Reveals Anticancer Mechanisms of Bufalin in Combination with Cinobufagin in..., Zhang [/bib_ref]. Studies have shown that Chansu can induce apoptosis and inhibit proliferation of MM cells. In the study by Wu et al. represents inhibitory effects, represents stimulative effects. FIGURE 6 | Action pathways involved in the anti-MM effects of berberine. represents inhibitory effects, represents stimulative effects. Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 13 | Article 818179 6 (2018), both bufalin and BF211 (a derivative of bufalin) were shown to suppress the progression of MM by inhibiting the IL-6/ JAK2/STAT3 pathway in vivo and in vitro. Inhibition of IL-6/ JAK2/STAT3 signaling pathway by BF211 led to increased levels of caspase-3, caspase-8, suppression of the expressions of Bcl-2 and Mcl-1, and showed stronger pro-apoptosis effects than bufalin. In addition, the cytocidal effect of bufalin in different MM cell lines was also mediated via inhibition of Akt/mTOR [bib_ref] MK2206 Enhances the Cytocidal Effects of Bufalin in Multiple Myeloma by Inhibiting..., Xiang [/bib_ref]. Results from [bib_ref] Targeting Poly (ADP-Ribose) Polymerase Partially Contributes to Bufalin-Induced Cell Death in Multiple..., Huang [/bib_ref] revealed that the active site of bufalin interacted with the catalytic domain of ploy (ADP-ribose) polymerase1 (PARP1), triggering decreased activity of PARP1 and cell apoptosis as well as G 2 -M phase cell cycle arrest in various MM cell lines. While PARP1 overexpression reversed bufalin-induced cell apoptosis. Besides, cinobufagin was shown to exhibit pro-apoptotic effects in U266 cells through activating ERK, JNK and p38MAPK pathways [bib_ref] Cinobufagin Exerts Anti-proliferative and Pro-apoptotic Effects through the Modulation ROS-Mediated MAPKs Signaling..., Baek [/bib_ref]. It should be noted that Chansu has a complex chemical composition, and its bioactive ingredients are highly toxic and may cause serious adverse events such as cardiac dysarythmia, tissue ischemia, and hypoxia. Whether Chansu is suitable for the treatment of MM warrants further investigations. ## Gambogic acid Gambogic acid [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref] is the major active component of gamboge derived from the Garcinia hanburryi tree. In TCM, gamboge (Teng Huang) has been used for relieving trauma-induced swelling and pain for thousands of years. It has been reported to exhibit potent anticancer activity against certain solid tumors. Recent studies have indicated the therapeutic potential of gambogic acid for the treatment of MM . [bib_ref] Effects of Gambogic Acid on the Activation of Caspase-3 and Downregulation of..., Yang [/bib_ref] found that gambogic acid can induce apoptosis of RPMI-8226 cells via ROS accumulation followed by caspase-3 activation, PARP cleavage, and SIRT1 downregulation. Hypoxic conditions in the bone marrow microenvironment have been implicated in the progression of angiogenesis, and chemotherapeutic resistance in MM [bib_ref] Impact of Hypoxia on the Pathogenesis and Therapy Resistance in Multiple Myeloma, Ikeda [/bib_ref]. This hypoxic condition may induce adaptive cellular responses mediated via hypoxia-inducible transcription factors (HIF) [bib_ref] The Emerging Role of Hypoxia, HIF-1 and HIF-2 in Multiple Myeloma, Martin [/bib_ref]. In the study by [bib_ref] Gambogic Acid Suppresses Hypoxia-Induced Hypoxia-Inducible Factor-1α/vascular Endothelial Growth Factor Expression via Inhibiting..., Wang [/bib_ref] , gambogic acid treatment suppressed MM progression and angiogenesis through inhibition of HIF1α/VEGF expression, which was associated with the inhibition of PI3K/Akt/ mTOR pathway. Osteoporosis and lytic lesions are also common in patients with MM [bib_ref] Myeloma Bone Disease: The Osteoblast in the Spotlight, Andrews [/bib_ref]. Stromal cell-derived factor 1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) signaling has been shown to be associated with osteoclastogenesis [bib_ref] Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis, Dong [/bib_ref]. Gambogic acid was shown to inhibit SDF-1α-induced chemotaxis of MM cells and downstream signaling of CXCR4. Further study demonstrated that the gambogic acid-induced inhibition of CXCR4 was caused by inhibiting the binding of NF-κB to the CXCR4 prompter, which in turn caused inhibition of IL-6 expression in MM cells. Moreover, gambogic acid also inhibited expression of IL-6 in macrophages, thus inhibiting osteoclastogenesis [bib_ref] Gambogic Acid Inhibits Multiple Myeloma Mediated Osteoclastogenesis through Suppression of Chemokine Receptor..., Pandey [/bib_ref]. Besides, gambogic acid and bortezomib at non-toxic concentration loaded with nanoparticles exhibited significant inhibitory effects by inducing G 2 /M phase cell cycle arrest and apoptosis via increasing expression of proapoptotic Bax, Caspase-3, and inhibiting anti-apoptotic PI3K/Akt pathway and Bcl-2 [bib_ref] Inducing Cell Cycle Arrest and Apoptosis by Dimercaptosuccinic Acid Modified Fe3O4 Magnetic..., Zhang [/bib_ref]. ## Ginsenoside Ginseng (Ren Shen) is a famous traditional Chinese medicine herb used in China for more than two thousand years. The medicinal value of Ginseng has been recognized globally. Rg3, one of the pharmacologically active ginsenoside saponins isolated from ginseng has shown anti-aging, anti-inflammatory, antitumor, cardioprotective and neuroprotective properties. Ginsenoside Rg3 has been studied for the different efficacy of two chemical forms, namely 20(R)-ginsenoside Rg3 [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref] and 20(S)-ginsenoside Rg3 [fig_ref] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM [/fig_ref]. Recent studies have FIGURE 7 | Action pathways involved in the anti-MM effects of gambogic acid. represents inhibitory effects, represents stimulative effects. Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 13 | Article 818179 shown the therapeutic potential of ginsenoside Rg3 in MM.reported that 20(S)-ginsenoside Rg3 suppressed the proliferation of U266 cells via partly inducing G 1 phase cell cycle arrest and apoptosis, as evidenced by increased levels of caspase-3, caspase-8, and caspase-9. In addition, the VEGF secretion by U266 cells was also downregulated. [bib_ref] Inhibition of Multiple Myeloma Cell Proliferation by Ginsenoside Rg3 via Reduction in..., Li [/bib_ref] demonstrated that the inhibitory effects of Rg3 on the proliferation of MM cells were associated with inhibition of the IGF-1/Akt/mTOR signaling pathway. Besides, ginseng, compound K, a metabolite of the ginsenoside was also shown to induce apoptosis in U266 cells via inhibiting the JAK1/STAT3 pathway [bib_ref] Inhibition of JAK1/STAT3 Signaling Mediates Compound K-Induced Apoptosis in Human Multiple Myeloma..., Park [/bib_ref]. ## Other bioactive components Other than the active ingredients discussed above, there are other bioactive components derived from TCMMs which have not been studied intensively, but could still provide new therapeutic options for MM patients. Icariin, a highly-potent active ingredient extracted from Epimedium (Ying Yang Huo), is a promising lead compound that has shown high efficiency in the treatment of various cancers. Jung et al.recently reported that icariin significantly potentiated the apoptotic effects of bortezomib via inhibiting JAK/STAT3 signaling pathway, and downregulating the downstream targets including Bcl-2, Bcl-xl, survivin, IAP-1/2, COX-2, VEGF, and MMP-9. Additionally, daidzin, extracted from Pueraria lobate (Ge Gen) showed inhibitory effects on STAT3 signaling cascade in MM cells; this agent is frequently used to treat a broad spectrum of disorders including pain, diabetes, neurodegenerative diseases, gastrointestinal diseases, cerebrovascular disorders, and cardiac dysfunction . Formononetin, another naturally-occurring isoflavone derived from Pueraria lobata and Astragalus membranaceus , can also suppress the DNA binding capacity and nuclear translocation of STAT3 and STAT5, thus triggering cell cycle arrest, inhibition of angiogenesis and proliferation, and induction of apoptosis in U266 and RPMI 8226 cells. Hedyotis diflfus (Bai Hua She She Cao) is a common ingredient in Chinese herbal medicine formulas used for cancer treatment. Lin et al.found that polysaccharides extracts from Hedyotis difus significantly suppressed the proliferation and induced apoptosis in RPMI 8226 cells, at least, partly via downregulating Akt and NF-κB signaling cascades. Scutellarein is a flavonoid derived from Scutellaria barbata (Ban Zhi Lian), a famous and expensive anticancer traditional Chinese herbal medicine. It was shown to induce mitochondrial-mediated intrinsic apoptosis in a variety of MM cell lines, and to greatly reduce MM xenograft tumor burden in nude mice [bib_ref] Scutellarein Selectively Targets Multiple Myeloma Cells by Increasing Mitochondrial Superoxide Production and..., Shi [/bib_ref]. ## Summary, conclusion, and future perspective Currently, bioactive components derived from TCMMs have been gradually drawing attention, and their cytotoxic effects on MM have been intensively validated. Collectively, the available evidence supports that curcumin, resveratrol, baicalein, berberine, bufadienolides, gambogic acid, ginsenoside, and other less investigated ingredients exhibit significant anti-MM effects via mainly suppressing angiogenesis and proliferation, by inducing cell apoptosis, cell cycle arrest and autophagy, as well as by inhibiting osteoclastogenesis. These findings indicate a promising role of bioactive components extracted from TCMMs as novel therapeutic agents for MM. However, several issues still need to be considered. First, studies that have investigated the cytotoxic effects of these ingredients on MM cell lines have largely been limited to assessing the effects on cell proliferation, apoptosis, or autophagy, while there is a paucity of investigations on the immune system or multiple myeloma bone marrow microenvironment. In addition, epigenetic modifications induced by these ingredients should also be studied since epigenetics modulation are risk factors including environmental and occupational exposures, as well as aging . Moreover, theses bioactive components may have a cytotoxic effect on normal cells, while most studies have been performed on MM cells. Whether and how these ingredients discriminate between normal and MM cells remains unknown. Secondly, the targeting proteins, metabolites, and signaling pathways involved in the regulation of angiogenesis, proliferation, apoptosis should be studied by utilizing multiomics technologies, including genomics, transcriptomics, proteomics, and metabolomics. This approach may provide more in-depth characterization of the mechanisms of actions of these bioactive components. Besides, network pharmacology and molecular docking analysis on molecular targets should also be considered to shed light on the underlying mechanisms of TCMMs. Additionally, the anti-MM impact of promising ingredients, such as curcumin and resveratrol, in the clinical context is still quite vague. Few clinical studies have evaluated treatment with these ingredients alone or in combination with other drugs in MM patients. Thus, further well-designed clinical trials with follow-up and in-vivo examinations should be performed. Third, many of these ingredients exhibit poor bioavailability and are rapidly metabolized, which may undermine their pharmacological activity. Therefore, discovery and utilization of drug delivery systems, such as nanoparticles and liposomes, as well as the novel synthetic analogs can enhance drug stability, prolong drug action, and enable a sustained and slow-release rate while reducing the side effects. # Author contributions C-CY and YL contributed equally to this work. C-CY and YL conceived the main ideas and wrote this paper. Z-JC, XW, and WM helped design the framework and illustrate figures. Y-WZ helped revise the manuscript. All authors contributed to the article and approved the submitted version. # Funding [fig] FIGURE 2 |: Chemical structures of active components extracted from TCMMs for treaing MM. [/fig] [fig] FIGURE 4 |: Action pathways involved in the anti-MM effects of resveratrol. [/fig] [fig] FIGURE 5 |: Action pathways involved in the anti-MM effects of baicalein. [/fig]

Targeting CD82/KAI1 for Precision Therapeutics in Surmounting Metastatic Potential in Breast Cancer # Introduction Approximately 2.26 million new female breast cancer cases were diagnosed globally in 2020. Furthermore, it has been estimated that 20-30% breast cancer patients treated at the primary tumor stage, will spread to other organs in the body. Hence, there is an urgent need to develop more effective treatment strategies for combating breast cancer, in particular, hindering breast cancer spread to distant sites such as the lungs and brain, as metastasis is known to be the leading cause of cancer-related deaths. Metastasis refers to the spread of cancer cells originating from a primary tumor, travelling through the bloodstream or lymphatic system, to form new tumors at a distant site in the body. The metastatic process is complex and involves many steps and, hence, it is an uphill task to elucidate the mechanisms involved in the different steps of metastasis. Traditionally, the concept for metastasis was believed to be the end-point of cancer progression, with the view that metastases only developed at the late stages of cancer, at which point the cancer is deemed incurable. In the linear progression model, heterogeneous clones undergo successive mutations and selection before acquiring metastatic properties. However, there have been reports indicating that cancer cells tend to show a 'metastatic phenotype' from the onset when the tumor is still small , as illustrated in the parallel progression model. Notably, these studies revealed that primary tumor cells and metastatic cells showed similar gene expression patterns, implying that the propensity of a tumor to metastasize is determined early during cancer development. The process of metastasis begins with intravasation (as shown in, where the tumor cells remodel the cytoskeleton to facilitate migration through actin remodeling. The extracellular matrix is degraded by matrix metalloproteinases (MMPs). The cancer cell(s) then dissociate from the primary tumor, commonly via an epithelial to mesenchymal transition (EMT). EMT causes a decreased cell-cell contact, loosened associations, and cells to attain migratory properties, in the process lowering the expression of E-cadherin and increasing the expression of mesenchymal markers. Cells, which then enter into blood vessels, are named circulating tumor cells (CTCs) and travel to the metastatic site. Lastly, extravasation occurs when cells form a pre-metastatic niche and alter the environment of the destination organ. the three stages, namely, intravasation of primary tumor cells, before circulating in the bloodstream as CTCs, and extravasation at a distant site giving rise to a metastatic deposit. Breast cancer metastasis is still a major clinical problem. Despite the availability of several therapeutic modalities, including chemotherapy, endocrine therapy, immunotherapy and targeted therapy, treatment outcome remains poor year overall survival rate <. Diagrammatic representation of the metastatic cascade showing the three stages, namely, intravasation of primary tumor cells, before circulating in the bloodstream as CTCs, and extravasation at a distant site giving rise to a metastatic deposit. Breast cancer metastasis is still a major clinical problem. Despite the availability of several therapeutic modalities, including chemotherapy, endocrine therapy, immunotherapy and targeted therapy, treatment outcome remains poor year overall survival rate < 30%). Hence, the efficacious treatment of early stage primary breast tumors and the prevention of metastatic spread, would significantly improve treatment outcome for breast cancer patients. In this regard, the concept of a precision medicine model where a group of breast cancer patients are selected based on a metastasis-related molecule which is subsequently, targeted (as elaborated in Section 5), seems to be an attractive therapeutic strategy. Precision medicine is an emerging approach for breast cancer therapy with the aim of selecting the optimal therapy for a specific group of cancer patients. The discovery and characterization of molecules that are able to suppress metastasis holds great value in providing biological insights into mechanisms involved in this multifaceted process. It is, therefore, not surprising that metastatic suppressors are often reduced in expression in metastatic tumors, and upon overexpression or re-expression, are able to inhibit the metastatic ability of cancer cells; thus, slowing or preventing tumor spread. This review will focus on CD82, a metastatic suppressor, and evaluate its potential usage as a therapeutic strategy in overcoming the distant spread of breast cancer cells. ## Cd82 glycoprotein CD82, also known as KAI1 (kangai1-anticancer in Chinese), is a glycoprotein that belongs to the tetraspanin family. The CD82 gene is located on human chromosome 11p11.2, and comprises 10 exons and 9 introns. The CD82 protein is 267 amino acids long, with its predicted structure based on previously known tetraspanins, coupled with modelling of the individual sections. Cancers 2021, 13, x FOR PEER REVIEW 3 of 17 30%). Hence, the efficacious treatment of early stage primary breast tumors and the prevention of metastatic spread, would significantly improve treatment outcome for breast cancer patients. In this regard, the concept of a precision medicine model where a group of breast cancer patients are selected based on a metastasis-related molecule which is subsequently, targeted (as elaborated in Section 5), seems to be an attractive therapeutic strategy. Precision medicine is an emerging approach for breast cancer therapy with the aim of selecting the optimal therapy for a specific group of cancer patients. The discovery and characterization of molecules that are able to suppress metastasis holds great value in providing biological insights into mechanisms involved in this multifaceted process. It is, therefore, not surprising that metastatic suppressors are often reduced in expression in metastatic tumors, and upon overexpression or re-expression, are able to inhibit the metastatic ability of cancer cells; thus, slowing or preventing tumor spread. This review will focus on CD82, a metastatic suppressor, and evaluate its potential usage as a therapeutic strategy in overcoming the distant spread of breast cancer cells. ## Cd82 glycoprotein CD82, also known as KAI1 (kangai1-anticancer in Chinese), is a glycoprotein that belongs to the tetraspanin family. The CD82 gene is located on human chromosome 11p11.2, and comprises 10 exons and 9 introns. The CD82 protein is 267 amino acids long, with its predicted structure based on previously known tetraspanins, coupled with modelling of the individual sections.. Large extracellular domain contains cysteine protein motifs (CCG, CSC, and EGC) as yellow boxes with disulfide bonds shown as yellow lines. N-glycosylation sites are portrayed as dark grey boxes. The green rectangle represents another cysteine residue, red box depicts an asparagine residue and light blue boxes denote glutamine residues. Tetraspanins are found on the cell membrane, and usually comprise four transmembrane domains, cytoplasmic N-and C-termini, a small and a large extracellular domain. The large extracellular domain contains asparagine residues that are N-glycosylated, and is divided into a constant region which contains α-helices, and a variable region which has cysteine residues that form intramolecular disulfide bonds. The variable. Large extracellular domain contains cysteine protein motifs (CCG, CSC, and EGC) as yellow boxes with disulfide bonds shown as yellow lines. N-glycosylation sites are portrayed as dark grey boxes. The green rectangle represents another cysteine residue, red box depicts an asparagine residue and light blue boxes denote glutamine residues. Tetraspanins are found on the cell membrane, and usually comprise four transmembrane domains, cytoplasmic N-and C-termini, a small and a large extracellular domain. The large extracellular domain contains asparagine residues that are N-glycosylated, and is divided into a constant region which contains α-helices, and a variable region which has cysteine residues that form intramolecular disulfide bonds. The variable region is known to contain most of the known protein-protein interaction sites. Cysteine residues proximal to the membrane in the cytoplasmic domains are palmitoylated. Each distinct domain of CD82 contributes to its overall activity. N-glycosylation is shown to play an essential role in the function of cell surface receptors and adhesion molecules. Transmembrane polar residues have also been found to be important for the anti-cancer effects of CD82, including migration, invasion, metastasis, and membrane protrusion, as well as being involved in both the physical interactions within the lipid bilayer, and maintaining the overall conformation of the protein. They also play an important role in intramolecular packing, and intermolecular interactions and interfaces. Palmitoylation of the cysteine residues proximal to the membrane contributes to the localization of CD82 at the cell membrane, as well as its participation in the tetraspanin-enriched microdomain, contributing to its suppression of cell motility and invasion. The small extracellular domain of CD82 has been found to be important for the anti-metastatic activity of CD82. CD82 anti-metastatic activity was observed to be lost upon the addition of negatively charged residues, or after perturbation of the secondary structure of the domain. The C-terminal of CD82 was discovered to play a significant role in its activity towards the epidermal growth factor receptor (EGFR). It was observed that upon the deletion of the C-terminus, the endocytic trafficking of CD82 was inhibited, which affected the regulation of the ligand-induced ubiquitylation of EGFR. ## Cd82 and cancer metastasis CD82 is known to inhibit cell motility, which is important in the intravasation and extravasation steps in the metastatic cascade (as shown in, and down-regulating cell-cell adhesion which would impede colonization at the metastatic site. Some mechanisms have been proposed regarding how cell motility is facilitated by tetraspanins, such as the regulation of cell adhesion molecules or growth factor receptors, thereby altering cellular behavior. In particular, CD82 attenuates cellular morphogenesis and EGFR signaling via downregulation of the associated integrin and EGFR. The other possible mechanism involves tetraspanins' cellular functions by signal initiation and transduction. For instance, CD82 is known to diminish lamellipodia formation and perturb actin organization by the deregulation of Rac1, RhoA, and their effectors cofilin and Rho kinase. Tetraspanins have also been reported to control various cell functions such as cell adhesion, migration and communication via the regulation of digitation junctions. CD82 has been observed to inhibit cell movement by hampering the formation and development of both cellular protrusions and retractions at the cellular level. CD82 was also reported to attenuate the activation of β1 integrin and downregulate outsidein signaling, decreasing cell adhesion and motility. Knockdown of liprin α1 was observed to lead to an upregulation of CD82, inhibiting the formation of microprotrusions. The CD82-mediated inhibition of tumor cell movement has been linked to its binding to cholesterol, and its coalescence with lipid rafts and tetraspanin-enriched microdomains. Other aspects of the functional roles of CD82, include innate immune signaling through association with Toll-like receptor 9 (TLR9) in the endoplasmic reticulum (ER) and post-ER, and the modulation of the TLR9-dependent nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) nuclear translocation. ## Significance of cd82 expression as a metastasis suppressor in different types of cancer ## Breast cancer CD82 was first reported as a potential marker for breast cancer metastasis by Yang et al., who demonstrated that the metastatic propensity of a variety of breast cancer cell lines was inversely correlated with CD82/KAIl mRNA expression. Yang and co-workers further showed in another study that a lower CD82 protein expression was associated with breast malignancy as analyzed by immunohistochemistry in breast tissues from 81 patients (comprising 7 normal tissues, 7 ductal carcinoma in situ and 67 breast cancer tissues). CD82 mRNA expression was also observed to be reduced in breast cancer tissues as compared to normal breast tissue. Malik et al.further reported a significant correlation of the CD82 transcripts with TNM staging and that a higher expres-Cancers 2021, 13, 4486 5 of 17 sion of CD82 was associated with more favorable survival in breast cancer patients. In addition, CD82 mRNA expression was observed to be significantly reduced in breast cancer metastases to the brain. CD82 protein expression detected by immunohistochemical staining, was reported to be significantly associated with the axillary lymph node status and advanced tumor stage, but no correlation was observed with the hormonal receptor (HR) or Human Epidermal Growth Factor-2 (HER-2) receptor status. In a study on 109 breast cancer patients, Huang et al.observed that CD82-negative tumors had significantly lower 5-year disease-free survival compared to their CD82-positive counterparts; thus, showing that the reduction in CD82 expression is correlated with tumor recurrence. In another clinicopathological study, Christgen et al.analyzed CD82 expression in 92 distant metastases from breast cancers, which included matched pairs of breast cancer and metachronous distant metastases. The same authors observed an association of CD82positive metastases with the estrogen receptor (ER)-negative phenotype, implying that CD82 is not a good determinant of cancer progression in this breast cancer subtype. However, more in vitro and in vivo experimentation, as well as a larger cohort of breast cancer patients, are required to validate that metastasis in ER-negative tumors is independent of CD82. Likewise, in vitro studies have revealed that CD82 plays an important role in cell adhesion, migration and invasion in breast cancer cells. The knockdown of CD82 expression in MDA-MB-231 breast cancer cells promoted cell migration and invasion, which was posited to be via the dysregulation of mitogen-activated protein kinase (MAPK) signaling, and the interaction of CD82 with EGFR. In addition, CD82 was observed to inhibit cell adhesion which could possibly be mediated via its interaction with integrins. The metastasis suppressive functions of CD82 have been reported to be abrogated by the splicing of the CD82 gene, where the CD82 spliced variant was observed to enhance cell migration and proliferation, concomitant with the activation of Src kinase in MDA-MB-231 breast cancer cells. Overexpression of Sulfatase 2 (Sulf 2) was also reported to promote cell migration and invasion with the concomitant downregulation of CD82 expression in MDA-MB-231 breast cancer cells. In the same study, Sulf 2 overexpressing MDA-MB-231 breast cancer cells were observed to invade surrounding muscle tissues, thereby demonstrating significant invasive ability in a mouse xenograft model. Interestingly, the institution of endocrine therapy was successful in re-inducing the expression of CD82 in ER-positive breast cancer patients treated with ER antagonists. In this same study, exposure to fulvestrant, a clinically approved ER antagonist was observed to upregulate CD82 expression in ER+ MCF-7 and T-47D breast cancer cells. In another clinically relevant study, Wang et al.noted higher expression of CD82 in the serum exosomes from breast cancer patients, as compared with patients who had benign breast disease and healthy controls. They postulated that a redistribution of the CD82 protein via exosomes from breast cancer tissues to blood occurs during breast cancer development. Hence, expression levels of CD82 measured in exosomes could be useful as a potential biomarker for determining the metastatic potential in breast cancer. ## Other cancers CD82 expression has also been reported in other cancer types, which include cancers of the prostate, lung and pancreas. CD82 was first discovered to be under-expressed in cell lines derived from metastatic prostate cancer cells in 1995. Another study further revealed that CD82 enhanced the shedding of E-cadherin through the suppression of disintegrin and metalloprotease 17 (ADAM17), while promoting motility, migratory, and invasive properties of prostate cancer cells. CD82 is also known to inhibit cancer invasion and metastasis in non-small-cell lung carcinoma (NSCLC) via multiple mechanisms. A higher expression of CD82 was reported in tumors which are better differentiated, less likely to metastasize to lymph nodes, and present at an earlier clinical stage in NSCLC. In addition, the survival period of NSCLC patients with low CD82 was significantly shorter than patients with a positive expression of CD82. A reduced expression of CD82 has been reported in pancreatic cancer metastases. A recent study showed that CD82 inhibits the EMT process in pancreatic cancer by increasing E-cadherin expression and reducing the expression of Snail, vimentin, MMP2, and MMP9, which are involved in different steps of the EMT process, thereby effectively reversing the EMT process. A lowered expression of CD82 corresponding to increased differentiation associated with metastasis has been observed in cervical cancer. Decreased CD82 expression was reported to be significantly correlated with advanced disease and poor prognosis in melanomas. CD82 was also observed to be a potential and promising therapeutic target for acute myelogenous leukemiaand oral cancer. Moreover, CD82 has been reported to inhibit invasion and metastasis of esophageal squamous cell carcinoma via the regulation of TGF-β1, and decrease colon cancer cell motility. The next section focuses on the therapeutic value of CD82 in curbing breast cancer metastasis, which is the cancer of interest in this review. ## Cd82 as a therapeutic target for personalized therapy in breast cancer Currently, therapeutic strategies for breast cancer depend essentially on the classification of the breast cancer subtypes, viz., HR+/HER2−, HER2+ and triple-negative (HR−/HER2−). For non-metastatic breast tumors, local treatment involves surgery (which may entail the removal of axillary lymph nodes) or radiotherapy. Systemic therapy instituted would depend on the breast cancer subtype. Basically, endocrine therapy (tamoxifen or aromatase inhibitors) is the mainstay of treatment for all HR+ tumors. Trastuzumab-based HER2 − directed monoclonal antibody therapy with chemotherapy is recommended for all HER2+ breast cancers (together with endocrine therapy in the presence of concurrent HR positivity). For triple negative breast cancer, chemotherapy is advocated, for instance the combination of cyclophosphamide with a taxane (docetaxel) or an anthracycline (doxorubicin), or combination with methotrexate and 5 Fluorouracil. More recently, immunotherapy, such as use of the monoclonal antibodies, pembrolizumab which inhibits programmed cell death 1 (PD-1) and atezolizumab, an inhibitor of the programmed cell death ligand 1 (PD-L1) that releases the suppression of the PD-1/PD-L1-mediated immune response in patients with triple negative breast cancer, has shown promise. Personalized treatment and precision medicine, tailored towards specific individuals and subgroups of selected patients, respectively, are gaining traction in oncology, where an emphasis is placed on the prevention of disease progression, and the treatment regime is selected to maximize the efficacy and minimize toxicity. As CD82 is known to have anti-metastatic properties (as elaborated in Section 4.1), this glycoprotein would be a good and suitable potential molecular target to be further explored for precision therapy in preventing breast cancer progression, when used in combination with systemic therapy according to the cancer subtype (as described in the above paragraph). ## Selection of suitable breast cancer patients for therapy Stage I-III non-metastatic breast cancer patients with a low expression of CD82 (such as those shown inbelow) can be first identified from patient biopsies, or potentially, exosomes from the bloodstream, which is an example of a liquid biopsy using a noninvasive approach. Selected patients could then undergo targeted therapy to restore CD82 in order to minimize the possibility of metastasis. ## Potential therapeutic options for upregulating cd82 in breast cancer ## Drugs known to target cd82 Tyrosine Kinase Inhibitors (TKIs) Imatinib, a Type 2A TKI, has been widely used in chemotherapy for chronic myeloid leukemia (CML), among others. Imatinib was the first kinase inhibitor to be approved by the FDA in 2001. Additionally known as "Gleevec" or "Glivec", imatinib was once hailed as the "magical bullet" that could cure cancer. Although imatinib is known to inhibit Abelson (ABL) tyrosine kinase, which is expressed in CML, its 'polypharmacology' has facilitated its use in the therapy of several types of cancers, such as gastrointestinal stromal tumors. However, imatinib when used singly, was found to lack clinical activity in Platelet-Derived Growth Factor Receptor-overexpressing metastatic breast cancer (MBC), with potential immunosuppressive effects. Interestingly, it was reported that imatinib up-regulated CD82 gene expression in human MCF-7 breast cancer cells, concomitant with a significant inhibition in cell proliferation. ## Potential therapeutic options for upregulating cd82 in breast cancer ## Drugs known to target cd82 tyrosine kinase inhibitors (tkis) Imatinib, a Type 2A TKI, has been widely used in chemotherapy for chronic myeloid leukemia (CML), among others. Imatinib was the first kinase inhibitor to be approved by the FDA in 2001. Additionally known as "Gleevec" or "Glivec", imatinib was once hailed as the "magical bullet" that could cure cancer. Although imatinib is known to inhibit Abelson (ABL) tyrosine kinase, which is expressed in CML, its 'polypharmacology' has facilitated its use in the therapy of several types of cancers, such as gastrointestinal stromal tumors. However, imatinib when used singly, was found to lack clinical activity in Platelet-Derived Growth Factor Receptor-overexpressing metastatic breast cancer (MBC), with potential immunosuppressive effects. Interestingly, it was reported that imatinib up-regulated CD82 gene expression in human MCF-7 breast cancer cells, concomitant with a significant inhibition in cell proliferation. However, there has not been much follow-up to explore its use for preventing breast cancer metastasis. However, there has not been much follow-up to explore its use for preventing breast cancer metastasis. TKIs such as lapatinib, neratinib, and tucatinib, which are HER-2-specific, have demonstrated efficacy in the management of MBC. In fact, lapatinib was the first FDA approved TKI to treat HER2−-positive MBC in combination with capecitabine, with neratinib being approved later on in 2020 for the same purpose. Neratinib has also been reported to be effective in treating early HER-2+ breast cancers as shown by the results from the ExteNET trial. Furthermore, tucatinib, yet another selective HER2 inhibitor, was also approved in 2020 for breast cancer patients who developed brain metastases. Currently, unlike imatinib, it is not known if lapatinib, neratinib, and tucatinib have the propensity to upregulate CD82 expression. It would be worthwhile to investigate if the latter three drugs have any effect on CD82 expression, so as to explore the possibility of repositioning these TKIs for treating primary breast cancers with the goal of eradicating metastasis. Etoposide DNA topoisomerases (categorized into DNA topoisomerase I and DNA topoisomerase II) are enzymes that play essential roles in DNA replication and transcription. Etoposide, a podophyllotoxin derivative, is a topoisomerase II inhibitor which was discovered to have increased antineoplastic activity and synthesized as etoposide (VP-in 1966. Since then, etoposide has been used in the treatment of small cell lung cancer, lymphomas, ovarian cancer, and breast cancer. TKIs such as lapatinib, neratinib, and tucatinib, which are HER-2-specific, have demonstrated efficacy in the management of MBC. In fact, lapatinib was the first FDA approved TKI to treat HER2 − positive MBC in combination with capecitabine, with neratinib being approved later on in 2020 for the same purpose. Neratinib has also been reported to be effective in treating early HER-2+ breast cancers as shown by the results from the ExteNET trial. Furthermore, tucatinib, yet another selective HER2 inhibitor, was also approved in 2020 for breast cancer patients who developed brain metastases. Currently, unlike imatinib, it is not known if lapatinib, neratinib, and tucatinib have the propensity to upregulate CD82 expression. It would be worthwhile to investigate if the latter three drugs have any effect on CD82 expression, so as to explore the possibility of repositioning these TKIs for treating primary breast cancers with the goal of eradicating metastasis. Etoposide DNA topoisomerases (categorized into DNA topoisomerase I and DNA topoisomerase II) are enzymes that play essential roles in DNA replication and transcription. Etoposide, a podophyllotoxin derivative, is a topoisomerase II inhibitor which was discovered to have increased antineoplastic activity and synthesized as etoposide (VP-16) in 1966. Since then, etoposide has been used in the treatment of small cell lung cancer, lymphomas, ovarian cancer, and breast cancer. The results for treatment of metastatic cancers using etoposide in combinational therapy have been promising for some metastatic cancers but mixed for MBC. Oral etoposide was assessed as a valuable and safe option for pre-treated MBC patients. Moreover, etoposide with apatinib has been reported to be effective and tolerable in heavily pretreated, metastatic HER2 − negative breast cancer patients. On the other hand, etoposide in combination with irinotecan in a Phase II trial for refractory MBC, was terminated as the interim analysis revealed severe toxicity effects. In fact, two decades ago, it was also reported that a chronic oral regimen of etoposide for 21 days produced significant toxicity. The results for treatment of metastatic cancers using etoposide in combinational therapy have been promising for some metastatic cancers but mixed for MBC. Oral etoposide was assessed as a valuable and safe option for pre-treated MBC patients. Moreover, etoposide with apatinib has been reported to be effective and tolerable in heavily pretreated, metastatic HER2−-negative breast cancer patients. On the other hand, etoposide in combination with irinotecan in a Phase II trial for refractory MBC, was terminated as the interim analysis revealed severe toxicity effects. In fact, two decades ago, it was also reported that a chronic oral regimen of etoposide for 21 days produced significant toxicity. Interestingly, etoposide has been previously observed to activate CD82 in a dosedependent manner via p53 and c-Jun, in human prostate cancer cell lines. Moreover, etoposide has been shown in breast cancer cell lines to induce p53 expression which could then upregulate CD82 expression. Thus, it may be meaningful to verify if etoposide upregulates CD82 expression in breast cancer patients, for repurposing as a therapeutic agent in treating a subset of non-metastatic breast cancer patients with low CD82 expression. This is especially since etoposide at a low dose is preferred by some clinicians because of its relatively lower cost compared to other chemotherapeutic drugs and oral route of administration (thereby avoiding visits to the clinic for drug infusions). ## Cd82 mimics Instead of targeting CD82, attempting to mimic the activity of CD82 could be another potential method of promoting the metastatic suppressing ability of CD82. Targeting protein-protein interactions that involve CD82 is a potential method of CD82 activation. Recombinant soluble long extracellular loops (LELs) mimicking the CD82 LEL (present in its transmembrane domain as shown in, could be a possible approach for activating pathways that involve CD82. Recombinant LELs have been utilized to inhibit infection of macrophages in HIV. Recently, a peptide mimicking the small extracellular domain (EC1) of CD82 has been used to treat various cancer cells, including colon, breast, prostate, and lung cancer cells. In this same study, the EC1 amino acid sequence mimic peptide of CD82 (CD82EC1-mP) was successful in inhibiting cell migration, invasion and adhesion in vitro, while also suppressing metastasis in lung cancer cells in mice. With respect to breast cancer, CD82EC1-mP was observed to enhance homotypic cell-cell aggregation and inhibit the cell migration and invasion in MDA-MB-231 breast cancer cells. Further mechanistic studies suggested that the suppression of metastasis was mediated via the inhibition of the EMT process through the modulation of the Wnt and Hippo pathways. Interestingly, etoposide has been previously observed to activate CD82 in a dosedependent manner via p53 and c-Jun, in human prostate cancer cell lines. Moreover, etoposide has been shown in breast cancer cell lines to induce p53 expression which could then upregulate CD82 expression. Thus, it may be meaningful to verify if etoposide upregulates CD82 expression in breast cancer patients, for repurposing as a therapeutic agent in treating a subset of non-metastatic breast cancer patients with low CD82 expression. This is especially since etoposide at a low dose is preferred by some clinicians because of its relatively lower cost compared to other chemotherapeutic drugs and oral route of administration (thereby avoiding visits to the clinic for drug infusions). ## Cd82 mimics Instead of targeting CD82, attempting to mimic the activity of CD82 could be another potential method of promoting the metastatic suppressing ability of CD82. Targeting protein-protein interactions that involve CD82 is a potential method of CD82 activation. Recombinant soluble long extracellular loops (LELs) mimicking the CD82 LEL (present in its transmembrane domain as shown in, could be a possible approach for activating pathways that involve CD82. Recombinant LELs have been utilized to inhibit infection of macrophages in HIV. Recently, a peptide mimicking the small extracellular domain (EC1) of CD82 has been used to treat various cancer cells, including colon, breast, prostate, and lung cancer cells. In this same study, the EC1 amino acid sequence mimic peptide of CD82 (CD82EC1-mP) was successful in inhibiting cell migration, invasion and adhesion in vitro, while also suppressing metastasis in lung cancer cells in mice. With respect to breast cancer, CD82EC1-mP was observed to enhance homotypic cell-cell aggregation and inhibit the cell migration and invasion in MDA-MB-231 breast cancer cells. Further mechanistic studies suggested that the suppression of metastasis was mediated via the inhibition of the EMT process through the modulation of the Wnt and Hippo pathways. ## Epigenetic drugs for treatment of breast cancer Long Non-Coding RNA (lncRNA)-Based Therapy lncRNAs are RNAs more than 200 nucleotides in length with a functional significance in transcriptional and post-transcriptional silencing. Recently, a novel lncRNA named SKAI1BC (Suppressor of KAI1 in Breast Carcinoma) has been shown to epigenetically inactivate the anti-oncogenic activity of KAI/CD82 in breast cancer. Hence, SKAI1BC could be a potential target for lncRNA-based therapy in preventing metastatic spread in breast cancer. The approach taken towards inhibiting oncogenic lncRNA in breast cancer, thus far, includes the use of antisense oligonucleotides, treatment with locked nucleic acids (DNA analogues), and nanoparticle-mediated RNA interference (RNAi) technology. ## Microrna (mirna) therapeutics MicroRNAs (miRNAs) are small RNA molecules that are involved in the regulation of gene expressionby inducing mRNA degradation and repressing translation in cells. Recently, studies on miRNA therapeutics have shown that miRNA delivery can be useful in treating diseases. Although rarer, translational upregulation by miRNAs has been observed either through direct activation via miRNA/miRNP, or the relief of repression, where the activity of a repressive miRNA or miRNP is negated. miRNA therapy, touted as new generation therapeutics, can be classified into oncogenic miRNA inhibition, and tumor-suppressor miRNA mimics. Tumor-suppressive miRNAs tend to be under-expressed in breast cancers. For instance, miR-124 expression was reported to be lower in higher grade breast tumor tissues and highly metastatic MDA-MB-231 cell line. Further studies have revealed that miR-124 could inhibit breast cancer invasion, and metastasis. miR-125a-5p was significantly downregulated in breast cancer, concomitant with a lower overall free survival and progression-free survival, and observed to suppress breast cancer cell proliferation and migration. Interestingly, miR-125a-5p was able to overcome chemoresistance when paired with chemotherapeutic drugs. miR-137, miR-139, and miR-145 were reported to be significantly downregulated in triple negative breast cancer and have been shown to be effective in increasing the susceptibility of breast cancer to chemotherapy, in addition to inhibiting cell proliferation and metastasis. miR-671-5p was observed to suppress cell proliferation and invasion, while sensitizing cells to radiotherapy. On the other hand, oncogenic miRNAs are usually overexpressed in breast cancers. miR-96 promotes cell proliferation, migration, and invasion, while hindering apoptosis and drug resistance. Additionally, miR-370 was noted to enhance metastasis and cell invasion. To date, there have been no reports in the literature on miRNAs that target CD82 in breast cancer. Interestingly, a search, using the miRWalk database, revealed that there are close to 500 miRNAs that are predicted to regulate the expression of the CD82 gene. In fact, all the miRNAs associated with breast cancer that were mentioned in the above paragraph have been predicted by the miRWalk database to regulate CD82 mRNA expression. With regard to other cancer types, miRNAs have also been observed to promote cancer metastasis via their modulation of CD82 expression in hepatocellular cancer, malignant melanoma and gastric cancer. As CD82 is a metastasis suppressor, oncogenic miRNAs which reduce its expression are up-regulated during the metastatic process, and can be targeted by the delivery of miRNA antagonists that are complementary to the targeted miRNA or decoying the target miRNA with sponge RNAs. The general steps in using this strategy would involve the selection of the miRNA candidate, the validation of its presence in a patient sample, designing the anti-miRNA inhibitor and developing a safe and effective delivery system. Although many miRNA-based therapies have been explored in breast cancer, research and trials concerning their clinical applications are still in their early stages, and more work needs to be conducted before miRNAs could become translatable in clinical practice as breast cancer therapeutics. Nonetheless, miRNAs remain as promising targets due to the wide range of pathways that they can affect and their ability to enhance the effects of current chemotherapeutic drugs. ## Challenges and future directions The fact that breast cancer harbors molecular and cellular heterogeneity poses a considerable challenge in the attempt to unravel the complex relationship between the molecular biology of cancer and response to a specific therapeutic strategy, since there could be many drivers leading to cancer progression. Hence, the need for innovative approaches such as precision oncology, especially in the case of triple-negative breast cancer which is known to be a very heterogeneous subset of breast cancer. The proposed TKIs and etoposide in Section 5.2.1 are all FDA-approved drugs for cancer treatment. The repurposing of existing drugs would, therefore, be especially beneficial for treating triple-negative breast cancer patients, as there is a lack of targeted therapies for this subtype of breast cancer. It is anticipated that with advancements in computational methods related to chemoinformatics and genomics, more existing (both approved and investigational) drugs would be repositioned to treat breast cancer. The development of CD82 mimics for therapy is still in the infancy stage. As the CD82 EC1 mimic is a natural peptide, it is unlikely to exert any immunogenic effects. However, the flip side is that since the CD82 protein is also present in normal tissues, it is essential to ascertain the safety profile of the peptide in the human body before clinical use. More preclinical investigations and clinical trials are needed to establish the optimal therapeutic window for the efficient and safe clinical application of such mimetics. For non-coding RNA-based therapeutics, the litmus test is to be able to identify the most efficacious lncRNA and miRNA candidates with good safety profiles and achieve an effective targeted delivery to the breast cancer tissues. In terms of miRNA therapeutics, the surge in the availability of genomic and proteomic data would facilitate the identification of major miRNA targets, which together with the recent development of efficient therapeutic miRNA delivery systems, could enhance miRNA-based therapeutics for cancer. Although the prevalent view used to be that tumor suppressors (which include metastasis suppressors) are un-targetable, there is now a paradigm shift since the recent report of Hsiue et al.on p53 mutant peptide-targeted immunotherapy, albeit an antibody-based therapeutic that targets the most commonly mutated tumor suppressor gene. # Conclusions CD82 is known to play a significant anti-metastatic role in multiple cancers, including breast cancer. Currently, treatment strategies involving CD82 as a therapeutic target to hinder breast cancer spread has not been fully exploited, even though potential drugs such as the TKIs, lapatinib, neratinib, and tucatinib have been approved for MBC treatment, while imatinib and etoposide have been investigated in clinical trials for MBC. The latter two drugs exhibit their anti-metastatic activity through the activation of CD82 via different pathways as mentioned earlier, while it is at present not known if the actions of lapatinib, neratinib, and tucatinib are also mediated in part via the CD82 glycoprotein. The reprofiling of these clinically approved drugs for treating non-metastatic breast cancer would also reduce patient safety issues, such as risks of adverse effects, and bring down the cost of treatment. Whether CD82 mimics or non-coding RNA therapeutics will be part of the arsenal in overcoming breast cancer metastasis remains to be further investigated. For precision therapeutics to be successfully implemented in breast cancer patients, clinical oncologists must overcome the inertia to change standard practice and be prepared to conduct precision oncology-based clinical trials to validate the efficacy of novel therapeutic approaches.

Receptor-Independent Interaction of Bacterial Lipopolysaccharide with Lipid and Lymphocyte Membranes; the Role of Cholesterol Lipopolysaccharide (LPS) is a major constituent of bacterial outer membranes where it makes up the bulk of the outer leaflet and plays a key role as determinant of bacterial interactions with the host. Membrane-free LPS is known to activate Tlymphocytes through interactions with Toll-like receptor 4 via multiprotein complexes. In the present study, we investigate the role of cholesterol and membrane heterogeneities as facilitators of receptor-independent LPS binding and insertion, which underpin bacterial interactions with the host in symbiosis, pathogenesis and cell invasion. We use fluorescence spectroscopy to investigate the interactions of membrane-free LPS from intestinal Gram-negative organisms with cholesterol-containing model membranes and with T-lymphocytes. LPS preparations from Klebsiella pneumoniae and Salmonella enterica were found to bind preferentially to mixed lipid membranes by comparison to pure PC bilayers. The same was observed for LPS from the symbiote Escherichia coli but with an order of magnitude higher dissociation constant. Insertion of LPS into model membranes confirmed the preference for sphimgomyelin/cholesterol-containing systems. LPS insertion into Jurkat T-lymphocyte membranes reveals that they have a significantly greater LPS-binding capacity by comparison to methyl-b-cyclodextrin cholesterol-depleted lymphocyte membranes, albeit at slightly lower binding rates. # Introduction Gram-negative bacteria, co-evolving alongside human hosts, have adapted to occupying available ecological niches as extracellular symbiotes, facultative or true intracellular pathogens. Bacterial interactions with the host reflect their role in the niche, allowing establishment of stable populations or host colonisation relying on evasion of the host immune system by immunomimicry [bib_ref] Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function, Hajishengallis [/bib_ref] [bib_ref] Microbial Hijacking of Complement-Toll-Like Receptor Crosstalk, Wang [/bib_ref] , epithelial disruption and invasion of host immune cells. The host response to environmental stimuli, associated with bacterial presence, is governed by cell surface receptor-activated cascades [bib_ref] Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster, Triantafilou [/bib_ref] [bib_ref] LPS/TLR4 signal transduction pathway, Lu [/bib_ref]. In addition, an important role in signalling has been attributed to phase heterogeneities in the host cell membranes, such as lipid microdomains or rafts [bib_ref] Lateral diffusion of Toll-like receptors reveals that they are transiently confined within..., Triantafilou [/bib_ref] [bib_ref] The Interaction of N-Acylhomoserine Lactone Quorum Sensing Signaling Molecules with Biological Membranes:..., Davis [/bib_ref]. Some true pathogens directly utilise lateral phase heterogeneities in host plasma membranes to invade host macrophages whilst silencing TLRmediated inflammatory response [bib_ref] Role of cholesterol and the ganglioside GM(1) in entry and short-term survival..., Naroeni [/bib_ref] [bib_ref] Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus, Kim [/bib_ref]. Lipopolysaccharide is the principal component of bacterial outer membranes and its chemical composition is highly speciesspecific. LPS is released as endotoxin in oligomeric and monomeric form during outer membrane renewal in Gramnegatives and plays an important role in pathogen-host signalling in activating immune response to bacterial presence through Tolllike receptor 4, TLR4 [bib_ref] Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene, Poltorak [/bib_ref] [bib_ref] Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: Evidence..., Hoshino [/bib_ref]. Receptor activation by LPS is indirect and involves a number of other proteins including LPSbinding protein (LPB) [bib_ref] Isolation of a lipopolysaccharidebinding acute phase reactant from rabbit serum, Tobias [/bib_ref] , CD14 and MD-2. LPB facilitates LPS binding to the GPI-anchored receptor CD14 [bib_ref] Lipopolysaccharide-binding protein-mediated complexation of lipopolysaccharide with soluble CD14, Tobias [/bib_ref] , which, in turn, stimulates TLR4 dimerisation and initiation of the cellular signalling cascade [bib_ref] CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding-protein, Wright [/bib_ref]. Independently of CD14, LPS can also bind to MD-2 either in solution or in association with TLR4 [bib_ref] MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4, Shimazu [/bib_ref]. Evolutionary adaptations in some bacteria have yielded modified LPS with a reduced ability to activate TLR4-mediated proinflammatory cascades. S. typhimurium, for example, produces LPS with an altered membrane-associated domain, lipid A, which reduces TNF-a expression by monocytes [bib_ref] Regulation of lipid a modifications by Salmonella typhimurium virulence genes phoP-phoQ, Guo [/bib_ref]. Mixed lipid membranes containing phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol have been shown to undergo lateral phase separation over a certain compositional and temperature ranges [bib_ref] Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: Boundaries and composition of lipid rafts, De Almeida [/bib_ref] into more ordered, detergent-resistant membrane (DRM) sphingomyelin/cholesterol-rich membrane domains or rafts and phosphatidylcholine-rich disordered membrane phase. Lateral mobility of molecules in the DRM phase is significantly lower than in the PC-rich phase [bib_ref] Lipid lateral diffusion in ordered and disordered phases in raft mixtures, Filippov [/bib_ref]. In cell membranes, this has been used to show that on LPS activation TLR4 partitions, at least transiently, into the less mobile phase [bib_ref] Lateral diffusion of Toll-like receptors reveals that they are transiently confined within..., Triantafilou [/bib_ref]. Detergent extraction of DRM domains from cells treated with LPS has revealed association of the TLR4/CD14/MAPK signalling complexes with lipid rafts [bib_ref] The role of lipid rafts in LPS-induced signaling in a macrophage cell..., Olsson [/bib_ref]. Additional role of lipid rafts as mediators of host invasion by Brucella abortis has been suggested after observation of class A scavenger receptor co-localisation with lipid rafts during SR-A-mediated internalisation of the pathogen [bib_ref] Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus, Kim [/bib_ref]. Besides receptor-mediated association with cellular surfaces, LPS has been shown to interact with membranes of pure PC and of CP/SM/cholesterol mixtures directly from solution [bib_ref] Spontaneous insertion of lipopolysaccharide into lipid membranes from aqueous solution, Alam [/bib_ref]. We hypothesize that such direct LPS interaction with membranes is important to facultative pathogens for host invasion in avoidance of triggering immune response but confers no particular advantage to symbiotes. Here, we report results from fluorescence spectroscopic analysis of binding and incorporation of smooth type LPS from a symbiote normally present in the intestinal microflora, Escherichia coli, and from facultative pathogens, Salmonella enterica and Klebsiella pneumoniae, with model membranes and with immortalised human lymphocyte lines. The interaction of each type of LPS with PC/SM/cholesterol membranes is compared to its interaction with pure PC membranes in a quantitative way to assay the role of membrane composition and lateral heterogeneity on LPS/membrane interactions. The role of membrane cholesterol in LPS binding to lymphocyte membranes is also investigated in Jurkat cells before and after treatment with methyl-bcyclodextrin, MbCD. The interactions of LPS with each of the membrane types were characterised using a novel fluorescence technique developed in our laboratories. The technique takes advantage of the charge on molecules that on binding and or insertion into membranes leads to small changes of the membrane electrostatic surface potential (see e.g. [bib_ref] Toward a mathematical model of the assembly and disassembly of membrane microdomains:..., Richardson [/bib_ref]. This leads to a change of the pK of a membrane surface located fluorescence acid-base indicator moiety that at constant pH is observed as changes of the fluorescence due to the binding/insertion interactions. One virtue of the technique is that it can be implemented with both model and living cell membranes [bib_ref] The fusion domain of HIV gp41 interacts specifically with heparan sulfate on..., Cladera [/bib_ref]. We also utilised a second and complementary fluorescence technique that measures an important membrane quantity known as the dipole potential. Our laboratories pioneered a fluorescent technique to measure membrane interactions that change as the result of changes of the membrane dipole potential. The advantage of using this approach is that this technique illuminates particularly the macromolecular insertion into the body of a membrane [bib_ref] Intramembrane molecular dipoles affect the membrane insertion and folding of a model..., Cladera [/bib_ref]. # Results The interaction of LPS with artificial lipid membranes LPS has been shown to insert spontaneously into lipid bilayers and can lead to membrane breakdown at high concentrations [bib_ref] Spontaneous insertion of lipopolysaccharide into lipid membranes from aqueous solution, Alam [/bib_ref]. To investigate the lipid specificity of LPS/membrane interactions and obtain quantitative measurements of the binding capacity of membranes for LPS, membranes of different composition were prepared with fluorescein phosphatidylethanolamine (FPE) at levels known not to have any influence on membrane interactions. LPS was added from aqueous solution to large unilamellar vesicle suspensions and fluorescence spectra were recorded. The integrity of FPE-labelled vesicles was assessed by comparing excitation-emission spectra acquired before and after LPS addition [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref]. No major changes in spectral line shape were observed, upon the LPS addition. Changes of the net fluorescence were observed to take place due to the molecular binding reactions and are in accordance with the established mode of action of the FPE reporting system [bib_ref] Toward a mathematical model of the assembly and disassembly of membrane microdomains:..., Richardson [/bib_ref] [bib_ref] The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide membrane..., Wall [/bib_ref]. As the there are no concomitant or slower changes of the spectrum however this indicates that the molecular environment of the FPE is not changed and so the membrane structure is not modified by the interaction of FPE. This indicates that LPS does not disrupt the liposomal membranes over the concentration ranges employed in this study. All types of LPS carry net negative charges. Addition of LPS to the model membrane preparations resulted in a decrease in fluorescence [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref] , lower panel), which is indicative of binding of negative charges to the lipid membranes as described by Wall et al. [bib_ref] Interactions of macromolecules with the mammalian-cell surface, Wall [/bib_ref]. Three types of endotoxin (S. enterica, K. pneumoniae and E. coli) were titrated separately against membranes of pure phosphatidylcholine, PC 100 , and mixed membranes of PC, SM and cholesterol: PC 55 SM 15 Chol 30 . This composition was chosen to approximate that of natural cell membranes and also model showing lateral phase properties at the temperature of our studies [bib_ref] Functional imaging of microdomains in cell membranes, Duggan [/bib_ref]. Each type of LPS was added cumulatively and the observed fluorescence increase diminishes at each addition as LPS accumulates on the membrane surface until saturation at its binding capacity, B max , as shown in [fig_ref] Figure 2: Binding isotherms showing changes in initial fluorescence intensity [/fig_ref]. The data were, also, fitted to a cooperative binding model (i.e. a sigmoidal function) and an F-test was carried out identify if a cooperative binding profile could best define the signal changes. The B max (prior to normalization) and K d values were obtained from the graphs and are presented in [fig_ref] Figure 2: Binding isotherms showing changes in initial fluorescence intensity [/fig_ref] ,C and numerically in [fig_ref] Table 1: The values of K d and B max for E [/fig_ref]. For all three types of LPS, B max is higher for endotoxin interactions with mixed lipid bilayer than with the plain PC 100 [fig_ref] Figure 2: Binding isotherms showing changes in initial fluorescence intensity [/fig_ref]. Assuming all types of vesicles were of uniform size and have the same overall surface area available for LPS binding, the results suggest that more negative charge from LPS can be accommodated on the membrane surface in the presence of lipid domains. In addition, K d values are observed to be smaller for LPS interactions with the PC 55 SM 15 Chol 30 membranes [fig_ref] Figure 2: Binding isotherms showing changes in initial fluorescence intensity [/fig_ref] for all three types of LPS, which indicates that LPS preferentially binds to phase separated mixed lipid membranes. In other words these data indicate that LPS exhibits a preference for membrane microdomain structures within the fluid mosaic lipid membrane. We have previously measured any preferential localisation of FPE between simple fluid-phase PC membranes and those which also contain cholesterol-rich microdomains [bib_ref] Functional imaging of microdomains in cell membranes, Duggan [/bib_ref]. Under the experimental conditions of the present study, however, no such preferential localisation takes place. In any event this would only have a bearing on our estimations of the relative binding capacity and not the K d which are independent of the total fluorescence signals. The measured values of B max are similar for all LPS types, thus indicating that the total number of 'binding' sites for the macromolecule are the same for each membrane type (perhaps expected as the total surface area of each membrane system utilised is the approximately the same). On the other hand, the membrane affinity (i.e. as K d ), measured for the E. coli LPS membrane interactions, is significantly higher than obtained from the S. enterica and K. pneumoniae studies. These observations indicate that there is a lower membrane affinity of LPS from the symbiote E. coli for either type of membrane when compared to LPS from the opportunistic pathogens. Another difference between LPS binding to PC 100 and PC 55 SM 15 Chol 30 membranes is that the interaction profile for the latter appears to exhibit a greater complexity as shown in [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref] , lower panel) than PC 100 (small inset in [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref] , lower panel). Two distinct stages can be resolved, first a fast initial binding of the negatively charged LPS (labelled A in the figure) is followed by a slower change, B. This latter excursion phase is equivalent to an apparent (slower) recovery of electropositive membrane surface potential. As no positive chares were added the simplest explanation is that it represents the 'loss' of negative charge from the immediate membrane surface. It is possible to separate these two phases kinetically by fitting simple rate equations to the time evolution of the signal changes that yield the extent of the signal changes associated with each concentration of added LPS. Thus the B max and K d values can be determined separately for each phase as in the simpler case with the PC 100 membrane system. There is a number of explanations for this phenomenon, such as membrane insertion of some of the charged regions of the macromolecule (as described by [bib_ref] Time resolution of binding and membrane insertion of a mitochondrial signal peptide:..., Golding [/bib_ref] or that the macromolecule located on the membrane surface is undergoing a structural re-arrangement, such that charges move nearer or farther away from the membrane surface and as such exert a greater or lesser effect on the membrane surface potential [bib_ref] The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide membrane..., Wall [/bib_ref]. Such a rearrangement on the membrane surface appears to have a timescale of minutes, during which a portion of the negative charge moves away from the vicinity of the fluorescent reporter at the lipid surface (labelled B). While a similar pattern is observed for pure PC 100 membranes, the fluorescence intensity rapidly reequilibrates on the timescale of seconds (inset to [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref] , lower panel). As lateral diffusion within the membrane is important in the re-equilibration process, one contributing factor to this complexity may be the presence of phase heterogeneity in the mixed lipid membranes and the associated different diffusion coefficients in the ordered and disordered phases [bib_ref] The effect of cholesterol on the lateral diffusion of phospholipids in oriented..., Filippov [/bib_ref]. It is unlikely that shape changes of the membrane vesicles play any role in these phenomena as we are using 100 nm monodisperse unilamellar vesicles, which are thermodynamically very stable under our experimental conditions rather than giant vesicles as in Alam et al. [bib_ref] Spontaneous insertion of lipopolysaccharide into lipid membranes from aqueous solution, Alam [/bib_ref]. Receptor-independent binding of LPS to membranes is important to opportunistic pathogens as a route to host invasion. To gain a better insight into this process, the equilibrium properties of initial binding and fluorescence re-equilibration following LPS/membrane interactions were analysed for smooth type LPS from S. enterica and K. pneumoniae. The binding/ equilibration curves are shown in [fig_ref] Figure 3: Two stages of S [/fig_ref] and the corresponding B max and K d values are compared in [fig_ref] Figure 3: Two stages of S [/fig_ref] ,C and summarised in. LPS from E. coli showed similar kinetics of binding to both types of membranes and is not included in the following analysis. The values of B max for the binding step are slightly higher than the re-equilibration step in both types of LPS. This suggests that only a fraction of the charges that bind the lipid bilayer is then rearranged in the second step. The K d values obtained from initial binding curves are significantly higher than from the re-equilibration step, which suggests that hydrophobic interactions play a significant role in LPS redistribution within the membrane. Differences in kinetic constants between S. enterica and K. pneumoniae LPS are minimal, which points to a common mechanism of host target engagement. ## Partial insertion of lps into phospholipid membranes We investigated the role of membrane composition on insertion of LPS into membranes using the fluorescent probe di-8-ANEPPS [formula] (4-[2-[6-(Dioctylamino)-2-naphthalenyl] [/formula] ethenyl]-1-(3-sulfopropyl)-pyridinium, inner salt), one advantage of this probe is that it can yield information about the penetration of macromolecules into the membrane interior [bib_ref] Intramembrane molecular dipoles affect the membrane insertion and folding of a model..., Cladera [/bib_ref]. Thus by measuring the emission at different excitations, the ratio (R 460/520 ) has been shown to be a good approximation of the level of the membrane dipole potential and any concomitant changes due to molecular interactions. Smooth LPS from S. enterica was titrated into di-8-ANEPPScontaining phospholipid vesicles made up of PC 100 and PC 55 SM 15 Chol 30 . Excitation was measured before and after endotoxin addition and subtracted to obtain from the difference spectra a red shift in fluorescence [fig_ref] Figure 4: Difference fluorescence spectraof di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol... [/fig_ref]. The magnitude of such shifts has been shown to be dependent on the concentration of the interacting species [bib_ref] Effects of the membrane dipole potential on the interaction of saquinavir with..., Asawakarn [/bib_ref] , thus as more LPS became bound to the membrane this led to greater changes of the membrane dipole potential with one interpretation being that the LPS penetrates the membrane interior. R 460/520 ratios were measured before and after LPS addition and differences were plotted against LPS concentration [fig_ref] Figure 4: Difference fluorescence spectraof di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol... [/fig_ref]. The experiments were carried out with pure lipid, PC 100 , and mixed lipid membranes, PC 55 SM 15 Chol 30 , to investigate the role of lateral phase separation and the presence of cholesterol. Comparison between the membrane binding of smooth LPS from opportunistic pathogens S. enterica and K. pneumoniae showed very similar kinetics thus we report only our studies of membrane insertion of S. enterica LPS. Difference spectra and kinetic constants of smooth S. enterica LPS binding to PC and to mixed lipid membranes are shown in [fig_ref] Figure 4: Difference fluorescence spectraof di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol... [/fig_ref] and summarised in compared to PC 100 . The K d from LPS binding to pure PC 100 was found to be larger than that found for the mixed bilayer. Combined, the FPE and di-8-ANEPPS data reveal a preference for LPS from all species to insert into membranes that would exhibit microdomains i.e. most likely as laterally segregated cholesterol-containing triple mixtures over the pure PC. ## The interactions of lps with t lymphocytes The role of cholesterol in binding of LPS from S. enterica to membranes of live cell and the putative role of lipid rafts were investigated in Jurkat cells, labelled with FPE. The lipid composition of cell membranes is approximated by the triple lipid mixture model preparation used in this study (PC 55 SM 15 Chol 30 system), which are known to undergo phase separation into raftlike domains (akin to cell membranes [bib_ref] Functional imaging of microdomains in cell membranes, Duggan [/bib_ref]. For comparison, treatment with MbCD depletes membranes of cholesterol and reduces membrane propensity to phase separate into raft-like lateral microdomains [bib_ref] Effects of the membrane dipole potential on the interaction of saquinavir with..., Asawakarn [/bib_ref]. LPS from S. enterica was titrated into a suspension of T lymphocytes and the membrane interactions monitored as changes in FPE fluorescence. Studies were also performed with Lymphocytes which had been pre-treated with MbCD to deplete the cell membrane cholesterol. Fluorescence intensity is shown in [fig_ref] Figure 5: Binding isotherms of LPS from S [/fig_ref] along with B max and K d . Experimental scatter in the LPS titration curves hindered differentiation between binding and charge rearrangement and so only the composite total signal profiles can be reported (i.e. comprising both the binding and rearrangement phases of the LPS interaction -defined in [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref]. The observed LPS binding capacity B max values are higher in MbCD-treated Jurkat cells, suggesting a greater LPS binding capacity to cholesterol-depleted membranes. By contrast, the corresponding dissociation constant is also higher, which shows a lower affinity for LPS after cholesterol depletion by comparison to untreated cells. The apparent increased LPS binding affinity observed for the MbCD treated cells is interesting and may be the result of combining the 2 interaction phases we define in [fig_ref] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles [/fig_ref]. Alternatively (or in addition) it may reside in the possibility that MbCD is only known to remove cholesterol whereas in cells microdomains may be stabilised by several other factors in addition to cholesterol. These factors may include cytoskeletal elements, ECM and other lipid components unaffected by MbCD treatment or modulations of the levels of the fluid membrane free volume by cholesterol [bib_ref] Lateral diffusion in the liquid phase of dimyristoylphosphatidylcholine cholesterol lipid bilayers -a..., Almeida [/bib_ref]. The lower K d values observed in untreated cells is consistent with our FPE fluorescence results from mixed lipid PC 55 SM 15 Chol 30 membranes (see earlier section) and supports the hypothesis of a greater LPS affinity for phase separated/lipid raft-containing membranes. ## Lps insertion into lymphocyte cell membranes The time-dependent insertion of LPS from S. enterica into the Jurkat lymphocyte cell membranes and into the cholesteroldepleted membranes (MbCD-treated) was investigated following changes in the fluorescence of membrane embedded di-8-ANEPPS and was compared to model systems PC 55 SM 15 Chol 30 and PC 100 , respectively [fig_ref] Figure 6: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat... [/fig_ref]. Following addition of LPS, significantly longer equilibration times on the order of 30 minutes were required to achieve a fluorescence steady state in the live system by comparison to the model membrane system. Such observations may arise due to structures absent in a model system compared to that of the cellular system. This strategy has been employed previously in other systems and allows some discrimination between purely lipid-based interactions and those that may involve receptor systems (see e.g. Asawakarn et al. [bib_ref] Effects of the membrane dipole potential on the interaction of saquinavir with..., Asawakarn [/bib_ref]. LPS insertion into membranes without lipid domains, either PC 100 or Jurkats treated with MbCD, was notably faster by comparison to PC 55 SM 15 Chol 30 and untreated cells. The rate constant of LPS insertion into the former is about 40 times higher than for PC 55 SM 15 Chol 30 phospholipid vesicles and about 4 times greater in the case of MbCD-treated Jurkat cells compared to untreated cells with correspondingly lower K d values [fig_ref] Figure 6: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat... [/fig_ref]. However, the binding capacity for LPS was greater both in cholesterol-containing, native, Jurkat membranes and in PC 55 SM 15 Chol 30 phospholipid vesicles, where raft-like lipid domains were predicted or present. Qualitatively, these results suggest that by contrast to LPS binding, LPS insertion occurs with higher affinity in pure PC 100 membranes and in cholesteroldepleted Jurkat cells albeit at lower binding capacity by comparison to PC 55 SM 15 Chol 30 membranes and native Jurkat cell membranes. These observations are paralleled by slower kinetics of LPS insertion in the presence of cholesterol. This suggests a role of lateral diffusion and lipid order in the organised lipid sub-phases as retardants of LPS reorganisation and insertion into mixed lipid membranes both in vitro and in vivo. # Discussion The role of membrane heterogeneities in host-pathogen interactions has been investigated for receptor-mediated pathogen recognition during host response to bacteria. Key mediators of the LPS response cascade, molecular complexes involving TLR4 [bib_ref] Mediators of innate immune recognition of bacteria concentrate in lipid rafts and..., Triantafilou [/bib_ref] and CD14 [bib_ref] The role of lipid rafts in LPS-induced signaling in a macrophage cell..., Olsson [/bib_ref] , are recruited to membrane lateral domains following exposure to bacterial LPS. Advantageous to some bacterial pathogens, activation of TLR4-mediated inflammatory cascades is suppressed and the host membrane is engaged away from pro-inflammatory receptor complexes and used in host cell invasion [bib_ref] Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus, Kim [/bib_ref] [bib_ref] Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate..., Barquero-Calvo [/bib_ref]. Here, we used fluorescence spectroscopy to investigate the interactions of LPS from E. coli, S. enterica and K. pneumoniae with model and live cell membranes and the role of cholesterol and lateral phase separation in this interaction. The interaction between LPS from all three bacterial species showed preference for raft-containing cholesterol-rich membranes, characterised by lower dissociation constant and higher binding capacity. Interestingly, K d for E. coli LPS was an order of magnitude higher than the corresponding values for the other K. pneumoniae and S. enterica. Lower affinity may reflect an evolutionary adaptation of E. coli, which is a normal resident of the intestinal microflora. The molecular mechanism behind this is likely to reflect differences in the oligosaccharide, as E. coli and S. enterica have very similar lipid A moiety, which includes asymmetric 4:2 acylation of the sugars. By contrast, the lipid A moieties of S. enterica and K. pneumoniae are asymmetrically 4:2 and symmetrically 3:3 acylated (for review see [bib_ref] Structure and function of lipopolysaccharides, Erridge [/bib_ref] , yet the two types of LPS show very similar K d values. The overall similarity of the lipid A moieties of the three types of LPS investigate, all hexa-acylated, is the likely reason for the similarity in B max . Yet, subtle differences in acylation between E. coli and S. enterica on the one hand and K. pneumoniae, on the other, may account for the marginally greater difference in B max in the former two by comparison to the latter. The asymmetric acylation of the former two may provide a less ordered acyl chain region near the di-acyl moiety and a putative binding site for cholesterol. Lipid reporters with an acid-base surface-localised fluorophore, such as FPE, is sensitive to changes in the electrostatic environment within the membrane lipid headgroup region and can detect charge association with the membrane surface, as well as movement of charges due to lateral redistribution or following insertion of charged species into the membrane interior [bib_ref] Interactions of macromolecules with the mammalian-cell surface, Wall [/bib_ref]. Dissociation constants, determined from initial changes in FPE fluorescence are markedly higher for all LPS types by comparison to K d values determined after equilibration. This is likely to reflect a multistep process, during which LPS oligomers associate with the membrane where conversion to monomeric form of LPS leads to final insertion into the membrane. Such process is likely to be lateral diffusion-limited and to require loner equilibration times in raft-containing membranes, where lipid lateral diffusion rates are lower than in pure PC membranes [bib_ref] Lipid lateral diffusion in ordered and disordered phases in raft mixtures, Filippov [/bib_ref]. Indeed, analysis of timedependent changes in di-8-ANEPPS fluorescence during LPS insertion [fig_ref] Figure 6: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat... [/fig_ref] shows higher rates of insertion into PC membranes and cholesterol-depleted cells by comparison to PC 55 SM 15 Chol 30 and native Jurkat membranes, respectively. However, the corresponding membrane insertion capacities in the absence of cholesterol are lower both in the model and in the cell systems, which is likely to reflect optimal packing of the lipid A acyl moieties within ordered, cholesterol-rich membrane domains in these systems. The latter observation has a profound implication on our understanding of LPS interactions with cell membranes. Experimental evidence points to transient re-localisation of LPSactivated TLR4 receptor complexes into lipid rafts during initiation of immune response [bib_ref] Mediators of innate immune recognition of bacteria concentrate in lipid rafts and..., Triantafilou [/bib_ref] , which implies steady state localisation of inactive TLR4 in the fluid membrane sub-phase. By contrast, immunosilent host invasion, during which TLR4 cascades remain silent, involves a direct interaction between invading bacteria and lipid raft within the host membrane [bib_ref] Role of cholesterol and the ganglioside GM(1) in entry and short-term survival..., Naroeni [/bib_ref]. Results in the present study suggest preferential insertion of LPS into membrane rafts, which may be an important part of or contribute to immunosilent host invasion by pathogenic bacteria. In summary, fluorescence spectroscopy was used to investigate the interactions between LPS from E. coli, S. enterica and K. pneumoniae with lipid membranes composed of PC alone or containing PC, sphimgomyelin and cholesterol. The role of cholesterol on LPS binding was also investigated in Jurkat cells or in cholesterol-depleted MbCD-treated jurkat cells. Biding of all types of LPS to model membranes was characterised by lower dissociation constants and similar or slightly higher capacity in the triple mixtures. The K d values determined for LPS from nonpathogenic E. coli were significantly higher than the corresponding values from S. enterica and K. pneumoniae. LPS insertion showed preference for triple-lipid membranes with little difference between species of origin. Native Jurkat membranes showed higher binding capacity for LPS by comparison to cholesteroldepleted cells, although with correspondingly higher K d values. LPS insertion into model membranes and cells showed slower kinetics, which correlates with slower lateral diffusion and suggests preference of LPS insertion for ordered, cholesterol-rich membrane domains. Therefore, association of previously reported LPSinduced activation complex with lipid rafts may follow or be coincidental with incorporation of LPS into cholesterol-rich lateral domains. # Materials and methods ## Liposome preparation Phosphatidylcholine bilayers (PC 100 ) were prepared using egg lecithin mixture (Lipid Products, UK) and mixed lipid bilayer (PC 55 SM 15 Chol 30 ) with detergent-resistant domains was prepared from phosphatidylcholine, sphingomyelin (SM) and cholesterol (Chol) at the molar ratio of 55:15:30. Sphingomyelin and cholesterol were supplied from Sigma Aldrich (Poole, UK) and used without further purification. Phospholipid vesicles were prepared as described previously [bib_ref] Techniques for encapsulating bioactive agents into liposomes, Mayer [/bib_ref] ; briefly desired volumes of lipid in chloroform:methanol (solvent ratio 5:1) were measured before the solvent was evaporated under a stream of oxygen free N 2 gas. The resulting lipid film was resuspended in LPS buffer (10 mM Tris, 1 mM EDTA and pH 7.4) before freeze-thawing 10 times with liquid N 2 and hot (50uC) water bath. The resulting multilamellar vesicles (MLVs) were extruded through polycarbonate filter of pore-size 100 mm (Nucleophore Filtration Products, USA) using N 2 gas and pressure extruder (Lipex Biomembranes Inc., Vancouver, Canada) to generate 13 mM unilamellar lipid vesicle suspensions. Lipopolysaccharides were purchased from Sigma, UK; and used without further purifications. LPS samples were prepared by suspending LPS powder in LPS buffer (10 mM Tris, 1 mM EDTA and pH 7.4) to a final concentration of 6.67 mg/ml. ## Cell culture Jurkat T-lymphocytes (E6-1 clone) were obtained from the European Collection of cell cultures (ECACC) and cultured in 90% RPMI 1640 medium with 10% heat inactivated foetal calf serum (FCS), L-glutamine (100 mm/ml) and Penstrep (Penicillin and streptomycin mixture, 100 mm/ml). Culture medium was replaced every 3 days and the colony was maintained at 37uC and 5% CO 2 with a cell density between 1610 5 cells/ml and 1610 6 cells/ml. ## Fluorescent labelling Fluoresceinphosphotidylethanloamine (FPE) was synthesised according to the published methods of Wall et al [bib_ref] Interactions of macromolecules with the mammalian-cell surface, Wall [/bib_ref]. Phospholipid vesicles were labelled with either FPE or di-8-ANEPPS (Molecular Probes, Leiden, The Netherlands) to a final concentration of 0.2% (molar) and in the presence of ethanol (1.5% (v/v)) by adding the fluorescent dye as an ethanol solution to the vesicles stock and incubating at 37uC for 1 h for FPE and 1.5 h for di-8-ANNEPS [bib_ref] Effects of the membrane dipole potential on the interaction of saquinavir with..., Asawakarn [/bib_ref]. In order to remove unbound probe, FPE-labelled vesicles were additionally filtered through a PD-10 column equilibrated with buffer (10 mM Tris, 1 mM EDTA and pH 7.4). As di-8-ANEPPs has a low quantum yield in water relative to a lipid environment, this step was not required for di-8-ANEPPs labelled vesicles. To label lymphocytes with FPE confluent cell cultures (1610 6 cells/ml) were harvested by gentle centrifugation (3006g for 5 min at 25uC) and resuspended in sucrose-Tris buffer (280 mM sucrose, 10 mM Tris, 1 mM EDTA and pH 7.4) to a concentration of 1610 6 cells/ml. FPE solution was added to a final concentration of 8.8 nmoles of FPE per 1610 6 cells and incubated for 1 h at 37uC. To label Lymphocytes with di-8-ANEPPS confluent cell cultures (1610 6 cells/ml) were harvested by gentle centrifugation (3006g for 5 min at 25uC) and resuspended in sucrose-Tris buffer (280 mM sucrose, 10 mM Tris, 1 mM EDTA, pH 7.4) to a concentration of 0.5610 6 cells/ml. Di-8-ANEPPS was added to a concentration of 6 nmoles per 1610 6 cells) and the sample was incubated for 1.5 h at 37uC. Cholesterol-depleted, fluorescently-labelled (either FPE or di-8-ANEPPS) T-lymphocytes were prepared using the aforementioned labelling protocols before briefly exposing the cells to MbCD solution (66 mg/ml) for 4 min. Cells were then harvested by gentle centrifugation (3006g for 5 min at 25uC) and returned to sucrose-Tris buffer. This MbCD treatment protocol is adapted from the method of [bib_ref] Revaluation of the role of cholesterol in stabilizing rafts implicated in T..., Rouquette-Jazdanian [/bib_ref]. ## Fpe fluorescence measurements FPE-experiments were conducted using labelled phospholipid vesicles at a concentration of 390 mM of total lipid and excitation scan (450-520 nm range, emission measured at 530 nm) followed by emission scan (measured emission over 520-590 nm range with excitation at 490 nm) were recorded at 37uC before and after LPS additions to inspect vesicle labelling efficacy. LPS was added cumulatively to a final concentration of 24 mg per mmole of total lipids and fluorescence was measured with a continuous excitation at 490 nm and emission detection at 520 nm. Changes in fluorescence were plotted as the inverse of the percentage change in signal and fitted to the hyperbolic equation (equation 1). [formula] DF~B max | LPS ½ K d z LPS ½ð1Þ [/formula] The FPE-experiments on labelled lymphocytes were carried at a concentration of 4610 4 cells/ml and LPS was added in 6 steps to a final concentration of 12 mg per 1610 4 cells. Data analysis was conducted as described previously for artificial membranes [bib_ref] The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide membrane..., Wall [/bib_ref]. ## Di-8-anepps fluorescence measurements Di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol 30 phospholipid vesicles were used at a concentration of 0.41 mmole of total lipid per ml and excitation scans were recorded at 37uC (400-550 nm range, emission measured at 590 nm). Fluorescence measurements were taken at 590 nm upon excitations at 460 and 520 nm (denoted F 460/590 and F 520/590 respectively). The ratio 460 nm/520 nm (R 460/520 ) was then calculated using the following equation: [formula] R 460=520~F 460=590 F 520=590ð2Þ [/formula] LPS from S. enterica was then added to six individual vesicle samples of both compositions (PC 100 and PC 55 SM 15 Chol 30 ) at a linear concentration range between 76 mg and 444 mg of LPS per 1 mmole of total lipid. Liposome samples were incubated with different concentrations of LPS for 25 min at 37uC before excitation scans and R 460/520 ratio were recorded. Excitation spectra were normalized before subtracting spectra in the presence of each concentration of LPS from those in the absence of the molecule in order to give difference spectra. In addition, changes in the R 460/520 ratio on addition of LPS were plotted against LPS concentration and fitted to a hyperbolic equation [bib_ref] Intramembrane molecular dipoles affect the membrane insertion and folding of a model..., Cladera [/bib_ref]. ## Time evolution of di-8-anepps fluorescence changes Di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol 30 phospholipid vesicles were used at a concentration of 0.41 mmole of total lipid per ml and a single concentration of LPS (0.3 mg LPS per 1 mmole of total lipid) was added to each membrane and R 460/520 ratio was measured as a function of time for approximately 25 min. Di-8-ANEPPS-labelled T-lymphocytes with and without MbCD pre-treatment were used at a concentration of 2610 4 cells/ml in sucrose-Tris buffer and a single concentration of LPS was added to each and R 460/520 ratio was measured as a function of time for approximately 15 min. [fig] Figure 1: Excitation-emission fluorescence spectra of FPElabelled phospholipid vesicles (top) before (dashed line) and after (solid line) LPS titration. Arrow indicates a small peak from residual, free FPE in solution; lower panel represents part of the LPS titration curve recorded over time for FPE-labelled PC 55 SM 15 Chol 30 phospholipid vesicles -initial drop, A, is followed by signal reequilibration, B. Binding curves(Figure 2) are obtained from measuring changes between the initial signal and the equilibrium state, C. Inset shows titration curve measured for pure PC 100 vesicles, with significantly smaller difference between the initial binding and re-equilibration stages. doi:10.1371/journal.pone.0038677.g001 [/fig] [fig] Figure 2: Binding isotherms showing changes in initial fluorescence intensity (cf.Figure 1) for three types of smooth LPS from S. enterica, (squares) K. pneumoniae (triangles) and E. coli (circles) upon binding to FPE-labelled PC 100 (dashed lines) or PC 55 SM 15 Chol 30 (solid lines) phospholipid vesicles. (A). In each case, the average of three repeats was used and the values are shown in comparisons to the data obtained for studies with PC 100 . Histograms of B max and K d values, corresponding to (A) but obtained from non-normalized data, are shown in panels (B) and (C), respectively and summarised in [/fig] [fig] Figure 3: Two stages of S. enterica LPS interaction with mixed PC 55 SM 15 Chol 30 membranes. (A): initial binding (hexagons) and conformational re-equilibration (diamonds). Both datasets were normalized to the starting fluorescent intensities for LPS binding to PC 100 and the values of B max are normalized to one for PC 100 (B) and values for K d are shown in (C). The average of three repeats is shown and fitted to Equation 1. Similar binding curves were obtained from K. pneumoniae LPS binding. The B max and K d values for both types of LPS are shown in (B) and (C) and summarised in [/fig] [fig] Figure 4: Difference fluorescence spectraof di-8-ANEPPS-labelled PC 100 and PC 55 SM 15 Chol 30 phospholipid vesicles before and after titration of LPS from S. enterica (A). The addition of endotoxin to both types of vesicles results in red shift. Changes in R460/520 ratio were plotted in (B) and fitted to Equation 1. The graphs are normalized to B max of the PC 100 and to the starting fluorescence intensity. The B max and K d values were estimated from the fits and are presented in (C) and (D), respectively. doi:10.1371/journal.pone.0038677.g004 [/fig] [fig] Figure 5: Binding isotherms of LPS from S. enterica to FPE-labelled Jurkat cells and to Jurkat cells, from which cholesterol has been removed with MbCD prior to addition of LPS (A). Both curves are normalized to initial fluorescence intensity of untreated cells. Values for B max and K d values are show in panel (B). doi:10.1371/journal.pone.0038677.g005 [/fig] [fig] Figure 6: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat cells exposed to a single concentration of S. enterica LPS. Panel (A) shows changes in di-8-ANEPPS fluorescence recorded over time from phospholipid vesicles and panel (B), form Jurkat cells; orange lines show membranes without lipid domains (either PC 100 or MbCD-treated Jurkat cells). The PC 55 SM 15 Chol 30 curve is normalised to PC 100 and Jurkats fluorescence is normalised to MbCD-treated cells. doi:10.1371/journal.pone.0038677.g006 [/fig] [table] Table 1: The values of K d and B max for E. coli LPS are approximated from the fits, as K d is greater than the concentration range investigated experimentally. Tolerances in K d and B max arise from fitting the data to Equation 1, while error bars in (A) show variance between runs. doi:10.1371/journal.pone.0038677.g002 [/table] [table] Table 3: The binding capacity (i.e. as B max ) for LPS is greater in PC 55 SM 15 Chol 30 membranes Table 1. Values of B max and K d obtained from fluorescence changes recorded in FPE-labelled PC 100 and PC 55 SM 15 Chol 30 phospholipid vesicles incubated with LPS of different bacterial origin: S. enterica, K. pneumoniae and E. coli. [/table]

Exploring experiences of quarantined people during the early phase of COVID-19 outbreak in Southern Nations Nationalities and Peoples’ Region of Ethiopia: A qualitative study BackgroundEthiopia enforced extremely rigorous contact tracing and mandatory quarantine for all suspected contact and travelers entering the country for a period of 14-days duration during the early phases of the COVID-19 outbreak. Several studies investigated the experience of quarantined people because of COVID-19 or previous outbreaks. However, quarantine is often perceived differently in different cultures because of its historical association with class, gender, ethnicity, politics, and prejudices. To our knowledge, there is limited literature on quarantine experience in Ethiopia related to either COVID-19 or other infectious diseases. Therefore, this study was aimed to explore quarantine experience of people in Southern Nations Nationalities and Peoples' Region (SNNPR) of Ethiopia during early phase of COVID-19 pandemic.MethodsThe study implemented an exploratory qualitative research design using a phenomenological approach. Face-to-face in-depth interviews were conducted with purposively recruited 29 respondents. Digitally recorded audio files have been listened to several times and verbatim transcriptions were done. The transcribed narratives were examined independently and content analysis was carried out through reading and re-reading the verbatim several times, open coding, grouping, categorizing, and abstracting the final themes.ResultsThree broad themes were identified and characterized the experiences of quarantined people due to COVID-19. These themes were a) handling of the suspected person, b) adverse effects of quarantine and c) coping strategies. In addition, quarantine refusals; injustice in PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.This study explored a range of complex experiences of quarantined people because of the COVID-19 outbreak in SNNPR. The quarantined people included in this study were adversely affected psychologically, physiologically, socially, and economically. They also experienced quarantine errors and injustice. There is a need to gather clear justification for close contact before forcing the suspect for mandatory quarantine. In addition, there is a need to develop risk communication strategy to approach suspected contacts for quarantine. Moreover, assessing psychological, physiological, social, and economic impacts of quarantine on the individuals while they are in quarantine and after release could be important. The use of personal and social coping strategies including psychosocial support may lessen the adverse impacts of the quarantine. [bib_ref] Social consequences of ebola containment measures in Liberia, Pellecchia [/bib_ref] [bib_ref] The experience of quarantine for individuals affected by SARS in Toronto, Cava [/bib_ref] [bib_ref] Lessons learned from the anti-SARS quarantine experience in a hospital-based fever screening..., Ching [/bib_ref] [bib_ref] Posttraumatic stress disorder in parents and youth after health-related disasters, Sprang [/bib_ref]. The issue of disparity in the impact of COVID-19 across culture, economy, and politics are also striking [bib_ref] Psychological and Social Impact of COVID-19, Osofsky [/bib_ref]. To our knowledge, there is a dearth of literature on the experience of quarantined people for COVID-19 or other contagious diseases in Ethiopia. Therefore, this study was aimed at exploring the experiences of quarantined people during the early phase of the COVID-19 outbreak in Ethiopia. # Methods ## Study design The study implemented an exploratory qualitative research design based on a phenomenological approach. A qualitative study design was recommended to investigate and describe all phenomena including all kinds of human experience, as the phenomena appear in their fullest breadth and depth [bib_ref] Phenomenology: a method for nursing research, Omery [/bib_ref]. Phenomenology allows exploration of the lived experience of an individual [bib_ref] Phenomenology in Its Original Sense, Van Manen [/bib_ref]. Therefore, qualitative design using a phenomenological approach was deemed to be the most appropriate for a deeper understanding of the phenomenon under investigation. ## Study setting and period The study was conducted in Southern Nations, Nationalities, and Peoples' Region (SNNPR), which is one of the ten regional states that formed the Federal Democratic Republic of Ethiopia. The region borders Kenya to the South, South Sudan to the Southwest and West, Oromia regional state to the North and East, and Gambela regional state to the West. The region is home to over 45 indigenous ethnic groups. SNNPR is the most densely populated region in Ethiopia. Quarantine centers in Gamo zone, Woloyta Zone, Halaba Zone, Hadiya Zone, Silte Zone, Konso Zone, and Hawasa city were purposively selected and included in the study. The study was conducted from June 20 to July 25, 2020. ## Eligibility criteria and population The eligibility criteria include: individuals aged 18 years or above, regardless of quarantine type, viz; forced, coerced, or voluntary quarantine, and stayed in quarantine center because of the following reason/s:having close contact with a confirmed COVID-19 positive person or [bib_ref] Understanding, compliance and psychological impact of the SARS quarantine experience, Reynolds [/bib_ref] having close contact with the suspected person for COVID-19 or (3) having travel history to COVID-19 positive reported area. ## Sampling and data collection procedures The investigators used a purposive sampling approach. Before conducting the interviews, participants' information and contact numbers were obtained from quarantine centers' registries. Participants with different cultural, religious, educational, professional, and economic backgrounds were purposively screened from the registry and contacted through the focal person of each quarantine center. Face-to-face in-depth interviews were conducted with eligible and interested participants under strict physical distancing and other preventive measures. A semistructured interview guide was prepared in English by reviewing previous works of literature [bib_ref] The psychological impact of quarantine and how to reduce it: rapid review..., Brooks [/bib_ref] [bib_ref] Is There a Case for Quarantine? Perspectives from SARS to Ebola, Barbisch [/bib_ref] [bib_ref] Social consequences of ebola containment measures in Liberia, Pellecchia [/bib_ref] [bib_ref] Exploring the experiences of healthcare providers who survived MERS-CoV in Saudi Arabia, Almutairi [/bib_ref] [bib_ref] The experience of quarantine for individuals affected by SARS in Toronto, Cava [/bib_ref] [bib_ref] Lessons learned from the anti-SARS quarantine experience in a hospital-based fever screening..., Ching [/bib_ref]. The interviews were held in Amharic and audio recorded. Before conducting the interview, the tool was translated to Amharic guided by WHO's tool translation guideline. Accordingly, the forward translation of the interview guide was done by ZA and ND, who were members of the research team, health care professionals, and fluent in Amharic. Expert and back translations were also done to meet the aims of the study. The sample required for this study was decided by the concept of theoretical saturation [bib_ref] Sample size in qualitative research, Sandelowski [/bib_ref]. Based on the suggestion, we explored lived experiences of 29 individuals as determined by the research team. Each interviewed individual was compensated 300 ETB (equivalent to 8.6 USD at the rate of data collection moment). The interviews were recorded for ranging from 22 to 47 minutes. # Data analysis Participants were invited to describe their experience of quarantine in detail including the situation before quarantine, experience at the center, and after being released from the center. Digitally recorded audio files have been listened to repeatedly, and verbatim transcriptions were done by AB, ND, and ZA. The transcribed narratives were examined independently by AB, ND, and ZA using inductive thematic content analysis. The analyses were carried out through reading verbatim several times, open coding, grouping, categorizing, and abstracting the final themes [bib_ref] The qualitative content analysis process, Elo [/bib_ref] [bib_ref] Analysing and presenting qualitative data, Burnard [/bib_ref]. To validate the analyses, multiple coding, peer review, and discussion were made until an agreement was reached [bib_ref] Checklists for improving rigour in qualitative research, Power [/bib_ref] [bib_ref] Rigour and qualitative research, Mays [/bib_ref]. # Ethical consideration The ethical clearance was obtained from the institutional review board (IRB) of Arba Minch University. A letter of support was written by Arbaminch University to respective institutions. The purposes and importance of the study were explained to the participants of the study and all participants provided written informed consent before the interview. The respondents were informed that participation in the study was voluntary. In addition, the respondent had the right to withdraw from the study at any time during an interview without the urge to explain the reason for withdrawal. The participants were assured that the data will be handled exclusively by the investigators, used only for the study, and no one will be able to recognize them in the report. The confidentiality of information collected from the participants was maintained. # Results A total of 29 in-depth interviews were carried out among 18 males and 11 females to explore experiences of quarantined people because of contact suspects for COVID-19 in SNNPR, Ethiopia. The participants' age ranged from 19 to 65 years. Individuals coming from different occupational backgrounds including farmers, drivers, merchants, daily laborers, students, nurses, doctors, and members of parliament were interviewed and included in the study. The majority of the participants experienced mandatory quarantine [fig_ref] Table 1: Characteristics of the study participants [/fig_ref]. The findings from content analyses were categorized into three main themes namely: handling of the suspected person, adverse effects of quarantine, and coping strategies. In addition, the findings were further categorized into the emerged sub-themes viz., the first and second themes each categorized into three and four sub-themes respectively, and, two sub-themes for the third theme. Details on each theme and sub-theme are provided below. ## First theme: handling of the suspected person This theme was classified into four sub-themes namely: quarantine refusal, injustice, and quarantine errors. Quarantine refusal. Quarantine refusal was experienced by the majority of the participants including health care providers. Denial, under communication, and lack of preparation for quarantine were reasons provided for violence with legal enforcing bodies. For example, one of the quarantined people during the early days of the first COVID-19 report in Ethiopia lamented as follows: "There was no ground base to force me for quarantine. My close contact already tested negative."~A young medical doctor. Contact denial was identified as the reason for quarantine refusal by several participants. Injustice. The study participants noted that disrespect, intimidation, harsh treatment, and detention were their frequent experiences related to quarantine. Several respondents blamed legal enforcement bodies for disrespect and harsh treatment. The following quotes were some of the examples given by the study participants: "The way the police officers forced me was similar to the situation we were experienced during the 'dergue' regime (a military government that ruled Ethiopia from 1974 to 1991). They (the police officer) use all available forces to take me to the quarantine center."~53 years old farmer "About ten police officers with a gun arrived at my house. I was scared and felt guilty because of the numbers and activities of police officers seemed I committed the worst crime such as homicide"~35 years old woman Intimidation by law enforcement bodies was reported by the majority of respondents who were forced into quarantine. One of the respondents narrated the situation as follows: ". . .I was traveling back home from University following the announcement of lockdown. At the gate to Hawasa city, police officers with guns loudly shout at us saying to get out of the car. Do not try to escape. If you do so, you will be hit by the bullet. I was shocked as I did not have information on what happened to us at the moment. Later they took us to the quarantine center and they told us someone escaped from Addis Ababa to Hawasa following tested positive using public transport and they were looking for the person"~19 years old boy who was a first-year university student Some of the participants were also detained at the rural police stations for days before reaching a nearby quarantine center. The way they were treated at police stations was similar to that of people sentenced for conducting crime according to their expression. One of the respondents narrated the situation as follows: "I was detained in rural prison like a person suspected for crime in rural prison for two days before they transferred me to quarantine center" a 31 years old man. Quarantine errors. Some respondents blamed the authorities for the quarantine mistake due to failure to confirm the identity of the suspect that need quarantine. In some cases, individuals were wrongly quarantined because of a wrong report to quarantine centers by the individuals who were in personal conflicts with the suspects. For example one of the respondents described her experience as follows: " I had a personal conflict with one of my neighbors. She holds a grudge and reported me to the task force (which was established following the COVID-19 outbreak in Ethiopia). I had no either travel history or close contact with suspected person or any clinical manifestations"~37 years old woman Second theme: Adverse effects of quarantine Four major themes that described the adverse effects of quarantines were identified in this study. These are psychological distress, physiological changes, social effects, and financial losses [fig_ref] Fig 1: Adverse effects of quarantine [/fig_ref]. Psychological distress. The causes of psychological distress were related to anxiety, feeling of guilt, confusion, anger, uncertainty about the future, aggression, obsessions, depression, fear of death, fear of death without mourning, fear of contracting the disease from the center, and suicide ideation were identified psychological and mental health effects of quarantine in this study. The stressors were varying from person to person, however, they had common predisposing causes in most of the cases which were fear of the virus. Uncertainty about the disease led some of the respondents to phycological distress such as anxiety and fear of death. A young medical doctor said that, Uncertainty about the disease also caused psychological distress to some respondents who came from a cultural background where property inheritance to children has cultural value and personal satisfaction. For example, one of the respondents said, ## "i heard that the disease is a killer. i feared that if the entire family and i could die of the virus, who would inherit my properties? am i be the last person of my clan without even having a person going to inherit me?"~46 years old farmer Fear of infection from the center was another concept that causes psychological distress to quarantined people. A 26 years old man said, "My stay at the center was quite boring. I shared a single room with six suspected men. Although I was confident regarding my status, I was too stressed because the chance of contracting the disease from another suspected person at the center was tremendous." Another concept that caused psychological distress was interpersonal conflicts at the quarantine centers. For example one of the respondents said, ## Plos one Experiences of quarantined people during early phase of COVID-19 outbreak in Ethiopia: A qualitative study ". . .My stay in quarantine was full of depression. I was asked to report my close contacts. I mentioned several people including my boss. She was a merchant at the local market in the area and I used to push a wheelbarrow for her. The police officers brought the woman to the center. She yelled at me, abused me verbally, and even stoned me. I was very irritated the day she cursed my newly born son saying 'let God kill the newborn!' I preferred staying in the room given to me than going outside fearing her verbal abuses."~a 23-year-old man Interpersonal conflicts were also reported between quarantined people and the authorities. For example, some of the participants reported that they conflicted with health care providers and law enforcement bodies for no justifiable reasons, which later ended with regret. One of the respondents lamented as follows: ". . .Unjustifiable disputes with the health care workers and security guards were my frequent experience at the quarantine center. I yelled at health care providers several times and regretted"~a mother of two and merchant The other cause of psychological distress was being in quarantine with my children. For example, one of the respondents lamented the situation as follows: "I was quarantined with my three children and all of them were less than 10 years old. They want to go outside but they were not allowed to do so. They never stayed in a little room for the whole day. Imagine a child coming from a rural area and being forced to stay in a little room. Sometimes my children cry for hours. The worst story was that my children were repeatedly asked for food that I cannot provide for them. They were repeatedly asking for food saying, 'Dad I need food, I am hungry. . .crying'. I do not want to remember those bad days (crying)." Physiological changes. Sleep disturbance, poor appetite, and weight loss were reported by the majority of the study participants. Fake news, uncertainty about the disease, anxiety, a new environment, and less preparation in the quarantine centers were identified factors that led quarantined people to physiological changes. For example, the following narratives showed how fake news led to physiological changes at the quarantine center: "I heard a bad story about how a dead body from COVID-19 was handled by one of the quarantine people. He said that the dead body is not allowed for burial, instead cremation (Ethiopians do not cremate dead bodies). Starting that night, I started to ask myself such questions. Am I going to be burned like wood? how my community is not allowed to bury me? Following the news, I had poor appetite, difficulty in falling asleep, and nightmares until I was informed tested negative"~45 years old man. Some respondents said that their weight loss was attributable to shortage and lack of food varieties at the center in addition to stress about the disease. For example, a 65 years old man described his experience as follows: "Food inadequacy at the center was a great challenge to me. The food they serve to me was not even sufficient to feed children." Another man lamented his experience as follows: "There was no food choice at the center. For breakfast, rice with bread was the only food provided by the center. We usually repeat the same food for lunch and dinner. Unfortunately, It was my first time eating rice and I was unable to adapt to eating rice. I asked the authority either to provide alternative food or allow me to buy food from outside. I was not allowed either."~46 years old farmer Social effects. Experience of discrimination, stigma, and loneliness was majorly reported as social effects of quarantine in this study. Other minor mentions were isolation from social life and missing social events. Stigma and discriminations were common experiences to quarantined people both at quarantine centers and after being released from the centers. One of the respondents lamented his experience at the center saying that, ## "it was ramadan month and i was on fasting the whole day. the 'iftar' time was up but the food was not served to me on time. i asked the security officers to buy food for me. however, he responded to me with an unexpected answer saying 'your money is contaminated, therefore, i cannot collect from you.' i was nervous and envy death at the moment"~a 45 years old man Perceived discrimination at the center was another experience reported by some of the participants. For example, "Food is served on a plate according to my culture. However, what I experienced at the quarantine center was odd to me. They served me with a thin plastic bag, which was disrespectful and discriminatory to me. Older people are highly respected in my community."~65 years old man. After being released from quarantine centers some respondents nicknamed COVID-19. Such experiences were mainly reported by individuals living in urban areas. For example, "Some people are calling me with the nickname 'COVID-19' in public"~A daily laborer and young man. In some cases, the discrimination was extended to the family of quarantined people. For example, ". . .My family members were prevented from entering the market by local militia following a loud shout from people in the market."~A 53 years old farmer. Loneliness was a common experience for several respondents at the quarantine center. Some respondents described quarantine as detachment from socialization. One of the respondents described his quarantine experience as follows: "Bismillah. . .prison is a better place. In prison, you get imprisoned with a human being and you share the room with people. However, in quarantine, I am forced to sleep alone in a single room. At the prison I used to talk to other people physically, in the quarantine center I was allowed to talk and play physically with others" 42 years old man. ## Financial losses. The financial loss associated with quarantine in this study was largely due to the restriction of the movement. However, some of the respondents blamed the sudden and forced quarantine of entire family members for their losses. For example, one of the respondents described the situation that led her to financial loss and debt as follows: "I am ensete (staple food in the area) products, retailer. No one was left at home as entire of my family members were forced into quarantine. After two week stay at the center, I returned home but the ensete estimated to be 20,000 birrs (equivalent to 600 USD) was already spoiled. I am in debt at this moment to support my family"~A 46 years old retailer woman Another respondent said: "I am a farmer and we wait a season to plow the land. I lagged to farmland. That two weeks were crucial to me. I left without seeding maize, millet, and sorghum on time."~A 53-yearold farmer In some situations, respondents there were respondents whose income was reduced after being released from quarantine due to discrimination from the community. For example, one of the respondents lamented his experience as follows: ## "i have a flour milling machine to run business. most of my customers banned the house hearing that i was in quarantine suspected of covid-19. since then, my income is reduced ## Third theme: coping strategies Both personal and social supports were identified as coping strategies among quarantined people in the present study. Personal supports. Strengthening of spiritual attachment with God or Allah has majorly mentioned coping strategy with quarantine in this study. Praying for God's mercy and salvation, and strengthening personal bonding with God were reported by most of the respondents. In addition, listening to spiritual music and reading spiritual scripts were minor mentioned by the respondents. The followings were practices of spirituality described by the respondents: "Praying helped me a lot during my stay in the quarantine. I frequently prayed for God's salvation, for the sin I did both intentionally and unintentionally."~A 35 years old woman "God was the only hope I rely on at the center. Truly speaking, quarantine further strengthens my relationship with God."~28 years old man ". . .It is a disease, it came from Allah. Consistent praying for the Mercy of Allah was the sole action I used to do at the center to cope with the encountered crisis."~47 years old man Another personal support identified in this study was problem-oriented coping strategies. The commonly mentioned strategies were reading books, taking shower, physical exercise, refraining from daily updates checking on news regarding COVID-19, preventing themselves from disclosing their quarantine stay. Browsing the internet, using social media, and phone calls with friends and family were also mentioned by some individuals particularly health care workers as coping strategies at a quarantine center. The followings are examples of quotes selected from the participants' experiences: "Listening to music, taking shower, and doing some kinds of physical exercises at the quarantine center was my frequent experience to forget about COVID-19"~a 19 years old female "I can say that accessing technology such as internet and phone reduced my stress at quarantine center"~A young medical doctor Psycho-social supports. Social support from friends and family, and psycho-social support from health care workers and volunteers helped most of the quarantined people during their stay at the centers. The respondents received psychosocial support both in formal and informal ways although the degree of support varies across respondents. For example, compared to non-healthcare providers, quarantined healthcare providers received less psychological support either from volunteers or healthcare providers. However, quarantined healthcare workers received more social support including access to the use of technologies, food services with varieties, and access to visitors maintaining physical distancing compared to non-healthcare providers in this study. The experiences of some participants are lamented as follows: "My first few days at the center were normal. I had access to the internet, mobile phone, and other essential pieces of stuff. Later, I experienced ignoring daily habitual actions such as brushing teeth, washing faces, and others. Friends of mine and co-workers noticed the situation and provided me some kind of psychological despite it being informal. My quarantine stay could have been worst without those encouragements and motivational speeches. I can witness that health care workers barely receive psychological support although they are at high risk to experience phycological distress with similar severity non-healthcare providers faces because of COVID-19 related quarantine"~26 years old Medical doctor "The first three days at the quarantine center were quite difficult as I had the habit of smoking. I received psychological support at quarantine including some counseling related to COVID-19 and smoking. It helped me to cope with quarantine and even reaching on a decision to stop cigarette smoking. I didn't smoke during the fourteen days stay in quarantine and after release as well" 26 years old man # Discussions The main goal of this study was to explore the experiences of quarantined people due to the COVID-19 Pandemic in SNNPR, Ethiopia. Three broad themes were identified that characterized the experiences of quarantined people due to COVID-19. These themes are a) Handling of the suspected person, b) adverse effects of quarantine, and c) coping strategies. Our findings revealed that there was mishandling of participants suspected to be infected with COVID-19. Some participants reported denial of and unpreparedness for being quarantined. Similar findings were reported in a qualitative study done in Uganda on people in institutional quarantine [bib_ref] Experiences of persons in COVID-19 institutional quarantine in Uganda: a qualitative study, Ndejjo [/bib_ref]. Additionally, poor communication, lack of transparency about the virus, and how to conduct themselves during the quarantine were experienced. Some participants were not given any information as to why they were being quarantined and others were given limited information which brought about a lot of confusion [bib_ref] Experiences of persons in COVID-19 institutional quarantine in Uganda: a qualitative study, Ndejjo [/bib_ref] [bib_ref] Psychological distress in quarantine designated facility during covid-19 pandemic in saudi arabia, Alkhamees [/bib_ref]. Another study was done in Kashmir also reported feelings of shock and denial about being quarantined among participants [bib_ref] Experiences of Quarantine for Individuals during the COVID -19 Outbreak in Kashmir:..., Maqbool [/bib_ref]. The participants also experienced police brutality in this study. A couple of African studies have shown that police, military, and law enforcement officers use intimidation, threats, and even imprisonment to strictly enforce COVID-19 quarantine and lockdown measures [bib_ref] Police Brutality and Solidarity during the COVID-19 Pandemic in Mathare during the..., Mutahi [/bib_ref] [bib_ref] Violence and discrimination among Ugandan residents during the COVID-19 lockdown, Katana [/bib_ref]. The possible reason for police brutality is probably because of the belief that instilling fear in people suspected to be infected with COVID-19 will enable compliance with quarantine measures [bib_ref] Quarantine experience of close contacts of COVID-19 patients in China: A qualitative..., Chen [/bib_ref] [bib_ref] Perceptions and experiences of the public regarding the COVID-19 pandemic in Nepal:..., Bhatt [/bib_ref]. However, brutality by law enforcement officers has been shown to cause more harm than good [bib_ref] The Philippines' COVID-19 Response: Securitising the Pandemic and Disciplining the Pasaway, Hapal [/bib_ref] and additionally, adherence to the quarantine measures mainly stems from inadequate or complete lack of information on COVID-19. The relationship between lack of knowledge of COVID-19 and poor adherence to quarantine measures has been reported in some studies [bib_ref] Do Quarantine Experiences and Attitudes Towards COVID-19 Affect the Distribution of Mental..., Lu [/bib_ref] [bib_ref] How to improve adherence with quarantine: rapid review of the evidence, Webster [/bib_ref]. A previous study conducted in Uganda reported that people were mistaken to be having COVID-19. Their neighbors called the police to investigate them, therefore leading to unnecessary quarantine because they did not have any contact with an infected person [bib_ref] Coronavirus disease-2019 epidemic response in Uganda: The need to strengthen and engage..., Besigye [/bib_ref]. This study also found that one participant's hateful neighbor called the police to her home to forcefully quarantine her even though she had no travel history or contact with an infected person. The findings of this study revealed that quarantine stations or centers were underprepared. Participants reported a shortage of food, water, and sanitary materials in quarantine centers, which made it difficult for them to withstand their time at the quarantine centers. Other studies reported that food and basic supply shortage at the quarantine centers caused frustration and anxiety among the participants [bib_ref] Experiences of persons in COVID-19 institutional quarantine in Uganda: a qualitative study, Ndejjo [/bib_ref] [bib_ref] Do Quarantine Experiences and Attitudes Towards COVID-19 Affect the Distribution of Mental..., Lu [/bib_ref] [bib_ref] How to improve adherence with quarantine: rapid review of the evidence, Webster [/bib_ref]. This study also found that participants experienced several adverse effects of being under quarantine for COVID-19. The quarantined people in this study experienced adverse mental health outcomes such as anger, stress, anxiety, feeling of guilt, confusion, aggression obsessions, and depression. The impact of COVID-19 on anxiety, anger, and depression was previously investigated and reported comparable results [bib_ref] The Psychological and Social Impact of Covid-19: New Perspectives of Well-Being, Saladino [/bib_ref] [bib_ref] Mental Health Effects of COVID-19, Roush [/bib_ref] [bib_ref] Experiences, impacts and mental health functioning during a COVID-19 outbreak and lockdown:..., Ló Pez-Castro [/bib_ref] [bib_ref] COVID-19 Pandemic: Psychological effects of quarantine on adults and, Frolli [/bib_ref]. Additionally, participants also had fear of death especially without mourning guaranteed, fear of infecting their families, the uncertainty of the future, and fear of contracting the disease from the quarantine center when they were confident they did not have the disease officially [bib_ref] Learning about COVID-19-related stigma, quarantine and isolation experiences in Finland, Lohiniva [/bib_ref] [bib_ref] Caring for Infectious Disease in the Prehospital Setting: A Qualitative Analysis of..., Alexander [/bib_ref] [bib_ref] In the eye of a quiet storm: A critical incident study on..., Durosini [/bib_ref]. Physiological effects such as insomnia, loss of appetite, and weight loss also contributed to negative experiences at the quarantine center. This mainly stemmed from the bad stories and news they heard about COVID-19 [bib_ref] Physical and mental health impacts of COVID-19 on healthcare workers: A scoping..., Shaukat [/bib_ref]. Social effects which included stigma, discrimination, and loneliness were reported by the participants. Several studies have also reported stigma and discrimination against quarantined participants [bib_ref] COVID-19-Related Stigma in COVID-19 Survivors in Kampala, Amir [/bib_ref] [bib_ref] The socio-economic and psychosocial impact of Covid-19 pandemic on urban refugees in..., Bukuluki [/bib_ref] [bib_ref] The coronavirus (COVID-19) pandemic's impact on mental health, Javed [/bib_ref]. In this study, participants also faced economic effects such as loss of jobs, reduced income, and inability to support families as a result of financial failure due to quarantine restrictions. Negative economic effects as a result of quarantine restrictions due to the COVID-19 pandemic have been reported by other studies [bib_ref] Effect of a COVID-19 on Social, Psychological, Economic and Health Conditions in..., Elgimati [/bib_ref] [bib_ref] Psychosocial, and Economic Impacts of the Covid-19 Pandemic on People with Chronic..., Singh [/bib_ref] [bib_ref] The impact of the COVID-19 pandemic on mental health in the general..., Knolle [/bib_ref]. Quarantined participants resorted to coping strategies to get through their time at the quarantine center; participants resorted to personal coping strategies such as spirituality and problem-oriented coping strategies. This study found that participants prayed and meditated more to cope with the quarantine situation. Similarly, a study done in the USA among college students reported that the participants resorted to meditation and spirituality to cope with the quarantine stress [bib_ref] Effects of COVID-19 on college students' mental health in the United States:..., Son [/bib_ref]. However, another study done across South Asian countries reported a decline in religious beliefs during quarantine [bib_ref] Positive and negative impacts of COVID-19, an analysis with special reference to..., Karunathilake [/bib_ref]. Social support such as friends, family, and health workers also helped participants to cope with the quarantine stress. A study done in Ethiopia reported that quarantined participants who had good social support were less likely to experience anxiety during quarantine [bib_ref] Mental Health Adverse Effects of COVID-19 Pandemic on Health Care Workers in..., Asnakew [/bib_ref]. ## Limitations of the study The current study has some limitations. The recruitment of the participants for the study was entirely from SNNPR. Although this study involved participants from different cultures and a wide geographical environment, the information used in this study are still does not representing entire parts of Ethiopia. Therefore, the generalization of these findings should be with caution as living environment and cultural differences could affect the quarantine experience. As the study was entirely qualitative, the study lacks quantifiable measurements. A quantitative study is suggested for future studies for a better understanding of the adverse effects of quarantine. # Conclusions This study explored a range of complex experiences of peoples quarantined for COVID-19 in SNNPR. The study identified experiences related to the handling of the suspected person including quarantine refusal; injustice in quarantine such as disrespect, intimidation, harsh treatment, and detention; and quarantine errors were identified as major experiences. In addition, adverse effects of quarantine include psychological distress such as anxiety, feeling of guilt, confusion, anger, uncertainty about the future, and aggression; physiological changes such as reduced appetite and sleep disturbance; social effects such as stigma, discrimination, and loneliness; and financial losses such as reduced income, debt, and loss of job were identified. Moreover, coping strategies with quarantine including personal and social support were identified. This study explored a range of complex experiences of peoples quarantined for COVID-19 in SNNPR. The quarantined people included in this study were adversely affected psychologically, physiologically, socially, and economically. They also experienced quarantine errors and injustice. ## Implications The present study findings indicate the need to gather clear justification on the suspected individual for close contact before forcing for mandatory quarantine. In addition, the findings indicate the need to develop a strategy for risk communication in approaching individuals for quarantine. There is also a need to assess the psychological, physiological, social, and economic impacts of quarantine on the individuals while they are in quarantine and after release. Furthermore, strengthening personal and social coping strategies including psychosocial support may lessen the adverse impacts of the quarantine. The current findings are useful for policymakers in Ethiopia and beyond as input in responding to evolving and emerging communicable diseases. Supporting information S1 File. Interview guide. [fig] Fig 1: Adverse effects of quarantine. https://doi.org/10.1371/journal.pone.0275248.g001 [/fig] [table] Table 1: Characteristics of the study participants. [/table]

The Bcl-2 Family in Host-Virus Interactions Members of the B cell lymphoma-2 (Bcl-2) family are pivotal arbiters of mitochondrially mediated apoptosis, a process of fundamental importance during tissue development, homeostasis, and disease. At the structural and mechanistic level, the mammalian members of the Bcl-2 family are increasingly well understood, with their interplay ultimately deciding the fate of a cell. Dysregulation of Bcl-2-mediated apoptosis underlies a plethora of diseases, and numerous viruses have acquired homologs of Bcl-2 to subvert host cell apoptosis and autophagy to prevent premature death of an infected cell. Here we review the structural biology, interactions, and mechanisms of action of virus-encoded Bcl-2 proteins, and how they impact on host-virus interactions to ultimately enable successful establishment and propagation of viral infections. # Introduction From the observation of specific changes in cell morphology upon cellular suicide, and ending in engulfment of the cell by phagocytes, [bib_ref] Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics, Kerr [/bib_ref] concluded that there must be a genetically programmed form of cell death responsible for cell deletion, that they called apoptosis [bib_ref] Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics, Kerr [/bib_ref]. From these early origins, it is now recognised that apoptosis is one of a spectrum of programmed cell death (PCD) pathways that includes not only apoptosis but also autophagy, necroptosis and more specialised forms of PCD such as pyroptosis, ferroptosis, anoikis, entosis, pathanatos, netosis, and cornification [bib_ref] Molecular definitions of cell death subroutines: Recommendations of the Nomenclature Committee on..., Galluzzi [/bib_ref]. The genetic and molecular basis of these different pathways are still being determined. Correct apoptosis regulation is key to homeostasis, and regulates the clearance of cells that are no longer required in development, and are damaged, dangerous, or infected [bib_ref] Deciphering the rules of programmed cell death to improve therapy of cancer..., Strasser [/bib_ref] [bib_ref] Multiple functions of BCL-2 family proteins, Hardwick [/bib_ref]. Apoptosis is an important regulator of the immune response, and pathogens have acquired genes that both prevent the cell from initiating apoptosis during their replicative stage, and initiating apoptosis to release their progeny. Using molecular mimicry of host proteins, pathogens have evolved mechanisms to overcome host cell defences. Such host-pathogen interactions are poorly understood, but several of these pathways are regulated by the activity of proteins of the B-cell lymphoma-2 (Bcl-2) family, a group of about 20 proteins, and numerous studies have been performed to further understand and characterise its mechanism [bib_ref] The BCL-2 protein family: Opposing activities that mediate cell death, Youle [/bib_ref] [bib_ref] The Bcl-2 family: Structures, interactions and targets for drug discovery, Kvansakul [/bib_ref] [bib_ref] The structural biology of BH3-only proteins, Kvansakul [/bib_ref] [bib_ref] Structural biology of the Bcl-2 family and its mimicry by viral proteins, Kvansakul [/bib_ref] [bib_ref] Thirty years of BCL-2: Translating cell death discoveries into novel cancer therapies, Delbridge [/bib_ref]. Ultimately, the caspase cascade is activated [bib_ref] The protein structures that shape caspase activity, specificity, activation and inhibition, Fuentes-Prior [/bib_ref] , enabling disassembly of the apoptotic cell followed by its phagocyte-mediated engulfment and elimination via lysosomes [bib_ref] Self-consumption: The interplay of autophagy and apoptosis, Marino [/bib_ref]. Bcl-2 proteins arose early in metazoan evolution [bib_ref] Comparative genomics: The evolutionary history of the Bcl-2 family, Lanave [/bib_ref] [bib_ref] Evolution of the animal apoptosis network, Zmasek [/bib_ref] , and are characterised by the existence of short conserved sequence regions, the Bcl-2 homology (BH) motifs [bib_ref] Evolution of Bcl-2 homology motifs: Homology versus homoplasy, Aouacheria [/bib_ref]. Two phylogenetically [bib_ref] Phylogenomics of life-or-death switches in multicellular animals: Bcl-2, BH3-Only, and BNip families..., Aouacheria [/bib_ref] and structurally [bib_ref] Structural biology of the Bcl-2 family and its mimicry by viral proteins, Kvansakul [/bib_ref] separate groups of proteins constitute the Bcl-2 family, . Pathways for Bcl-2 protein action. In mammals, a tripartite mechanism regulated by the Bcl-2 family controls the integrity of the mitochondrial outer membrane (MOM). Activation of Bax and Bak leads to MOM permeabilisation (MOMP), and escape of factors such as cytochrome c from the mitochondrial intermembrane space to initiate the caspase cascade that is the defining step in apoptosis. BH3-only proteins either activate Bax and/or Bak either by removal of their inhibition by pro-survival proteins, or by direct interaction. The BH3-only protein Bid is activated by proteolytic cleavage that releases its BH3-motif for interaction. Viral Bcl-2 (vBcl-2) orthologues can act on the BH3-only proteins, or directly block the action of Bax and Bak to prevent apoptosis initiation. Activation steps are shown as arrows and inhibition as bars. Bcl-2 proteins are not only pivotal in higher organisms such as mammals, worms and flies, but have been identified in evolutionary ancient species such as sponges [bib_ref] Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293..., Wiens [/bib_ref] [bib_ref] Cell death in Porifera: Molecular players in the game of apoptotic cell..., Wiens [/bib_ref] and hydra [bib_ref] Hydra and the evolution of apoptosis, David [/bib_ref]. Key molecular and mechanistic features appear to be well preserved, with the identification of pro-survival Bcl-2 and pro-apoptotic Bak in sponges [bib_ref] Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal..., Wiens [/bib_ref] , along with representatives of pro-survival Bcl-2, as well as BH3-only proteins and Bak like proteins in hydra [bib_ref] The molecular cell death machinery in the simple cnidarian Hydra includes an..., Lasi [/bib_ref]. The key role of the membrane is preserved, and interactions between the pro-survival Bcl-2 and Bax-like proteins are conserved in hydra, with the Bcl-2 orthologue HyBcl-2 co-localising with HyBax on the periphery of mitochondria [bib_ref] Monomeric red fluorescent protein variants used for imaging studies in different species, Muller-Taubenberger [/bib_ref]. The Bcl-2 family probably forms part of a primitive immune response for cnidarians, and Bax has been shown to be upregulated in response to disease in the coral Acropora hyacinthus [bib_ref] In situ hybridisation detects pro-apoptotic gene expression of a Bcl-2 family member..., Ainsworth [/bib_ref]. The demosponge Geodia cydonium Bcl-2 orthologue, BHP2, is the most ancient Bcl-2 protein to be described at a molecular level to date [bib_ref] Structural insight into an evolutionarily ancient programmed cell death regulator -the crystal..., Caria [/bib_ref]. Structurally, the Bcl-2 fold and the BH3 motif-in-groove interaction is conserved, as shown for the sponge Bcl-2 BHP2 [fig_ref] Figure 2: Structures of Bcl-2 family members [/fig_ref] , although subtle differences allow BHP2 to discriminate between most human pro-apoptotic Bcl-2 proteins to be selective for its sponge counterparts [bib_ref] Structural insight into an evolutionarily ancient programmed cell death regulator -the crystal..., Caria [/bib_ref]. Bcl-2 proteins are not only pivotal in higher organisms such as mammals, worms and flies, but have been identified in evolutionary ancient species such as sponges [bib_ref] Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293..., Wiens [/bib_ref] [bib_ref] Cell death in Porifera: Molecular players in the game of apoptotic cell..., Wiens [/bib_ref] and hydra [bib_ref] Hydra and the evolution of apoptosis, David [/bib_ref]. Key molecular and mechanistic features appear to be well preserved, with the identification of prosurvival Bcl-2 and pro-apoptotic Bak in sponges [bib_ref] Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal..., Wiens [/bib_ref] , along with representatives of pro-survival Bcl-2, as well as BH3-only proteins and Bak like proteins in hydra [bib_ref] The molecular cell death machinery in the simple cnidarian Hydra includes an..., Lasi [/bib_ref]. The key role of the membrane is preserved, and interactions between the pro-survival Bcl-2 and Bax-like proteins are conserved in hydra, with the Bcl-2 orthologue HyBcl-2 co-localising with HyBax on the periphery of mitochondria [bib_ref] Monomeric red fluorescent protein variants used for imaging studies in different species, Muller-Taubenberger [/bib_ref]. The Bcl-2 family probably forms part of a primitive immune response for cnidarians, and Bax has been shown to be upregulated in response to disease in the coral Acropora hyacinthus [bib_ref] In situ hybridisation detects pro-apoptotic gene expression of a Bcl-2 family member..., Ainsworth [/bib_ref]. The demosponge Geodia cydonium Bcl-2 orthologue, BHP2, is the most ancient Bcl-2 protein to be described at a molecular level to date [bib_ref] Structural insight into an evolutionarily ancient programmed cell death regulator -the crystal..., Caria [/bib_ref]. Structurally, the Bcl-2 fold and the BH3 motif-in-groove interaction is conserved, as shown for the sponge Bcl-2 BHP2 [fig_ref] Figure 2: Structures of Bcl-2 family members [/fig_ref] , although subtle differences allow BHP2 to discriminate between most human pro-apoptotic Bcl-2 proteins to be selective for its sponge counterparts [bib_ref] Structural insight into an evolutionarily ancient programmed cell death regulator -the crystal..., Caria [/bib_ref]. It is now emerging that the apoptotic machinery is closely associated with other cellular regulatory pathways such as autophagy, the unfolded protein response, and endoplasmic reticulum (ER) stress signalling [bib_ref] BCL-2 family: Integrating stress responses at the ER to control cell demise, Pihan [/bib_ref]. Viral subversion of the cellular Unfolded Protein Response (UPR) is a mechanism that is increasingly recognised as being fundamental for host immunity [bib_ref] Emerging functions of the unfolded protein response in immunity, Janssens [/bib_ref]. The UPR is a multimodal response to perturbed ER function ("ER stress") that results from unfolded proteins accumulating in the ER faster than they are able to be folded, leading to shut down of translation, an increase in the rate of protein folding, activation of degradation pathways of the ubiquitin-proteasome or autophagy, and ultimately apoptosis if the stress is unrelieved. Thus, ER stress, autophagy, and apoptosis are all tightly linked and regulated by viruses. The gatekeepers of mitochondrial integrity are the pro-apoptotic proteins Bax and Bak that have overlapping roles [bib_ref] The combined functions of proapoptotic Bcl-2 family members Bak and Bax are..., Lindsten [/bib_ref]. Bax and Bak are necessary for instigation of apoptosis; however, the details of their mode of action are still disputed. In mammals, after apoptotic stimuli, cytosolic Bax migrates to the mitochondrial outer membrane (MOM) to generate pores in the mitochondrial outer membrane that allows the escape of apoptogenic factors that have activated the caspase cascade . The apoptotic programme is not conserved in all aspects; for example, apoptotis in Caenorhabditis elegans differs from that in mammals. In C. elegans, there is a single folded Bcl-2 protein (CED-9) present in this organism that is associated with mitochondria [bib_ref] Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death, Chen [/bib_ref] , and it interacts with the caspase activator CED-4 to inhibit apoptosis. The CED-9:CED-4 interaction is antagonised by the BH3-only protein, Egl-1, to release CED-4 and activate the caspase CED-3 and the caspase cascade. ## An expanding family of viral bcl-2 orthologues has been discovered The importance of apoptosis and the Bcl-2 proteins in immune cell regulation and innate immunity responses has created an evolutionary pressure for viruses to acquire the genes for the pro-survival Bcl-2 proteins [bib_ref] How do viruses control mitochondria-mediated apoptosis?, Neumann [/bib_ref]. There are a multitude of large DNA viruses that mimic pro-survival Bcl-2 (vBcl-2) proteins, hijacking the intrinsic apoptotic pathway for their benefit; these are summarised in [fig_ref] Table 1: Pro-survival Bcl-2 proteins encoded by viruses [/fig_ref]. ## Membrane interactions The accumulation and oligomerisation of Bax and Bak at the intracellular membrane is the key event and the point of no return in apoptosis, and it is the least well understood at a molecular level [bib_ref] Pore formation by dimeric Bak and Bax: An unusual pore?, Uren [/bib_ref]. vBcl-2 orthologues also play a role here, with many localising to intracellular membranes such as the mitochondrial membrane, ER and nuclear envelope in the host cell. The presence of a putative hydrophobic transmembrane (TM) region for many of the Bcl-2 family indicates the importance of this interaction, though the exact molecular mechanisms remain ill-defined, Bax and Bak accumulate on the mitochondrial surface and ultimately lead to its disruption and leakage. Viral control over membrane disruption is thus crucial to maintaining the host cell viability for replication [bib_ref] Epstein-Barr virus provides a new paradigm: A requirement for the immediate inhibition..., Altmann [/bib_ref]. The folded Bcl-2 proteins are partitioned between the cytosol and intracellular membranes and trafficking between the two environments occurs. Differences in cytosol-membrane partitioning are dependent on their rate of translocation, which is in turn dependent on their TM regions [bib_ref] Differential retrotranslocation of mitochondrial Bax and Bak, Todt [/bib_ref]. Bak and Bcl-2 are predominantly membrane-associated [bib_ref] Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis, Zong [/bib_ref] [bib_ref] Multiple subcellular localization of Bcl-2: Detection in nuclear outer membrane, endoplasmic reticulum..., Akao [/bib_ref] , while others, including Bax and Bcl-x L are predominantly cytosolic but become membrane integrated after an apoptotic stimulus [bib_ref] Movement of Bax from the cytosol to mitochondria during apoptosis, Wolter [/bib_ref] [bib_ref] Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis, Hsu [/bib_ref]. Trafficking of Bax and Bak between the membrane and cytosol is a process dependent on Bcl-x L and Bcl-2 [bib_ref] Differential retrotranslocation of mitochondrial Bax and Bak, Todt [/bib_ref] [bib_ref] Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol, Edlich [/bib_ref] [bib_ref] The C-terminal helix of Bcl-x(L) mediates Bax retrotranslocation from the mitochondria, Todt [/bib_ref] [bib_ref] Bax exists in a dynamic equilibrium between the cytosol and mitochondria to..., Schellenberg [/bib_ref]. In addition to the requirement for the TM region the interaction between the pro-survival and proteins requires an exposed BH3-motif on Bax and Bak, a process that necessarily requires a conformational change from their solution conformation where the key residues of the BH3-motif are buried [bib_ref] Structure of Bax: Coregulation of dimer formation and intracellular localization, Suzuki [/bib_ref]. This suggests an interaction of the BH3 motif of Bax or Bak in the groove of the pro-survival protein, although this was one of the first interactions observed in the Bcl-2 family [bib_ref] Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed..., Oltvai [/bib_ref] there remains a deficit of structural data on this interaction. BH3-only proteins are also associated with intracellular membranes (see [bib_ref] The structural biology of BH3-only proteins, Kvansakul [/bib_ref] for a discussion), some such as Bik bear hydrophobic C-terminal regions suggestive of membrane interacting proteins. The interaction with BH3-only proteins releases the TM region from the BH3-binding groove in pro-survival proteins [bib_ref] Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its..., Wilson-Annan [/bib_ref] [bib_ref] The structure of Bcl-w reveals a role for the C-terminal residues in..., Hinds [/bib_ref] , potentially releasing it for membrane binding. Full biological activity of pro-survival Bcl-2 is not observed if the C-terminal region is truncated from these molecules even though binding to their BH3-targets is improved [bib_ref] Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its..., Wilson-Annan [/bib_ref]. The same behaviour has been observed for viral Bcl-2 orthologues, where C-terminal deletions reduce the pro-survival activity (See Opgenorth et al. 1992 [bib_ref] Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L..., Opgenorth [/bib_ref] for M11L truncation). Similarly, deletion of the C-terminal TM region of Bax impairs its membrane localisation and biological activity [bib_ref] Conformation of the Bax C-terminus regulates subcellular location and cell death, Nechushtan [/bib_ref]. A combination of biophysical, biochemical and genetic studies have shown that there is multiple redundancy in the interactions [bib_ref] Mechanisms of action of Bcl-2 family proteins, Shamas-Din [/bib_ref] [bib_ref] Mitochondrial regulation of cell death, Tait [/bib_ref] [bib_ref] Control of apoptosis by the BCL-2 protein family: Implications for physiology and..., Czabotar [/bib_ref]. Several models have been put forward for BH3-only protein PCD activation but most BH3-only proteins are able to activate Bax or Bak [bib_ref] Bid chimeras indicate that most BH3-only proteins can directly activate Bak and..., Hockings [/bib_ref]. Thus, the membrane interaction is critical to the pro-survival or pro-apoptotic activity of the Bcl-2 family and further complicates an already complex multilevel-redundant regulation mechanism for mammalian apoptosis. Many vBcl-2 orthologues, including the first viral Bcl-2 orthologue found, adenovirus E1B 19K, though without an obvious hydrophobic C-terminal region, are closely associated with intracellular membranes, the ER and nuclear envelope [bib_ref] Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of..., White [/bib_ref] and the association with membranes is required for its function [bib_ref] The E1B 19K protein associates with lamins in vivo and its proper..., Rao [/bib_ref]. Frog virus 3 Bcl-2 orthologue 97R localises to the ER and deletion of the C-terminal 29 residues inactivates the protein [bib_ref] Frog virus 3 open reading frame 97R localizes to the endoplasmic reticulum..., Ring [/bib_ref]. The African swine fever virus Bcl-2 orthologue A179L, is closely associated with viral factories, and though it lacks an obvious TM region, it is associated with the ER and mitochondrial membranes. However, the mutant G85A A179L loses its ability to keep cells alive, but also associates with ER membranes [bib_ref] A179L, a new viral Bcl2 homolog targeting Beclin 1 autophagy related protein, Hernaez [/bib_ref] , probably indicating that a competent BH3-binding is required for ER association, as this mutant destroys binding to the BH3-only proteins [bib_ref] Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis, Banjara [/bib_ref]. EBV BHRF1 is associated with membranes [bib_ref] Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B..., Henderson [/bib_ref]. Combined, these features attest to the importance of membrane association or integration to the activity of the folded Bcl-2 proteins, including those encoded by viruses. Though many questions remain about the exact nature of the molecular assemblies of Bax and Bak that disrupt the MOM, a more consistent mechanism is now emerging where Bax and Bak undergo a series of conformational changes to form high-order aggregates to create the membrane disrupting pores [bib_ref] Bax and Bak Pores: Are We Closing the Circle?, Cosentino [/bib_ref]. However, in solution Bax is a monomeric and relatively rigid protein with little evidence of conformational mobility [bib_ref] Structure of Bax: Coregulation of dimer formation and intracellular localization, Suzuki [/bib_ref] , a finding consistent with fluorescence cross correlation studies showing that Bax associates with mitochondria prior to oligomerisation [bib_ref] Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes..., Bleicken [/bib_ref] into ring-like pores [bib_ref] Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in..., Grosse [/bib_ref] [bib_ref] Bax assembly into rings and arcs in apoptotic mitochondria is linked to..., Salvador-Gallego [/bib_ref]. In a defined system consisting of only cBid, Bax and Bcl-x L , Bleicken et al. showed that the interactions between these apoptotic regulators is spatially regulated. When embedded in the membrane, Bax is proposed to form a positive feed-back loop recruiting Bax, and Bcl-x L inhibits this process by preventing Bax oligomer growth and translocating membrane Bax to the cytosol [bib_ref] Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes..., Bleicken [/bib_ref]. Accumulating evidence suggests that the TM regions are intermolecular interaction sites. Andreau-Fernandez et al. showed that the TM region of Bax interacts with the TM regions of Bcl-2 and Bcl-x L [bib_ref] Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes, Andreu-Fernandez [/bib_ref]. Earlier structural studies on Bcl-w where a near-full-length sequence was well behaved [bib_ref] The structure of Bcl-w reveals a role for the C-terminal residues in..., Hinds [/bib_ref] , showed that like Bax [bib_ref] Structure of Bax: Coregulation of dimer formation and intracellular localization, Suzuki [/bib_ref] the C-terminal tail lies in the BH3-binding groove. Furthermore, the presence of the TM region in Bcl-x L [bib_ref] Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its..., Wilson-Annan [/bib_ref] [bib_ref] Conformation of BCL-XL upon Membrane Integration, Yao [/bib_ref] and Bcl-w [bib_ref] The structure of Bcl-w reveals a role for the C-terminal residues in..., Hinds [/bib_ref] reduces their affinity for BH3-motifs. In the case of Bcl-x L is dimeric when the TM region is present [bib_ref] Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes..., Bleicken [/bib_ref] and monomeric in its absence, as shown by structural studies [bib_ref] X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell..., Muchmore [/bib_ref]. Nuclear Magnetic Resonance (NMR) investigation of the Bcl-x L :membrane interaction showed that it has an α-helical C-terminal tail that anchors the folded globular Bcl-2 domain head to the membrane [bib_ref] Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL, Yao [/bib_ref]. BH3-ligand displacement of the C-terminal residues of the α9 residues from the groove of Bcl-w renders them unstructured in solution [bib_ref] The structure of Bcl-w reveals a role for the C-terminal residues in..., Hinds [/bib_ref] , and a likely mechanism to drive Bcl-w to the membrane [bib_ref] Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its..., Wilson-Annan [/bib_ref]. Biochemical studies showed that like Bcl-x L , Bak has a transmembrane C-terminal anchor [bib_ref] Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the..., Iyer [/bib_ref]. Combined, these studies indicate that the C-terminal region of the Bcl-2 fold are not by-standers, and play an important role in not only membrane targeting and anchoring, but also the protein-protein interactions of these molecules. The observation that the viral pro-survival proteins mimic these membrane localisation and activities suggests the importance of modulating PCD in infected cells. Other models for the membrane oligomerisation include an initial dimerisation of Bax or Bak [bib_ref] Bak activation for apoptosis involves oligomerization of dimers via their α6 helices, Dewson [/bib_ref] prior to their oligomerisation at the membrane surface by unfolding an interaction. In this model, it is proposed that membrane rupture is caused by disordered clustering of Bak or Bax dimers. A "hit and run" mechanism has been suggested, where an initial weak interaction induces subsequent conformational changes in Bax or Bak. A second site has been proposed for binding BH3-only proteins on Bax, though it is a low affinity interaction that initiates apoptosis [bib_ref] BAX activation is initiated at a novel interaction site, Gavathiotis [/bib_ref]. Structural investigation of detergent treated Bax in the presence of BH3-motifs gave a symmetrical Bax dimer with the BH3 bound in the groove; this was proposed to be the active from of Bax that further oligomerises to form pores [bib_ref] Bax crystal structures reveal how BH3 domains activate Bax and nucleate its..., Czabotar [/bib_ref]. Structures of Bim and Bid BH3-motifs in the groove of domain swapped dimer have been determined [bib_ref] Crystal structure of Bax bound to the BH3 peptide of Bim identifies..., Robin [/bib_ref] , similar domain-swapped dimers have been observed for Bcl-x L , that also retain the ability to bind BH3 motifs [bib_ref] BCL-XL dimerization by three-dimensional domain swapping, O&apos;neill [/bib_ref]. A caveat on these studies is that they were performed with C-terminally truncated Bax and may not reflect membrane interactions in their entirety [bib_ref] Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes..., Bleicken [/bib_ref]. Further studies will be required to elucidate the exact mechanisms. ## Viral bcl-2-mediated subversion of programmed cell death Considering the importance of Bcl-2 proteins in regulating apoptosis, as well as autophagy in higher organisms [bib_ref] Self-consumption: The interplay of autophagy and apoptosis, Marino [/bib_ref] [bib_ref] Control of apoptosis by the BCL-2 protein family: Implications for physiology and..., Czabotar [/bib_ref] , it is unsurprising that numerous viruses have acquired sequence, functional and structural homologs of Bcl-2 to subvert host apoptosis as well as autophagy signalling for their own ends. Prevention of premature host cell death during the initial stages has been shown to be critical for successful infection of EBV [bib_ref] Epstein-Barr virus provides a new paradigm: A requirement for the immediate inhibition..., Altmann [/bib_ref] and demonstrates the pivotal role that disarming of host apoptotic defences plays in preventing clearance of virus to enable successful infection and propagation. However, whilst prevention of premature host cell apoptosis is highly desirable, ultimately, viruses also rely on host cell apoptosis at a later stage to aid viral dissemination, for example an avian reovirus triggers apoptosis to enable optimal release and dissemination of viral progeny [bib_ref] Avian reovirus-triggered apoptosis enhances both virus spread and the processing of the..., Rodriguez-Grille [/bib_ref]. The earliest identified virus encoded Bcl-2 homologs were E1B 19K from adenovirus and BHRF1 from EBV which both display substantial sequence identity (18% and 16% identical to human Bcl-2 respectively) to mammalian Bcl-2 and contain the hallmark BH1 and BH2 motifs. Functional studies of E1B 19K determined that it is a potent inhibitor of apoptosis that is induced by stimuli including Fas ligand, TNFα, and adenoviral infection. Mechanistically, E1B 19K engages Bax [bib_ref] The E1B 19K protein blocks apoptosis by interacting with and inhibiting the..., Han [/bib_ref] , Bak [bib_ref] Cloning of a Bcl-2 homologue by interaction with adenovirus E1B 19K, Farrow [/bib_ref] and Bik [bib_ref] E1B 19,000-molecular-weight protein interacts with and inhibits CED-4-dependent, FLICE-mediated apoptosis, Han [/bib_ref] , and is functionally interchangeable with Bcl-2 during adenovirus infection and transformation [bib_ref] Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the..., Chiou [/bib_ref]. One mechanism for modulating apoptosis is to manipulate the BH3-only and Bax family through interaction in the binding groove [fig_ref] Figure 2: Structures of Bcl-2 family members [/fig_ref]. This interaction has now been extensively studied and binding affinities measured , but the implications of binding specific BH3-motif bearing proteins is not always clear. For example, Bim is a universal Bcl-2 binder and binds all mammalian pro-survival proteins with relatively high affinity, yet is not bound by all vBcl-2 proteins (for example the variola virus F1L). Some viral Bcl-2 proteins are capable of binding nearly all pro-apoptotic proteins (e.g., A179L, FPV039), while others have a much more specific ligand range . The specificities of the viral Bcl-2 proteins for their BH3-targets have been determined , and in general, these interactions are of lower affinity than the pro-survival Bcl-2, probably reflecting a balancing act by the virus, as they need to block apoptosis during their replicative stage; however apoptosis is necessary for the escape of viral progeny on maturation. The cell type specificity of the virus also probably plays a role in deciding which host Bcl-2 proteins are inhibited, and this is an area for further investigation. ## Herpesviridae-encoded bcl-2 homologs Many members of the herpesviridae encode Bcl-2 like proteins. Epstein-Barr virus (or human herpesvirus 4) is a large DNA virus belonging to the γ-herpesviridae and harbours two Bcl-2 homologs, BHRF1 and BALF1. BHRF1 was shown to be an enhancer of cell survival [bib_ref] Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B..., Henderson [/bib_ref]. Biochemical and structural studies revealed that BHRF1 adopts a Bcl-2 fold [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref] [bib_ref] Solution structure of the BHRF1 protein from Epstein-Barr virus, a homolog of..., Huang [/bib_ref] and is bound to the BH3-only proteins Bim, Bid, and Puma, as well as Bak and Bax [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref] [bib_ref] BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2, Flanagan [/bib_ref]. Mechanistically, BHRF1 was shown to rely on the sequestration of Bim [bib_ref] The Epstein-Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a..., Desbien [/bib_ref] and Bak [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref] to inhibit apoptosis, and to confer chemoresistance in a Burkitt lymphoma mouse model, similar to Bcl-2 [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref]. BHRF1 was also shown to be constitutively overexpressed in a sub-set of EBV transformed B-cells, thus rendering them resistant to apoptosis [bib_ref] An Epstein-Barr virus anti-apoptotic protein constitutively expressed in transformed cells and implicated..., Kelly [/bib_ref]. The function of a second EBV-encoded Bcl-2 homolog, BALF1, remains controversial. Initial data suggested that BALF1 acts as a pro-survival Bcl-2 protein [bib_ref] Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits..., Marshall [/bib_ref] , however a second report showed that BALF1 is pro-apoptotic and inhibits the other EBV-encoded pro-survival protein BHRF1 [bib_ref] Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2..., Bellows [/bib_ref]. Subsequently, others reported that both BHRF1 and BALF1 are required for successful EBV-induced B-cell transformation [bib_ref] Epstein-Barr virus provides a new paradigm: A requirement for the immediate inhibition..., Altmann [/bib_ref]. The identification of BHRF1 in transformed B-cells sparked interest in developing antagonists against BHRF1 for targeted cancer therapy, and the feasibility of such an approach was recently demonstrated via the use of an engineered protein that bound BHRF1 with picomolar affinity [bib_ref] A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis..., Procko [/bib_ref]. No small molecule antagonists for BHRF1 have been reported yet; however their development is underway. Kaposi's sarcoma herpesvirus (KSHV or human herpesvirus 8) is also a large DNA virus and a member of the γ-herpesviridae. KSHV encodes a Bcl-2 homolog, Ks-Bcl-2 [bib_ref] Kaposi's sarcoma-associated herpesvirus encodes a functional Bcl-2 homologue, Sarid [/bib_ref] that adopts a Bcl-2 fold [bib_ref] Solution structure of a Bcl-2 homolog from Kaposi sarcoma virus, Huang [/bib_ref] and is able to bind Bim, Bid, Bik, Bmf, Hrk, Noxa, and Puma [bib_ref] BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2, Flanagan [/bib_ref]. Conflicting data exist for binding of Bax and Bak, with one report indicating that neither bind Ks-Bcl-2 [bib_ref] A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits..., Cheng [/bib_ref] , whereas a subsequent study revealed a high affinity interaction for Bak (50 nM) and moderate affinity for Bax (980 nM) [bib_ref] Solution structure of the BHRF1 protein from Epstein-Barr virus, a homolog of..., Huang [/bib_ref]. During viral infection, Ks-Bcl-2 appears to play a pivotal role in completion of the lytic cycle, as a Ks-Bcl-2 deletion virus of KSHV does not complete the lytic replication cycle. Interestingly, the Ks-Bcl-2-related ORF16 from rhesus rhadinovirus is able to functionally replace Ks-Bcl-2 during the lytic cycle, in contrast to other endogenous mammalian Bcl-2 proteins, such as Bcl-x L or other herpesvirus-encoded vBcl-2 proteins including M11 and vMIA [bib_ref] The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share..., Gallo [/bib_ref]. Another rhadinovirus, herpesvirus saimiri, also encodes a Bcl-2 homolog named ORF16 [bib_ref] Herpesvirus saimiri encodes a functional homolog of the human Bcl-2 oncogene, Nava [/bib_ref] , which was shown to be anti-apoptotic and bound Bak and Bax in pull-down assays. Murine γ-herpesvirus 68 encodes M11and was identified as an inhibitor of Fas and TNF induced apoptosis [bib_ref] The murine gammaherpesvirus-68 M11 protein inhibits Fas-and TNF-induced apoptosis, Wang [/bib_ref] [bib_ref] Murine γherpesvirus M11 gene product inhibits apoptosis and is expressed during virus..., Roy [/bib_ref] , but biochemical studies demonstrated binding to Bim, Bid, Bmf, Noxa, Puma, and Hrk, as well as Bak and Bax via the canonical ligand binding groove of its Bcl-2 fold [bib_ref] Structural and biochemical bases for the inhibition of autophagy and apoptosis by..., Ku [/bib_ref]. Thus, M11 can inhibit the major mitochondrial pathways to apoptosis, however functional studies [bib_ref] Structural and biochemical bases for the inhibition of autophagy and apoptosis by..., Ku [/bib_ref] [bib_ref] Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus..., Sinha [/bib_ref] indicate that the mitochondrial pathway may not be the primary target for M11 (see below). Cytomegalovirus (CMV or human herpesvirus 5) is a large DNA virus belonging to the β-herpesviridae. CMV encodes proteins that directly target host pro-apoptotic proteins Bax and Bak, but appear to be neither sequence nor structural homologs of Bcl-2. Human CMV encodes vMIA, which has been shown to inhibit Bax [bib_ref] A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2, Goldmacher [/bib_ref] and Bak oligomerisation [bib_ref] Role of Bax and Bak in mitochondrial morphogenesis, Karbowski [/bib_ref] [bib_ref] Cytomegalovirus proteins vMIA and m38.5 link mitochondrial morphogenesis to Bcl-2 family proteins, Norris [/bib_ref]. Interestingly, the interaction of vMIA with Bax does not involve the canonical Bcl-2 ligand binding groove. Unexpectedly, vMIA bound to an alternative binding site distinct from the canonical BH3 binding groove in Bax, which was mapped using NMR to define an interaction site comprising primarily of the loops connecting α1-α2, α3-α4, and α5-α6. Furthermore, the vMIA-Bax interaction was of high affinity with a K d of 22 nM [bib_ref] Structural mechanism of Bax inhibition by cytomegalovirus protein vMIA, Ma [/bib_ref]. Whilst human CMV vMIA appears to be able to neutralise both Bax and Bak, in mouse CMV Bax and Bak are neutralised by two proteins with single specificity [bib_ref] Cytomegaloviruses inhibit Bak-and Bax-mediated apoptosis with two separate viral proteins, Cam [/bib_ref]. MCMV-encoded m38.5 has been shown to be mitochondrially localised, and to inhibit Bax activation [bib_ref] Virally mediated inhibition of Bax in leukocytes promotes dissemination of murine cytomegalovirus, Manzur [/bib_ref] [bib_ref] The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated..., Arnoult [/bib_ref] , whereas Bak inhibition is achieved via m41.1 [bib_ref] MCMV-mediated inhibition of the pro-apoptotic Bak protein is required for optimal in..., Fleming [/bib_ref] [bib_ref] Viral inhibition of BAK promotes murine cytomegalovirus dissemination to salivary glands, Handke [/bib_ref]. Amongst the α-herpesviruses, a virus-encoded homolog of the endogenous turkey pro-survival Bcl-2 protein NR13 [bib_ref] Role of Nr13 in regulation of programmed cell death in the bursa..., Lee [/bib_ref] , Bcl-B [bib_ref] Bcl-B, a novel Bcl-2 family member that differentially binds and regulates Bax..., Ke [/bib_ref] , Boo, Diva, or NRH [bib_ref] Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an..., Aouacheria [/bib_ref] in mammals, has a Bcl-2 fold [bib_ref] The restricted binding repertoire of Bcl-B leaves Bim as the universal BH3-only..., Rautureau [/bib_ref] [bib_ref] The structure of Boo/Diva reveals a divergent Bcl-2 protein, Rautureau [/bib_ref] and has been termed vnr-13 [bib_ref] Characterization of vNR-13, the first alphaherpesvirus gene of the bcl-2 family, Aouacheria [/bib_ref]. Though little is known about vnr-13, it was shown to localise to the outer mitochondrial membrane, and inhibit apoptosis after serum deprivation [bib_ref] Characterization of vNR-13, the first alphaherpesvirus gene of the bcl-2 family, Aouacheria [/bib_ref]. . Affinities (in nM) of different pro-survival Bcl-2 proteins for peptides spanning the BH3 motif of endogenous pro-apoptotic Bcl-2 family members or Beclin-1 (measurements taken from: [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref] [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref] [bib_ref] Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis, Banjara [/bib_ref] [bib_ref] Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds..., Kvansakul [/bib_ref] [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref] [bib_ref] Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a..., Okamoto [/bib_ref] [bib_ref] Structural basis of Deerpox virus-mediated inhibition of apoptosis, Burton [/bib_ref] [bib_ref] Structural basis of apoptosis inhibition by the fowlpox virus protein FPV039, Anasir [/bib_ref] [bib_ref] BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2, Flanagan [/bib_ref] [bib_ref] An insight into the mechanistic role of Beclin 1 and its inhibition..., Ku [/bib_ref] [bib_ref] Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein, Aoyagi [/bib_ref] [bib_ref] Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary..., Chen [/bib_ref] [bib_ref] Structural plasticity underpins promiscuous binding of the prosurvival protein A1, Smits [/bib_ref] [bib_ref] Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until..., Willis [/bib_ref] [bib_ref] Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax, Fletcher [/bib_ref] n/a n/a 2300 n/a n/a n/a MVA = modified vaccinia virus Ankara, VAR = variole virus, n/a = not available, NB = no binding. ## Poxviridae-encoded bcl-2 homologs The poxviridae encompass a number of families that encode for Bcl-2 proteins. Vaccinia virus is a large DNA virus, the prototypical member of the orthopoxviruses, and encodes F1L, a potent inhibitor of intrinsic apoptosis [bib_ref] Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis, Wasilenko [/bib_ref] [bib_ref] Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and..., Fischer [/bib_ref] that displays no recognisable sequence identity to Bcl-2. Structural studies revealed that F1L adopts an unusual Bcl-2 fold featuring a domain-swapped dimer configuration, in marked contrast to mammalian pro-survival Bcl-2 proteins, which are all monomeric [bib_ref] Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds..., Kvansakul [/bib_ref] [bib_ref] Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L, Campbell [/bib_ref]. Furthermore, F1L only bound a highly restricted subset of pro-apoptotic Bcl-2 including Bim [bib_ref] Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and..., Fischer [/bib_ref] [bib_ref] The vaccinia virus protein F1L interacts with Bim and inhibits activation of..., Taylor [/bib_ref] and Bak [bib_ref] Interaction of F1L with the BH3 domain of Bak is responsible for..., Postigo [/bib_ref] [bib_ref] The vaccinia virus F1L protein interacts with the proapoptotic protein Bak and..., Wasilenko [/bib_ref] , and was shown to inhibit Bak activation by functionally replacing Mcl-1 during infection [bib_ref] Vaccinia virus F1L interacts with Bak using highly divergent Bcl-2 homology domains..., Campbell [/bib_ref]. Although F1L is also able to inhibit Bax-mediated apoptosis [bib_ref] The vaccinia virus protein F1L interacts with Bim and inhibits activation of..., Taylor [/bib_ref] , this activity is likely via an indirect mechanism as F1L does not engage Bax in a cellular context. Like other Bcl-2 family proteins F1L is localised to mitochondrial membranes through its C-terminal residues, and this region is necessary for full pro-survival activity [bib_ref] Vaccinia virus F1L protein is a tail-anchored protein that functions at the..., Stewart [/bib_ref]. Mechanistically, the interaction of F1L with Bim was identified as the primary mechanism underlying F1L-mediated inhibition of apoptosis in the context of a live viral infection [bib_ref] Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L, Campbell [/bib_ref]. Interestingly, the F1L homolog in variola virus, the causative agent of smallpox and another member of the orthopoxviridae, appears to utilise a different mechanism for apoptosis inhibition, despite adopting a near identical structure and sequence [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref]. Unlike its vaccinia virus counterpart, variola virus F1L only binds Bid, Bak, and Bax, and not Bim , and only inhibits Bax-mediated apoptosis [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref]. A homolog of vaccinia virus F1L found in another orthopoxvirus, Ectromelia virus EMV025, was also shown to be anti-apoptotic by inhibiting Bax and Bak activation by directly engaging Bim and Bak [bib_ref] Ectromelia virus encodes an anti-apoptotic protein that regulates cell death, Mehta [/bib_ref]. Myxomavirus is a member of the leporipoxviridae and encodes for M11L, another potent inhibitor of intrinsic apoptosis lacking detectable sequence similarity with Bcl-2 [bib_ref] M11L: A novel mitochondria-localized protein of myxoma virus that blocks apoptosis of..., Everett [/bib_ref]. M11L is able to engage several host pro-apoptotic Bcl-2 proteins including Bak [bib_ref] Myxoma virus M11L prevents apoptosis through constitutive interaction with Bak, Wang [/bib_ref] , Bax, Bim, and Bid [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref]. Structural studies showed that M11L adopts a compact, monomeric Bcl-2 fold [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref] [bib_ref] Structure of M11L: A myxoma virus structural homolog of the apoptosis inhibitor,..., Douglas [/bib_ref] where the canonical ligand binding groove is used to engage pro-apoptotic Bcl-2 proteins [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref]. Interestingly, functional studies revealed that M11L primarily acts by sequestering Bak and Bax [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref] , in contrast to vaccinia virus F1L which acts primarily via Bim sequestration [bib_ref] Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L, Campbell [/bib_ref]. Orf virus is a parapoxvirus and encodes a readily identifiable Bcl-2 homolog, ORFV125, that potently inhibits intrinsic apoptosis [bib_ref] A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF..., Westphal [/bib_ref]. Functional studies revealed that ORFV125 interacts with several BH3-only proteins including Bim, Puma, Hrk, Bik, and Noxa as well as active Bax but not Bak [bib_ref] The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding..., Westphal [/bib_ref]. Among the avipoxviridae, both fowlpoxvirus FPV039 and canarypoxvirus CNP058 have been shown to suppress apoptosis. FPV039 inhibits apoptosis [bib_ref] The fowlpox virus BCL-2 homologue, FPV039, interacts with activated Bax and a..., Banadyga [/bib_ref] after overexpression of all BH3-only proteins [bib_ref] The fowlpox virus BCL-2 homologue, FPV039, interacts with activated Bax and a..., Banadyga [/bib_ref] , and was shown to adopt a Bcl-2 fold and engage all major host pro-apoptotic Bcl-2 proteins [bib_ref] Structural basis of apoptosis inhibition by the fowlpox virus protein FPV039, Anasir [/bib_ref]. Interestingly, the closely related canarypoxvirus CNP058 also inhibits apoptosis in transfected cells, but engaged a different subset of pro-apoptotic Bcl-2 proteins, largely with weaker affinities than FPV039 [bib_ref] Structural basis of apoptosis inhibition by the fowlpox virus protein FPV039, Anasir [/bib_ref]. Other Bcl-2 proteins-encoded by poxviruses include sheeppoxvirus SPPV14 and deerpoxvirus DPV022. SPPV14 displayed a broader spectrum of pro-apoptotic Bcl-2 interactions by binding Bim, Bid, Bmf, Hrk, Puma, as well as Bax and Bak [bib_ref] Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a..., Okamoto [/bib_ref]. In contrast, DPV022 only engaged Bim, Bax and Bak [bib_ref] Structural basis of Deerpox virus-mediated inhibition of apoptosis, Burton [/bib_ref]. Intriguingly, DPV022 also adopted a domain-swapped Bcl-2 fold similar to those observed for vaccinia and variola virus F1L [bib_ref] Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds..., Kvansakul [/bib_ref] [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref] [bib_ref] Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L, Campbell [/bib_ref] , suggesting that this particular topology for Bcl-2 proteins may be more widely found in nature. ## Asfarviridae and iridoviridae-encoded bcl-2 homologs African swine fever virus is a large double stranded DNA virus, the only member of the asfarviridae, and encodes A179L [bib_ref] An African swine fever virus gene with similarity to the proto-oncogene Bcl-2..., Neilan [/bib_ref]. A179L adopts a Bcl-2 fold [bib_ref] Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis, Banjara [/bib_ref] , and displays extreme promiscuity by binding all host pro-apoptotic Bcl-2 proteins [bib_ref] Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis, Banjara [/bib_ref] [bib_ref] A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and..., Galindo [/bib_ref]. A179L localised to mitochondria [bib_ref] A179L, a new viral Bcl2 homolog targeting Beclin 1 autophagy related protein, Hernaez [/bib_ref] and potently inhibits apoptosis in cell culture assays [bib_ref] African swine fever virus gene A179L, a viral homologue of bcl-2, protects..., Brun [/bib_ref]. Amongst the iridoviridae, grouper iridovirus was shown to encode GIV66, which inhibited apoptosis in a grouper kidney cell culture model [bib_ref] Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection, Lin [/bib_ref]. However, the structural and functional basis of GIV66-mediated apoptosis inhibition has not been established. ## Other functional roles of viral bcl-2 homologs Although the vast majority of virus-encoded Bcl-2 proteins primarily interfere with host cell intrinsic apoptosis signalling by targeting endogenous pro-apoptotic host Bcl-2 proteins, a number of studies have revealed that vBcl-2 proteins also harbour other activities. Several vBcl-2 proteins have been shown to inhibit autophagy. Murine γ-herpesvirus 68-encoded M11 utilises the canonical Bcl-2 ligand binding groove to bind the BH3 motif of Beclin-1, a key autophagy regulator [bib_ref] Structural and biochemical bases for the inhibition of autophagy and apoptosis by..., Ku [/bib_ref] [bib_ref] Molecular basis of the regulation of Beclin 1-dependent autophagy by the γ-herpesvirus..., Sinha [/bib_ref]. Another member of the herpesviridae, KSHV, also targets Beclin-1 using Ks-Bcl-2 [bib_ref] Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy, Pattingre [/bib_ref]. However, this ability to engage Beclin-1 is not limited to the herpesviridae, with African swine fever A179L displaying autophagy inhibitory activity in addition to anti-apoptotic activity [bib_ref] A179L, a new viral Bcl2 homolog targeting Beclin 1 autophagy related protein, Hernaez [/bib_ref]. Adenoviral E1B 19K was also shown to bind Beclin-1, and thus inhibit autophagy [bib_ref] The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected..., Piya [/bib_ref]. However, to date, no autophagy inhibitor has been identified amongst the poxviridae. Another virus-encoded Bcl-2 protein with multiple functionalities is the vaccinia virus F1L. In addition to the anti-apoptotic activity mediated by sequestering Bim, F1L was also shown to inhibit inflammasome activation [bib_ref] Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation, Gerlic [/bib_ref] via an unusual unstructured N-terminal extension prior to the Bcl-2 fold [bib_ref] The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically..., Caria [/bib_ref]. In addition to the ability to mediate inflammasome activation, the N-terminus of F1L was also proposed to act as a caspase-9 inhibitor [bib_ref] Structural determinants of caspase-9 inhibition by the vaccinia virus protein, F1L, Yu [/bib_ref] [bib_ref] Vaccinia virus protein F1L is a caspase-9 inhibitor, Zhai [/bib_ref] , however, a subsequent study suggested that the F1L N-terminus is not involved in apoptosis inhibition [bib_ref] The N Terminus of the Vaccinia Virus Protein F1L Is an Intrinsically..., Caria [/bib_ref]. F1L is not the only vaccinia virus-encoded Bcl-2 protein with dual functionality. N1L was shown to adopt a Bcl-2 fold (albeit lacking a TM anchoring region) and inhibit both intrinsic apoptosis by targeting several pro-apoptotic Bcl-2 proteins as well as modulating NF-κB signalling. Interestingly, N1L also adopts a Bcl-2 fold with dimeric topology; however, dimerisation is not achieved via a domain swap as seen in F1L and DPV022, but rather via a novel interface centering on the α1 and α6 helices [bib_ref] Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein, Aoyagi [/bib_ref] [bib_ref] Functional and structural studies of the vaccinia virus virulence factor N1 reveal..., Cooray [/bib_ref]. Furthermore, the ability to manipulate both apoptosis and NF-κB signalling is mediated via two discrete sites on N1L [bib_ref] Inhibition of Apoptosis and NF-κB Activation by Vaccinia Protein N1 Occur via..., De Motes [/bib_ref]. In addition to N1L, several other NF-κB inhibitors that adopt Bcl-2 folds have now been identified in vaccinia virus. These include B14 [bib_ref] Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have..., Graham [/bib_ref] andA52 [bib_ref] Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have..., Graham [/bib_ref] , as well as A46 [bib_ref] Characterization and structure of the vaccinia virus NF-κB antagonist A46, Fedosyuk [/bib_ref] [bib_ref] Structure of vaccinia virus A46, an inhibitor of TLR4 signaling pathway, shows..., Kim [/bib_ref] , A49 [bib_ref] Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma..., Neidel [/bib_ref] and K7 [bib_ref] Poxvirus K7 protein adopts a Bcl-2 fold: Biochemical mapping of its interactions..., Kalverda [/bib_ref]. Whilst all four proteins inhibit NF-κB, they are distinguished by substantial differences in mechanism, cellular activity, and structure. Similar to N1L, B14 and A52 form dimers utilising an interface involving α1 and α6 helices, with small but significant differences in the orientation of monomeric chains with each other within the dimers amongst the three proteins [bib_ref] Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have..., Graham [/bib_ref]. Furthermore, A46 also forms dimers, however, this involves an interface formed by α4 and α6 helices of the C-terminal Bcl-2 like domain. Intriguingly, A46 harbours an additional N-terminal domain that mediates tetramerisation of A46, thus adding an additional layer of quaternary structure-based regulation. In contrast, A49 and K7 are monomeric in solution. Unlike N1L, B14, A52 and K7 do not bind pro-apoptotic Bcl-2 proteins. However, K7 harbours dual functionality that is similar to N1L, and in addition to inhibition of NF-κB, it also binds to the human DEAD-box RNA helicase DDX3 to inhibit induction of the IFN-β promoter [bib_ref] Structural basis for targeting of human RNA helicase DDX3 by poxvirus protein..., Oda [/bib_ref] [bib_ref] Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated..., Schroder [/bib_ref]. ## Concluding remarks Virus-encoded Bcl-2 proteins have demonstrated the remarkable adaptability of the Bcl-2 fold, and its ability to modulate signalling that involves several cell death-associated pathways via multiple mechanisms. Whilst mammalian pro-survival Bcl-2 proteins display several distinct rules of engagement for their interactions with pro-apoptotic Bcl-2, the picture is not as clear amongst the virus-encoded homologs of Bcl-2. In mammals, key interactions between pro-survival and pro-apoptotic Bcl-2 are typically characterised by high affinities, with some such as Bcl-x L :Bim interactions straying into picomolar affinities. The caveat with the binding studies is that they have been performed with C-terminally truncated molecules, and probably overestimate true affinities [bib_ref] Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its..., Wilson-Annan [/bib_ref] [bib_ref] The structure of Bcl-w reveals a role for the C-terminal residues in..., Hinds [/bib_ref]. Furthermore, all mammalian pro-survival Bcl-2 proteins target Bim, the sole universal pro-apoptotic BH3-only protein [bib_ref] The restricted binding repertoire of Bcl-B leaves Bim as the universal BH3-only..., Rautureau [/bib_ref] and either Noxa or Bad, but not both [bib_ref] Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary..., Chen [/bib_ref]. In contrast, key interactions between virus-encoded pro-survival proteins tend to display weaker affinities, typically with dissociation constants (K d ) in the nanomolar range, although the dimeric poxvirus-encoded pro-survival proteins F1L [bib_ref] Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds..., Kvansakul [/bib_ref] [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref] and DPV022 [bib_ref] Structural basis of Deerpox virus-mediated inhibition of apoptosis, Burton [/bib_ref] only display low nanomolar or micromolar K d values. It remains to be determined if these markedly weaker affinities are related to the different oligomeric state of the virus-encoded proteins. Furthermore, the Bad/Noxa dyad does not apply to virus-encoded pro-survival Bcl-2, with fowlpoxvirus FPV039 [bib_ref] Structural basis of apoptosis inhibition by the fowlpox virus protein FPV039, Anasir [/bib_ref] and ASFV A179L [bib_ref] Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis, Banjara [/bib_ref] binding both Noxa and Bad. Indeed, both of these proteins bind all major pro-apoptotic Bcl-2 proteins, another feature not previously observed amongst their mammalian counterparts. Lastly, Bim is not a universal target amongst virus-encoded Bcl-2, with variola virus F1L showing no affinity for Bim, and instead displaying weak binding to Bid [bib_ref] Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus..., Marshall [/bib_ref] whilst being a potent inhibitor of Bax-mediated apoptosis in cellular assays. Overall the virus-encoded Bcl-2 pro-survival proteins display weaker affinities for host pro-apoptotic proteins. This could suggest that only small perturbations of the overall balance between pro-survival and pro-apoptotic proteins in a host cell are sufficient to impede apoptosis progression; however, the functional relevance of these lower affinities remains to be clarified. When considering mechanisms and the long-standing debate on the precise mechanism of action of cellular pro-survival Bcl-2, current models and mechanisms have not been entirely resolved [bib_ref] Control of apoptosis by the BCL-2 protein family: Implications for physiology and..., Czabotar [/bib_ref]. BH3-only proteins antagonise the pro-survival Bcl-2 family that in turn keep Bax and Bak in check, but they can also activate Bax and Bak . Direct binding to Bax and Bak has been observed though the affinities are generally low, for example, Bim binds full length Bax with a K d of 3.1 µM compared with K d in low nanomolar ranges for the pro-survival proteins [bib_ref] The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins, Merino [/bib_ref]. While the exact details of the membrane pore generated by Bak and Bax remain under investigation, it is clear that the interactions at the membrane are crucial to the apoptotic response in mammals, and viruses also appear to exploit this. vMIA [bib_ref] An anti-apoptotic viral protein that recruits Bax to mitochondria, Poncet [/bib_ref] and other viral Bcl-2 proteins translocate to the MOM, and a possible role for them would be to inhibit pore formation by either preventing pore growth through sequestration or retrotranslocation of the components into the cytosol as in the case of Bcl-x L [bib_ref] Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol, Edlich [/bib_ref] [bib_ref] Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes..., Bleicken [/bib_ref]. It is becoming apparent for the best understood virus-encoded Bcl-2 proteins that, in contrast to mammalian apoptosis, multiple mechanisms of action exist, though many of these need to be clarified with quantitative structure and binding studies that are complemented by live viral infection models. Whereas myxomavirus M11L was shown to act by sequestering Bax and Bak [bib_ref] A structural viral mimic of prosurvival Bcl-2: A pivotal role for sequestering..., Kvansakul [/bib_ref] , vaccinia virus F1L was shown to only require neutralisation of Bim in a viral infection setting [bib_ref] Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L, Campbell [/bib_ref]. For EBV BHRF1, a combination of neutralisation of Bim [bib_ref] The Epstein-Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a..., Desbien [/bib_ref] and Bak [bib_ref] Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1, Kvansakul [/bib_ref] was required. When considering the role of membranes in Bcl-2 activity, and the observation that it is Bak and Bax accumulation at the membrane that is critical for intrinsic apoptosis to proceed, the association of virus-encoded Bcl-2 proteins with membranes is not unexpected. Nearly all apoptosis inhibitory vBcl-2 proteins harbour transmembrane anchoring regions to direct their subcellular localisation, chiefly to the MOM. Interestingly, different vBcl-2 proteins appear to inhibit different stages of Bax activation and translocation to the outer mitochondrial membrane [bib_ref] Viral pro-survival proteins block separate stages in Bax activation but changes in..., Cross [/bib_ref]. E1B 19K and BHRF1, are examined for their ability to block Bax activation at different steps and thereby reveal the timing of mitochondrial changes during apoptosis. BHRF1 inhibited Bax activation but not upstream of apoptotic signalling events, whereas E1B19K permitted the initial stages of Bax activation to proceed, but prevented the subsequent oligomerisation of Bax. Furthermore, CMV-encoded m38.5 and vMIA appear to block Bax downstream of translocation to mitochondria, when Bax has already undergone structural changes [bib_ref] Cytomegalovirus proteins vMIA and m38.5 link mitochondrial morphogenesis to Bcl-2 family proteins, Norris [/bib_ref]. These data suggest that no universal mechanism exists that enables virus-encoded Bcl-2 to subvert premature host cell apoptosis, and that the precise mechanism reflects the unique circumstances under which viral infection takes place. In particular, the mechanism of action may be heavily influenced by the initial site of contact and tissue type. A pertinent example is CMV-encoded m38.5, with an m38.5 deletion virus showing no overt signs of impaired replication in visceral organs, whereas in salivary glands a 10-100 fold difference was observed [bib_ref] Virally mediated inhibition of Bax in leukocytes promotes dissemination of murine cytomegalovirus, Manzur [/bib_ref]. This suggests that particular tissues may be more prone to infection; however, this aspect and the observation that expression patterns of pro-apoptotic Bcl-2 proteins vary amongst tissues has not been adequately addressed for the vast majority of viruses. Interestingly, viruses can manipulate the host cell apoptosis program on many levels, with evidence emerging that viruses can manipulate the caspase cascade [bib_ref] Viral hijacking of host caspases: An emerging category of pathogen-host interactions, Connolly [/bib_ref] and the endogenous levels of the host Bcl-2 family members [bib_ref] Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation..., Collins-Mcmillen [/bib_ref]. These findings add an additional layer of complexity to the quest of identifying the precise molecular mechanism of action of vBcl-2 proteins, and ultimately suggest that more sophisticated approaches may be required to answer these questions. [fig] Figure 2: Structures of Bcl-2 family members. (A) Human Bcl-xL:Bim complex [33] (PDB ID 1PQ1); (B) G. cydonium BHP2:LB-Bak-2 complex [32] (PDB ID 5TWA); (C) EBV BHRF1:Bim complex [34] (PDB ID 2WH6); (D) Myxomavirus M11L:Bak complex [35] (PDB ID 2JBY); (E) African swine fever virus A179L:Bid complex [36] (PDB ID 5UA4); (F) Murine γ−herpesvirus 68 M11:Beclin-1 complex [37] (PDB ID 3BL2); (G) Vaccinia virus F1L:Bim complex [38] (PDB ID 4D2M); (H) Vaccinia virus A52 [39] (PDB ID 2VVW). [/fig] [table] Table 1: Pro-survival Bcl-2 proteins encoded by viruses. [/table]

Quality of life and pain multidimensional aspects in individuals with HTLV-1 Quality of lifeHTLV-1 a b s t r a c t HTLV-1 creates a chronic health condition that involves moderate to severe pain with a negative impact on quality of life (QoL). There is no consensus on which attitudes to pain are more related to the worsening of QoL in HTLV-1 infected patients. The aim of this study was to investigate the correlation between QoL and multidimensional aspects of pain in patients with HTLV-1. A cross-sectional study was conducted in Salvador, Bahia, Brazil. The study included individuals diagnosed with HTLV-1. The Short Form 36 Questionnaire was used to analyze QoL, and the Brief Pain Inventory was used to assess multidimensional aspects of pain. The mean pain intensity was 4.88 ± 3.06 on the visual pain scale, and the average impact on QoL corresponded to a loss of approximately 40%. Moderate to high correlations between pain intensity and all domains of QoL were observed and compared reaction attitudes for general activity, mood, ability to walk, ability to work, relationships, sleep, and ability to enjoy life (r > 0.40; p < 0.05). Moderate correlations were found between all domains of QoL, pain intensity, and reactive attitudes to pain. The greatest pain intensity impacts involved difficulty to walk and to work, and interpersonal relationships in the emotional aspect of QoL. (M.C. Macêdo). to 1.76% of the population. 4 Only 5% of patients with HTLV-1 develop symptoms associated with such retroviruses. It frequently manifests with spasticity, gait disturbances, http://dx. weakness and stiffness of the lower limbs, impaired dynamic balance, and pain. [bib_ref] Clinical pathophysiology of human T-lymphotropic virus-type 1-associated myelopathy/tropical spastic paraparesis, Yamano [/bib_ref] [bib_ref] Neurological outcomes analysis of HTLV-1 seropositive patients of the Interdisciplinary Research HTLV..., Reiss [/bib_ref] In Salvador, 84.3% of infected individuals experience pain, which is more than twice the prevalence in the general population (41.4%). [bib_ref] Pain is highly prevalent in individuals with tropical spastic paraparesis, Mendes [/bib_ref] [bib_ref] Chronic pain and gender in Salvador population, Sá [/bib_ref] The pain in these individuals mainly affects the lower back and legs, worsens with long periods in one position and physical effort, and presents daily. The pain, lack of bladder control, and changes in gait patterns lead to limitations that have a high impact on activities of daily living (ADLs), contributing to a reduced quality of life (QoL). [bib_ref] Disability profile of patients with HTLV-I associated myelopathy/tropical spastic paraparesis using the..., Franzoi [/bib_ref] [bib_ref] Quality of life in patients with HTLV-I associated myelopathy/tropical spastic paraparesis, Martins [/bib_ref] Because HTLV-1 is a neglected health condition, aspects such as the impact on the QoL of infected individuals have been poorly explored, especially using specific tools for delineation of multidimensional aspects of pain and assessment of different domains of QoL. Studies on QoL can guide education and health promotion programs focusing on self-care and the functional independence of individuals affected by HTLV-1. [bib_ref] Characteristics of chronic pain and its impact on quality of life of..., Neto [/bib_ref] As the impact of different sensory and reactive characteristics of pain on QoL of infected individuals remain unclear, this study aimed to correlate the multidimensional aspects of pain and levels of QoL for individuals with HTLV-1. A cross-sectional observational study was conducted in the Advanced Physical Therapy Clinic (CAFis) of the Bahia School of Medicine and Public Health (EBMSP) in Salvador, Bahia, Brazil. The study included men and women aged 18 and older who were diagnosed with HTLV-1 according to criteria defined by the World Health Organization, classified as defined and probable for HAM/TSP, and able to remain upright without support. Patients additional disorders such as arthritis, orthopedic impairments, and other neurological disorders, or who had difficulty understanding the questionnaires were excluded. The clinical team responsible for the Integrative Care and Research Center selected the participants. Given the scarcity of studies in the literature that served as the basis for this study, the sample size was calculated based on a correlation coefficient of 0.5. A sample of 43 subjects was estimated with a power of 80% and an alpha of 0.05. This sample was increased by 20% due to the large biological variability in this condition. Data were collected in a standardized manner after the participants signed an Informed Consent form. The project was approved by the Ethics Committee in Research of the Bahia School of Medicine and Public Health (registration number CAAE 13568213.8.0000.5544). Social class was defined according to criteria of the Brazilian Association of Survey Companies (ABEP), which divides the population into six socioeconomic strata namely A, B1 (high), B2, C1 (middle), C2, D, and E (low). The Brief Pain Inventory (BPI) was used to assess pain intensity and interference. [bib_ref] Validation of the Brief Pain Inventory for chronic nonmalignant pain, Tan [/bib_ref] To assess pain intensity we used two of the four sensory questions of this instrument (maximum and average pain intensity in the last 24 h). To assess pain interference we used the seven items of the pain interference section of the BPI (general activity, mood, walking ability, normal work, relations, sleep, and enjoyment of life). The Short Form 36 (SF-36) Questionnaire was used to assess quality of life according to the following domains: functional capacity, physical aspect, pain, emotional aspect, general health, social aspect, mental health, and vitality. [bib_ref] Tradução para a língua portuguesa e validação do questionário genérico de avaliação..., Ciconelli [/bib_ref] After assuring data had normal distribution, the Spearman test was used to assess the correlation of each of the two sensory items of the BPI with the seven interference aspects of the BPI and to each domain of the SF-36. Numerical variables were expressed as mean and standard deviation. All analyses were performed using the Statistical Package for Social Sciences (SPSS) version 14.0 for Windows (Chicago, USA). Of the 56 participants, 66.1% were female. The mean age was 52 ± 11.1 years old, and the body mass index (BMI) was 24.5 ± 4.5 kg/m 2 . There was a higher proportion of nonwhite individuals (92.9%) and individuals belonging to class C (51.8%). Of these, 42.9% used a device such as a cane or crutch for walking, and 26.8% made systematic use of muscle relaxants for pain relief. The mean disease duration was 10.7 ± 7.9 years. The mean most pronounced pain in the last 24 h was reported to be 4.88 ± 3.06 in intensity, while the mean pain score was reported to be 4.66 ± 3.75. The sites most affected by pain were the lumbar region, reported by 37 participants (66.1%); the knee, reported by 33 (58.9%); the leg, reported by 18 (32.1%); and the feet, reported by 26 (48.4%). [fig_ref] Table 1 -: Correlation between reaction and sensory aspects of pain, according to the Brief... [/fig_ref] shows how sensory aspects of pain impacted interference aspects. Moderate to high correlations were observed between the level of pain intensity in the last 24 h and the average pain in the last 24 h with all areas of pain interference. A correlation analysis between the most pronounced level of pain and average pain in the last 24 h with the domains of the SF-36 showed that a higher intensity of pain corresponded to worse scores in different domains of the QoL . shows correlations between the SF-36 domains and the BPI interference of pain items. All but the social aspects of the SF-36 domains were negatively correlated with pain interference. This study confirmed the hypothesis that pain appears to be moderate in this population; with increased pain intensity, Spearman correlation test, alpha 5%. [bib_ref] Pain assessment through the brief pain inventory in a low socio-economic level..., Santos [/bib_ref] investigated the pain profile in 191 subjects of low socioeconomic status in the same city, using the BPI. They found that pain was of moderate intensity in 46.8%, most often in the knees (46.1%) and the lumbar spine (42.4%). These findings correspond to results of the present study. It is possible that low socioeconomic status increases the frequency of pain in the lower back and knees because of environmental conditions, labor, and transportation, regardless of the health conditions generated by HTLV-1. [bib_ref] HTLV-I in the general population of Salvador, Brazil: a city with African..., Dourado [/bib_ref] [bib_ref] Pain is highly prevalent in individuals with tropical spastic paraparesis, Mendes [/bib_ref] The moderate inverse correlation between mean pain intensity and the ability to work can be explained by the low socioeconomic status of our sample. This socioeconomic condition is associated with physically demanding jobs and menial work, which generally is limited by physical dysfunction associated with high intensity pain. [bib_ref] Human T-cell lymphotropic virus type 1-associated myelopathy in São Paulo, Brazil. Epidemiologic..., Milagres [/bib_ref] Ratifying this negative influence of pain on the performance of menial labor activities, Herrero et al. [bib_ref] Influencia de variables sociodemográficas en la respuesta terapéutica y la productividad laboral, Herrero [/bib_ref] demonstrated improved productivity of workers in manual labor after drug therapy for pain relief. ## Table 2 -correlation between domains of quality of life (sf-36) with the intensity of pain and reaction to pain attitudes front (ibd). The impact of pain in the functional capacity of the SF-36 and the correlation between the most pronounced pain level and general activity of the BPI were moderate. Perhaps these findings are justified by characteristics of the sample, which largely consisted of people who were not wheelchair-bound or used assistive devices, and of people who needed only one crutch. In addition, we included only patients who had been diagnosed with or were probable of HAM/TSP and who were likely able to stand by themselves. Martins et al. [bib_ref] Quality of life in patients with HTLV-I associated myelopathy/tropical spastic paraparesis, Martins [/bib_ref] found a high impact of the disease in functional capacity and observed lower scores in wheelchairbound patients compared to the subgroup of those who still could walk. It is possible that those patients who are not wheelchair-bound feel able to implement most daily activities, even with compensation of movement. Furthermore, myelopathy does not affect function of the upper limbs, which enables patients to participate in many tasks. Reinforcing the idea that locomotion itself is not the main factor interfering with functional capacity in HTLV-1, Franzoi et al. [bib_ref] Disability profile of patients with HTLV-I associated myelopathy/tropical spastic paraparesis using the..., Franzoi [/bib_ref] showed that the loss of bladder control is worse than loss of locomotion. The physical aspect domain showed a moderately negative impact from pain. This domain assesses how daily activities and work are affected in relation to the intensity of the activity, the number of tasks, and the level of effort. This population presents reduced muscle and cardiorespiratory endurance, and thus susceptible to an increased sedentary lifestyle. The degree of fatigue increases in individuals who remain in a bent posture, such as people with HAM/TSP, and those with spinal cord injury because of muscle weakness. [bib_ref] Postural profile of individuals with HAM/TSP, Macêdo [/bib_ref] [bib_ref] Clinical correlates of fatigue in spinal cord injury, Fawkes-Kirby [/bib_ref] [bib_ref] Fatigue and spinal cord injury: a qualitative analysis, Hammell [/bib_ref] Severe fatigue associated with spinal cord injury can have negative implications in QoL and activities of daily living. 9, [bib_ref] Fatigue and spinal cord injury: a qualitative analysis, Hammell [/bib_ref] We observed a weak correlation between the level of pain and the social aspect domain. Although the assessment tools used to measure the social aspect domain have certain weaknesses, this result might be related to a sample consisting of lower social class of individuals who often have higher levels of resilience. [bib_ref] Quality of life issues in individuals with spinal cord injury, Tate [/bib_ref] In this study we did not assess the subgroups of patients with neuropathic pain, nociceptive pain, and both types of pain separately. As neuropathic pain is often associated with more severe impact in QoL, it is possible that our results cannot be extended to all patients with HTLV-1. We also did not identify patients with urinary and sexual dysfunction, which are also leading causes of decreased QoL in this population. Future studies should address these points in longitudinal studies to collect more consistent data about the impact of those aspects in the QoL of people infected by HTLV-1. The SF-36 and BPI, although generic assessment tools, were shown to be adequate in evaluating the issues faced by patients with HTLV-1. The quality of life and reactive attitudes to pain were inversely correlated. The greatest impacts of pain intensity involved difficulties with walking and working. In addition, difficulties in interpersonal relationships undermined the emotional aspect of quality of life. ## Conflicts of interest The authors declare no conflicts of interest. [table] Table 1 -: Correlation between reaction and sensory aspects of pain, according to the Brief Pain Inventory. [/table]

The Chemical Variability, Nutraceutical Value, and Food-Industry and Cosmetic Applications of Citrus Plants: A Critical Review Citation: Ben Hsouna, A.; Sadaka, C.; Generalić Mekinić, I.; Garzoli, S.; Švarc-Gajić, J.; Rodrigues, F.; Morais, S.; Moreira, M.M.; Ferreira, E.; Spigno, G.; et al. The Chemical Variability, Nutraceutical Value, and Food-Industry and Cosmetic Applications of Citrus Plants: A Critical Review.Abstract: Citrus fruits occupy an important position in the context of the fruit trade, considering that both fresh fruits and processed products are produced on a large scale. Citrus fruits are recognized as an essential component of the human diet, thanks to their high content of beneficial nutrients such as vitamins, minerals, terpenes, flavonoids, coumarins and dietary fibers. Among these, a wide range of positive biological activities are attributed to terpenes and flavonoids derivatives. In this review, a list of bibliographic reports (from 2015 onwards) on the phytochemical composition, beneficial effects and potential applications of citrus fruits and their by-products is systematically summarized. In detail, information regarding the nutraceutical and medicinal value closely linked to the presence of numerous bioactive metabolites and their growing use in the food industry and food packaging, also considering any technological strategies such as encapsulation to guarantee their stability over time, were evaluated. In addition, since citrus fruit, as well as its by-products, are interesting alternatives for the reformulation of natural cosmetic products, the sector of the cosmetic industry is also explored. More in-depth knowledge of the latest information in this field will contribute to future conscious use of citrus fruits. # Introduction The genus Citrus belongs to the angiosperm subfamily Aurantioideae of the family Rutaceae. Rutaceae are a family of flowering plants with about 160 genera. The most economically important members of the family are Citrus, which include the orange (Citrus sinensis), lemon (Citrus limon), grapefruit (Citrus paradisi) and lime (mainly Citrus aurantifolia). In general, Citrus represents one of the most important fruit crops in the world, and is grown mainly in tropical and subtropical climates of the world [bib_ref] Citrus origin, diffusion, and economic importance, Zhong [/bib_ref]. There are hundreds of different citrus cultivars. Some varieties were discovered accidentally from populations, Citrus production is one of the world's leading sectors of agricultural fruit production, with species such as oranges, lemons, limes, grapefruit, and tangerines having the greatest industrial importance. [fig_ref] Table 1: Cont. [/fig_ref] provides a literature overview of the recent studies on the chemical composition of these major citrus cultivars, their plant parts, products and processing by-products. Coumarin, Ascorbic acid, Citric acid, Linoleic acid, Limonoid, Malic acid, D-Limonene, β-Carotene. [bib_ref] Characterization of Antioxidant Property and Chemical Composition of Lemon (Citrus lemon L.)..., Aziz [/bib_ref] Peel (Indonesia) Ethanolic, n-hexane, ethyl acetate extracts Gallic acid (23.9 mg/L), 1,2-dihydroxybenzene (23.0 mg/L), Total phenolics (9-15.2 µg GAE/g), Flavonoids (25-29 µg QE/g).Peel EO, British Pharmacopoeia D-Limonene (82.9%), β-Phellandrene (1.6%), β-Pinene (1.5%), γ-Terpinene (9.9%), β-Cymene (1.3%), α-Limonene diepoxide (1.2%). [bib_ref] Hristova-Ivanova, Y. Chemical composition, antioxidant and antimicrobial activity of essential oils from..., Denkova-Kostova [/bib_ref] Peel (Ethiopia) EO, Clevenger-type apparatus Limonene (49.7%), β-Pinene (17.1%), γ-Terpinene (7.5%), o-Cymene (2.2%), β-Bisabolene (2.4%), β-Caryophyllene (1.5%). [bib_ref] Extraction of essential oil from lemon and orange peel by Clevenger apparatus:Comparative..., Zeleke [/bib_ref] Peel (Nigeria) Osbeck EO, Clevenger-type apparatus Limonene (85.9%), Sabinene (3.9%), Myrcene (3.1%), Linalool (0.5%). [bib_ref] Chemical composition of Citrus limon (L.) Osbeck growing in Southwestern Nigeria: Essential..., Owolabi [/bib_ref] Peel (Iran) EO, Clevenger-type apparatus Limonene (61.4%), β-Pinene (13.1%), γ-Terpinene (11.3%), α-Pinene (2.4%), Sabinene (2.3%), Myrcene (1.6%), Geranial (1.5%), Neral (1.1%). [bib_ref] Chemical composition and radical scavenging activity of Citrus limon peel essential oil, Ghoorchibeigi [/bib_ref] Peel (Algeria) EO, Cold-pressing Limonene (64.8%), γ-Terpinene (11.7%), β-Pinene (11.2%), α-Pinene (1.9%), β-Myrcene (1.7%), Geranial (1.7%), β-Bisabolen (1.0%). [bib_ref] Evaluation of the quality and composition of lemon (Citrus limon) peel essential..., Benoudjit [/bib_ref] Peel (Algeria) Eureka EO, Clevenger-type apparatus Limonene (61.3%), β-Pinene (9.7%), α-Citral (4.2%), γ-Terpinene (3.8%), cis-Citral (2.4%), β-Elemene (2.2%). [bib_ref] Chemical composition of Citrus limon (Eureka variety) essential oil and evaluation of..., Himed [/bib_ref] Peel (India) Burf EO, Clevenger-type apparatus Limonene (55.4%), Neral (10.4%), trans-Verbenol (6.4%), Decanal (3.3%), Ethyl cinnamate (2.2%), Ethyl p-methoxycinnamate (2.2%), cis-α-Bergamotene (1.6%), Geraniol (1.5%), trans-Carveol (1.3%), Nonanal (1.2%), Linalool (1.2%), α-Terpineol (1.1%). [bib_ref] Chemical composition of Citrus limon L. Burmf peel essential oil from North..., Paw [/bib_ref] Root (Cameroon) Methanolic extract Clausarin, Xanthyletin, Suberosin, E-suberenol, E-Methoxysuberenol, Thamnosmonin, Angelitriol, Hopey-hopin, Formlylumbelliferone, Atalantaflavone, Limonin, 1- [bib_ref] Polyphenols Effect on Circulating Lipids and Lipoproteins: From Biochemistry to Clinical Evidence, Cicero [/bib_ref] [bib_ref] Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (Citrus sinensis) peel, Li [/bib_ref] [bib_ref] By-products from different citrus processes as a source of customized functional fibres, Marín [/bib_ref] [bib_ref] Valorisation of citrus processing waste: A review, Zema [/bib_ref] [bib_ref] Citrus by-products as ruminant feeds: A review, Bampidis [/bib_ref] [bib_ref] Citrus waste derived nutra-/pharmaceuticals for health benefits: Current trends and future perspectives, Mahato [/bib_ref] [bib_ref] Current applications of citrus fruit processing waste: A scientific outlook, Suri [/bib_ref] abeo-7α-acetoxy-10 β-hydroxyisoobacunoic acid-3,10-lactone. [bib_ref] Chemical composition and synergistic antimicrobial effects of a vegetatively propagated cameroonian lemon,..., Nsangou [/bib_ref] Root (Cameroon) EO, Hydrodistillation Hexadecanoic acid, methyl ester (39.3%), β-Bisabolene (10.1%), (E)-9-octadecenoic acid, methyl ester (9.3%), α-Santalene (8%), Elemol (6.2%), (E)-5-Octadecene (6.1%), 1-Octadecene (5.7%). [40] Limonene (39.7%), β-Pinene (25.4%), α-Terpineol (7.3%), Nerolidol (6.9%), Farnesol (4.3%), Linalyl acetate (3.0%), Geranyl acetate (3.0%), Linalool (2.2%), Neryl acetate (1.7%). [ [bib_ref] Chemical composition and synergistic antimicrobial effects of a vegetatively propagated cameroonian lemon,..., Nsangou [/bib_ref] Leaf (Cameroon) Osbeck Ethanolic, acetone, water extract Alkaloids (12.2%), Saponins (5.5%), Total phenolics (208-289 mg GAE/g), Total flavonoids (447-1053 mg QE/g). [bib_ref] Phytochemical content and antioxidant potential of leaf extracts of Citrus limon (L.)..., Ehiobu [/bib_ref] Leaf (Nigeria) Osbeck EO, Clevenger-type apparatus Limonene (31.5%), Sabinene (15.9%), Linalool (4.6%), (E)-β-Ocimene (3.9%), Myrcene (2.9%), α-Pinene (1.2%). [bib_ref] Chemical composition of Citrus limon (L.) Osbeck growing in Southwestern Nigeria: Essential..., Owolabi [/bib_ref] Leaf (Iran) EO, Clevenger-type apparatus Linalool (30.6%), Geraniol (15.9%), α-Terpineol (14.5%), Linalyl acetate (13.8%), Geranyl acetate (6.7%), β-Pinene (4.5%), Neryl acetate (4.2%). [bib_ref] Chemical composition and biological activities of lemon (Citrus limon) leaf essential oil, Hojjati [/bib_ref] Leaf (China) EO, Hydrodistillation Citronellal (75.3%), (R)-(+)-limonene (11.4%), Citronellol (6.7%), Citronellyl acetate (1.7%).Orange (C. sinensis L.) ## Pulp (spain) osbeck Juice, Mechanical squeezing, HS-SPME 1-Octanol , α-Pinene , β-Mircene (693-1340), Limonene (4310-5210), α-Terpinolene (54-106), Linalool , Valencene (698-1200)-units are ion peak areas divided by 10 6 . [49] ## Plant part (origin) variety ## Isolate/isolation method major chemical components reference Peel (Egypt) Water and ethanolic extracts Narirutin (29 µg/g), Naringin (27 µg/g), Hesperetin (17 µg/g), Hesperetin-7-O-rutinoside naringenin (15 µg/g), Quinic acid (13 µg/g), Datiscetin-3-O-rutinoside (11 µg/g), Sakuranetin (9 µg/g). [50] Peel (Nigeria) Navel Decoct Quercitrin (22.6 mg/g), Rutin (17.9 mg/g), Quercetin (14.0 mg/g), Catechin (12.5 mg/g), Epicatechin (6.1 mg/g), Luteolin (5.9 mg/g), Naringin (5.7 mg/g), Kaempferol (3.8 mg/g), Caffeic acid (3.6 mg/g). Fruit (Iran) Volatiles, Headspace single-drop microextraction D-Limonene, β-Myrcene, α-Pinene, β-Pinene. [80] [94] Flavedo, albedo, juice sacs (China) Baishi, Cuixiangtian, Guanxi Ethanolic extract, UAE Limonin, Nomilin, Limonin glucoside.Peel (Vietnam) EO, Clevenger-type apparatus, Co-extraction, using citric acid Limonene (87.9%), β-Pinene (2.7%), α-Phellandrene (1.3%), γ-Terpinene (0.5%), Linalool (0.26%), trans-β-ocimene (0.24%), trans-linalool oxide (0.18%), α-Terpinene (0.16%), cis-linalool oxide (0.12%), β-Citronellol (0.09%), trans-p-mentha-2,8-dien-1-ol (0.08%). Pectines (24%). EO-essential oil, MAHD-microwave-assisted hydrodistillation, MAE-microwave-assisted extraction, UAE-ultrasonic-assisted extraction, ASE-accelerated solvent extraction, SFE-solid-phase extraction, HS-SPME-headspace solid-phase microextraction, GAE-gallic-acid equivalents, QE-quercetin equivalents, CE-catechin equivalents, RE-rutin equivalents. ## Nutraceutical value of citrus The nutraceutical and medicinal value of citrus fruits is mainly attributed to their richness in dietary fiber and bioactive compounds such as citric acid, polyphenols, terpenoids, vitamins, minerals, and essential oils. Their diverse phytochemical profile gives them high potential for the prevention and treatment of various diseases. Although citrusplant extracts are widely used in the food, cosmetic, and nutraceutical industries, their pharmacological potential still needs to be confirmed by clinical trials. ## Dietary fibers Citrus fruits contain soluble (such as pectin, fructans, and psyllium) and insoluble (such as cellulose, hemicellulose and lignin) fibers in varying amount in peel, pulp, and juices [bib_ref] Citrus peel as a source of functional ingredient: A review, Rafiq [/bib_ref]. Soluble fibers can resist digestion in the small intestine, being fermented into a gel-like substance by the microflora in the large intestine, whereas insoluble fibers keep their structure intact, move through the gastrointestinal tract, and prevent constipation problems by softening the stool [bib_ref] The composition of dietary fibre-rich extrudates from oat affects bile acid binding..., Drzikova [/bib_ref]. The consumption of dietary fiber has been shown to regulate physiological functions, and is linked with a lower risk of cancer, cardiovascular diseases, diabetes, obesity, and gastrointestinal disorders [bib_ref] Health benefits of dietary fiber, Anderson [/bib_ref] [bib_ref] PEGylation: Posttranslational bioengineering of protein biotherapeutics, Veronese [/bib_ref]. Furthermore, in a randomized cross-over clinical trial in women, consumption of dietary-fiber concentrates from citrus fruits over a 4-week period was shown to reduce both total serum cholesterol levels and high-density lipoprotein cholesterol levels [bib_ref] Effects of dietary fiber and its components on metabolic health, Lattimer [/bib_ref]. Effective fiber intake can be obtained from natural dietary sources such as fruits, especially citrus fruits, vegetables, and some cereals [bib_ref] Effects of dietary fiber and its components on metabolic health, Lattimer [/bib_ref]. However, in certain groups of people, such as the elderly, those with low dietary intake and those suffering from gastrointestinal disorders, or diagnosed with Parkinson's disease, it is difficult to rely solely on dietary sources as the only source of fibers. Therefore, fiber supplements, including those containing fibers derived from citrus fruits, are used and available as over-the-counter capsules and powders. In addition, some citrus-fiber supplements can be added to low-fiber and fiber-free foods (e.g., commercial snacks, baked goods, and bread) without affecting the sensory properties of the food in question. ## Citric acid The sugar/acid ratio is the main indicator of the quality and ripeness (maturity) of citrus fruits. Citrus fruits contain mainly citric, malic, succinic, tartaric, and oxalic acids, with citric and malic acids being the most important constituents [bib_ref] Biochemical characterization of blood orange, sweet orange, lemon, bergamot and bitter orange, Moufida [/bib_ref]. The commercial use of citric acid has led to an increasing demand for it. Citric acid is widely used in the food, beverage, pharmaceutical, nutraceutical, and cosmetic industries as an acidulant, preservative, emulsifier, sequestrant, flavoring and buffering agent [bib_ref] Citric acid: Emerging applications of key biotechnology industrial product, Ciriminna [/bib_ref]. Citric acid can be produced through fermentation from yeast and molds, while natural citric acid is obtained from citrus fruits [bib_ref] Citric acid production patent review, Anastassiadis [/bib_ref]. Since citric acid is abundant in citrus fruits, especially in C. aurantium from bitter oranges, it can be used as a commercial substitute source for citric-acid production [bib_ref] Biochemical characterization of blood orange, sweet orange, lemon, bergamot and bitter orange, Moufida [/bib_ref]. ## Polyphenolics: flavonoids Polyphenolics are the most abundant bioactive constituents of citrus fruits, but their concentration varies among citrus species, and this variation is also influenced by environmental conditions [bib_ref] Citrus Polyphenols in Brain Health and Disease: Current Perspectives, Pontifex [/bib_ref]. Polyphenolics can be divided into diferuloylmethanes, stilbenes, flavonoids, phenolic acids, and tannins [bib_ref] Dietary flavonoids: Bioavailability, metabolic effects, and safety, Ross [/bib_ref]. The most important class of polyphenolics is flavonoids, which are divided into several subgroups, including chalcones, flavones, flavanones, flavonols, and isoflavones [bib_ref] Flavonoids: An overview, Panche [/bib_ref]. These subgroups have different dietary sources. Citrus fruits such as oranges, bergamots, lemons, and grapefruit are an important source of flavanones [bib_ref] Flavonoids: An overview, Panche [/bib_ref]. Like most monomeric flavonoids in nature, citrus flavanones occur as glycosides bound to various sugars. Flavonoid glycosides are distributed in different parts of citrus, but the largest amount is found in the solid parts: flavedo, albedo, and membranes [bib_ref] Bioavailability and metabolism of citrus fruit beverage flavanones in humans, Tomás-Navarro [/bib_ref]. It is well-known that phenolic acids and flavonoids are great antioxidants. Recently, the use of polyphenolics as natural antioxidants in foods to prevent lipid oxidation and increase the nutritional value of foods has attracted great interest [bib_ref] Citrus Polyphenols in Brain Health and Disease: Current Perspectives, Pontifex [/bib_ref]. In addition to their antioxidant potential, numerous studies suggest that citrus polyphenolics have antiviral, antimicrobial, antiallergenic, anti-ageing, anticarcinogenic, antidiabetic, cardioprotective and neuroprotective potential [bib_ref] History, Global Distribution, and Nutritional Importance of Citrus fruits, Liu [/bib_ref] [bib_ref] Naringin inhibits matrix metalloproteinase-9 expression and AKT phosphorylation in tumor necrosis factor-alpha-induced..., Lee [/bib_ref] [bib_ref] Bioavailability and metabolism of citrus fruit beverage flavanones in humans, Tomás-Navarro [/bib_ref] [bib_ref] Anti-inflammatory effects of flavonoids: Genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and..., Hamalainen [/bib_ref] [bib_ref] Antihypertensive effect of sweetie fruit in patients with stage I hypertension, Reshef [/bib_ref] [bib_ref] Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-alpha induced human aortic..., Gimenez-Bastida [/bib_ref] [bib_ref] Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in..., Raso [/bib_ref] [bib_ref] Suppression of nitric oxide synthase and the down-regulation of the activation of..., Tsai [/bib_ref]. Polyphenolics from citrus also have anti-inflammatory effects by interacting with the nucleotide binding sites of regulatory enzymes that play a key role in the cellular inflammatory process, including receptor binding and cellular activity during inflammation. These compounds have been shown to regulate signaling at the molecular level, giving them the potential to prevent and treat diseases such as cancer, diabetes, neurodegenerative and cardiovascular diseases, and ageing [bib_ref] Citrus Polyphenols in Brain Health and Disease: Current Perspectives, Pontifex [/bib_ref] [bib_ref] Anti-inflammatory effects of flavonoids: Genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and..., Hamalainen [/bib_ref] [bib_ref] Nutraceutical value of Citrus flavanones and their implications in cardiovascular disease, Testai [/bib_ref] [bib_ref] Fruit and vegetable consumption and risk of stroke: A meta-analysis of cohort..., Dauchet [/bib_ref] [bib_ref] Fruit and vegetable consumption and risk of coronary heart disease: A meta-analysis..., Dauchet [/bib_ref]. In addition, citrus polyphenolics have prebiotic potential, especially for metabolic diseases [bib_ref] Metabolomics Elucidates Dose-Dependent Molecular Beneficial Effects of Hesperidin Supplementation in Rats Fed..., Guirro [/bib_ref] , and improve blood glucose levels and lipid profiles. A small proportion of deglycosylated flavonoids are taken up by intestinal bacteria, while most of them are degraded to short-chain fatty acids, which supports microbiota homeostasis, among other beneficial effects on the human metabolism. In addition, citrus flavonoids appear to improve mitochondrial dysfunction and lead to a reduction in acetylcholinesterase activity [bib_ref] Naringenin protects AlCl 3 /D-galactose induced neurotoxicity in rat model of AD..., Haider [/bib_ref]. Hesperidin inhibits pleurisy, hesperetin limits increases in triacylglycerol and cholesterol levels in the liver, naringenin decreases plasma cholesterol levels and stimulates DNA repair after oxidative damage in human-prostate cancer cells, and naringin increases superoxide dismutase and catalase activity, thus playing an important role in regulating antioxidant capacity. Furthermore, naringin blocks H 2 O 2 -induced cytotoxicity and apoptosis and may affect H 2 O 2 -induced expression of apoptosis-associated genes or proteins [bib_ref] Bioavailability and metabolism of citrus fruit beverage flavanones in humans, Tomás-Navarro [/bib_ref]. The neuroprotective and neuromodulatory effects of citrus polyphenolics such as hesperetin, naringenin and their in-vivo metabolites can be explained by their ability to cross the blood-brain barrier [bib_ref] Polyphenols and human health: Prevention of disease and mechanisms of action, Vauzour [/bib_ref]. Given the numerous examples of their health-promoting effects in the scientific literature, the citrus polyphenolics are valued for their considerable nutraceutical value [bib_ref] Nutraceutical value of Citrus flavanones and their implications in cardiovascular disease, Testai [/bib_ref] , and their use represents a potentially useful approach for the prevention and treatment of a wide range of diseases. Although some clinical and preclinical studies have been conducted, much experimental and human research data are still needed to apply citrus polyphenolics in human medicine. One of the main problems of flavonoids that affect their nutraceutical efficacy is their low bioavailability. This is due to their solubility in citrus juice [bib_ref] Nutraceutical value of Citrus flavanones and their implications in cardiovascular disease, Testai [/bib_ref] , differences in preparation techniques (fresh/natural vs. preserved/commercial) [bib_ref] Pharmacokinetics of flavanone glycosides after ingestion of single doses of fresh-squeezed orange..., Silveira [/bib_ref] , flavonoid metabolism and the gut microbiota responsible for this metabolism [bib_ref] Nutraceutical value of Citrus flavanones and their implications in cardiovascular disease, Testai [/bib_ref]. To increase their bioavailability, some citrus products, such as fruit juice, are fortified with more enzymatically stable citrus flavanones [bib_ref] Nutraceutical value of Citrus flavanones and their implications in cardiovascular disease, Testai [/bib_ref]. ## Terpenoids: carotenoids The major terpenoids in citrus fruits are carotenoids and limonoids, which are mainly found in peel and pulp. The color of citrus fruits is primarily due to their carotenoid pigments, which are tetraterpenes which exhibit yellow, orange, and red colors [bib_ref] Towards a better understanding of carotenoid metabolism in animals, Von Lintig [/bib_ref]. On the other hand, limonoids, which are oxygenated terpenoids, impart a distinctively bitter taste to the fruit. These terpenoids affect the color and taste of citrus species, which, in turn, influences the consumer acceptance. Due to their antioxidant activity, carotenoids have the potential to reduce the risk of cardiovascular disease, eye disease, as well as cancer, including skin cancer [bib_ref] Potential role of carotenoids as antioxidants in human health and disease, Fiedor [/bib_ref]. Several randomized controlled trials have investigated the effect of beta-carotene supplementation on cancer incidence, concluding that betacarotene supplementation is not recommended as it has no beneficial effect on cancer prevention [bib_ref] Beta-carotene supplementation and cancer risk: A systematic review and metaanalysis of randomized..., Druesne-Pecollo [/bib_ref]. While nutritional supplementation with carotenoids to prevent heart and eye disease is commercially available, further in-depth studies are needed to define a preventive and therapeutic strategy. Citrus carotenoids, especially extracts of C. reticulata, also exhibit antimicrobial activity. Nevertheless, the antimicrobial potential of citrus carotenoids needs further validation by in-vivo and clinical studies. ## Vitamins Citrus fruits are known to be rich in vitamin C, which is mainly contained in their peel. It has also been shown that citrus fruits contain significant amounts of vitamin A and B complex [bib_ref] 100% citrus juice: Nutritional contribution, dietary benefits, and association with anthropometric measures, Rampersaud [/bib_ref]. Some of the commercially available vitamin-C supplements and skin-care cosmetic products (such as anti-ageing creams and serums) are derived from citrus fruits. ## Minerals Calcium, potassium, sodium, magnesium, iron, copper, manganese and zinc have been reported to be present in citrus [bib_ref] 100% citrus juice: Nutritional contribution, dietary benefits, and association with anthropometric measures, Rampersaud [/bib_ref]. However, to the best of our knowledge, there is no commercially available nutraceutical or pharmaceutical containing any of the above minerals derived from citrus fruits. ## Essential oils Essential oils are isolates of plant volatile metabolites containing various lipophilic substances. They are oily liquids with a strong odor, usually lightly colored and insoluble in water and have lower density than water. Essential oils are usually defined as extracts obtained by the distillation of different plant parts; however, in the case of citrus oils, they can be obtained by cold pressing from peel. Although conventional methods such as hydrodistillation, steam distillation, and cold pressing are most commonly used for the isolation of EOs [fig_ref] Table 1: Cont. [/fig_ref] , novel techniques such as supercritical fluid extraction, microwave-assisted hydro-distillation and hydro-diffusion, etc., have also been used recently. The applied EOs isolation method has a great influence on the chemical profile of the EO, but factors such as species, harvest time and location, cultivation method, fruit storage conditions, climate, soil type, plant organs used, and plant vegetative cycle stage also play a role [bib_ref] Current and potential use of Citrus essential oils, Palazzolo [/bib_ref] [bib_ref] Citrus essential oils: Extraction, authentication and application in food preservation, Mahato [/bib_ref]. From the data presented in [fig_ref] Table 1: Cont. [/fig_ref] , it can be seen that the scientific literature usually reports the chemistry of essential oils of different citrus species (fruits, leaves, roots, and flowers, etc.) obtained by hydro-distillation using a Clevenger-type apparatus. The yield of citrus-peel EO ranges from 0.5 to 5.0% (w/v) [bib_ref] Current and potential use of Citrus essential oils, Palazzolo [/bib_ref] and they are complex mixtures of chemical compounds from different classes, generally divided into the volatile fraction (85-99%) and the non-volatile compounds (2-6%, long-chain hydrocarbons, fatty acids, waxes, carotenoids, sterols, and coumarins, etc.). The volatile fraction is dominant in citrus EOs and consists mainly of monoterpenes, sesquiterpenes and their oxygenated derivatives, but other compounds such as aliphatic alcohols, aldehydes and esters can also be found [bib_ref] Citrus essential oils: Extraction, authentication and application in food preservation, Mahato [/bib_ref]. [fig_ref] Table 1: Cont. [/fig_ref] lists the main compounds detected in EOs of major citrus species from recent studies, and it can be seen that limonene, an aliphatic hydrocarbon (cyclic monoterpene), is the main component in citrus EOs. However, its content varied from sample to sample due to the influence of various parameters discussed above. Its content in lemon peels ranged from 40 to 86%, in orange peels from 46 to 95%, in lime peels from 13 to 77%, in tangerine peels from 55 to 88%, in grapefruit peels from 86 to 93%, and in pomelo peels from 54 to 97%. This compound was also found in other Citrus plant parts in appreciable amounts. Among others, γ-terpinene, β-myrcene, pinene, ocimene, linalool, α-terpinene, and sabinene were detected in most samples. Citrus essential oils are generally considered safe and are known for their numerous beneficial biological effects, such as anesthetic, sedative, analgesic, antimicrobial and anti-inflammatory activities, and are, therefore, used in pharmaceuticals; however, in addition, they have also been used for food and beverage flavoring, food packaging, and in different cosmetic products [bib_ref] Valorization of pomelo (Citrus grandis Osbeck) peel: A review of current utilization,..., Tocmo [/bib_ref]. ## Citrus uses in the food industry Nowadays, consumers are more aware of the relationship between diet and health. In fact, the global consumption pattern has shifted toward foods that provide both nutritional value and health benefits. Therefore, the demand for functional foods has experienced an exponential growth in recent years [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref]. Citrus fruits are excellent sources of natural bioactive compounds with well-known health-promoting properties. As previously described, the main citrus phytochemicals include polyphenols (mainly flavonoids), carotenoids, vitamins, organic acids, dietary fiber, and essential oils, which have promising biological activities due to their anti-oxidative, anti-inflammatory, and anti-carcinogenic properties [bib_ref] Current applications of citrus fruit processing waste: A scientific outlook, Suri [/bib_ref]. These health benefits associated with citrus bioactive compounds are one of the main reasons the citrus-based food industry is expanding [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref]. The mentioned citrus phytochemicals are mostly found in citrus-fruit wastes, namely, citrus peels, seeds, pomace, and pulp [fig_ref] Table 1: Overview of the studies [/fig_ref] , which accounts for about 50 to 60% of the total weight of the fruit. Large amounts of processing waste are generated during the production of orange and other citrus juices, mainly the peel, cores, and segment membranes. OP is the main by-product, accounting for about half of the fruit mass. In fact, the citrus-processing industry generates more than 60 million tons of waste worldwide. In the USA alone, juice processing of oranges and grapefruits generates more than 5 million tons of citrus waste [bib_ref] Synthesis and characterization of Citrus limonum essential oil based nanoemulsion and its..., Kaur [/bib_ref] and 700,000 tons of OP annually [bib_ref] Phenols in Citrus Peel Byproducts. Concentrations of hydroxycinnamates and polymethoxylated flavones in..., Manthey [/bib_ref]. In India, about 2.15 million tons of citrus peels out of 6.28 million tons of citrus fruits are generated from citrus juice processing annually. ## Extraction of bioactive compounds for food applications In recent years, considerable attention has been paid to the extraction of bioactive compounds from citrus waste for further use as food additives, encapsulants, nanoparticles, prebiotics, or as a source of pectin, essential oils [fig_ref] Figure 2: By-products created as a result of industrial processing of citrus-fruit wastes [/fig_ref] , polyphenols, carotenoids, or dietary fibers [bib_ref] Technical and Economic feasibility of a stable yellow natural colorant production from..., Ciriminna [/bib_ref]. The extraction, isolation, and characterization of the mentioned bioactive components from citrus waste represent crucial steps in their recovery. Appropriate extraction conditions must be tested, optimized, and used to ensure that no degradation or loss of the bioactive compounds occurs. In fact, the solvent selection and extraction technique are the most crucial steps to maximize the yield of bioactive compounds [bib_ref] Current applications of citrus fruit processing waste: A scientific outlook, Suri [/bib_ref] [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref]. Recently, When OP goes unused, they become waste and are a possible cause of environmental pollution. Therefore, it is urgent to investigate and find solutions to convert these wastes into economically valuable products. OP were traditionally dried and marketed as livestock feed or used in the food industry [bib_ref] Enzymatic Hydration of (4R)-(+)-Limonene to (4R)-(+)-α-Terpineol, Cadwallader [/bib_ref] [fig_ref] Figure 2: By-products created as a result of industrial processing of citrus-fruit wastes [/fig_ref]. They have been used to produce animal feed [bib_ref] Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (Citrus sinensis) peel, Li [/bib_ref] [bib_ref] Citrus waste recovery: A new environmentally friendly procedure to obtain animal feed, Tripodo [/bib_ref] , single-cell proteins, fibers, pectinase/cellulose [bib_ref] Hydrolysis of grapefruit peel waste with cellulase and pectinase enzymes, Wilkins [/bib_ref] , immobilization support [bib_ref] Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for..., Plessas [/bib_ref] , ethanol, and bio-sorbents for heavy-metal removal [bib_ref] Pectin-rich fruit wastes as biosorbents for heavy metal removal: Equilibrium and kinetics, Schiewer [/bib_ref]. Currently, the extraction of phenolic compounds from OP has attracted considerable scientific interest for use as natural antioxidants, especially in foods to prevent rancidity and the oxidation of lipids [bib_ref] Radical scavenging activity of various extracts and fractions of sweet orange peel..., Anagnostopoulou [/bib_ref] [bib_ref] An industrial approach in the search of natural antioxidants from vegetable and..., Peschel [/bib_ref] [bib_ref] Synthesis of potential biologically active 1,2-Benzothiazin-3-ylquinazolin-4(3H)-ones, Zia-Ur-Rehman [/bib_ref]. ## Food industrial applications ## Functional food ingredient In recent years, consumer attention is moving towards consuming dietary-fiber-enriched foods. Considering that citrus fruits are excellent sources of antioxidants and dietary fibers, their inclusion in daily consumed foods such as baked goods, meat, and dairy products has become a hot topic of scientific research. Several authors have reported the potential of using citrus powder or flour in bakery and confectionery products as a functional ingredient [bib_ref] Utilization of citrus plant waste (peel) for the development of food product, Iftikhar [/bib_ref]. For example, Caggia et al.developed a low-fat bakery product (brioches) fortified with proportions (30, 50 and 70%) of debittered orange fibers, which improved the stability and nutritional properties of the developed product. The results obtained demonstrated that the addition of 50% debittered orange fiber resulted in a fat content of 4.5% in the products, in comparison to the 10% fat in the control sample. Furthermore, due to the antimicrobial properties of these natural extracts, food product safety was also ensured. In another study, the positive effects of citrus albedo addition on bread shelf life due to the high pectin and fiber content was confirmed. The authors demonstrated that the partial replacement of wheat flour with dried fruit-peel powder provided a higher ability to bind large amounts of water. However, changes in the mechanism of staling, as well as structure modification as a consequence of fortification, should be further investigated. Iftikhar et al. [bib_ref] Utilization of citrus plant waste (peel) for the development of food product, Iftikhar [/bib_ref] also demonstrated that Citrus ## Extraction of bioactive compounds for food applications In recent years, considerable attention has been paid to the extraction of bioactive compounds from citrus waste for further use as food additives, encapsulants, nanoparticles, prebiotics, or as a source of pectin, essential oils [fig_ref] Figure 2: By-products created as a result of industrial processing of citrus-fruit wastes [/fig_ref] , polyphenols, carotenoids, or dietary fibers [bib_ref] Technical and Economic feasibility of a stable yellow natural colorant production from..., Ciriminna [/bib_ref]. The extraction, isolation, and characterization of the mentioned bioactive components from citrus waste represent crucial steps in their recovery. Appropriate extraction conditions must be tested, optimized, and used to ensure that no degradation or loss of the bioactive compounds occurs. In fact, the solvent selection and extraction technique are the most crucial steps to maximize the yield of bioactive compounds [bib_ref] Current applications of citrus fruit processing waste: A scientific outlook, Suri [/bib_ref] [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref]. Recently, several authors have focused their efforts on describing, in detail, the extraction techniques as well as the conditions employed to recover the different bioactive compounds from citrus wastes to improve the extraction yield [bib_ref] Citrus pectins: Structural properties, extraction methods, modifications and applications in food systems-A..., Singhal [/bib_ref] [bib_ref] Valorization and extraction optimization of Citrus seeds for food and functional food..., Zayed [/bib_ref]. Traditionally, the most commonly applied techniques include maceration, hot-water extraction, solvent extraction, and alkaline extraction [bib_ref] Technical and Economic feasibility of a stable yellow natural colorant production from..., Ciriminna [/bib_ref] [bib_ref] Valorization and extraction optimization of Citrus seeds for food and functional food..., Zayed [/bib_ref]. Dar et al. [bib_ref] Evaluation of Different Techniques for Extraction of Antioxidants as Bioactive Compounds from..., Dar [/bib_ref] employed a maceration method to recover bioactive compounds from citrus peels, testing different solvent compositions (ethanol, water and 50% aqueous ethanol) for 8 h at room temperature. These authors reported that the extraction yield was higher with aqueous ethanol, followed by ethanol and water (29.28, 23.65 and 6.53%, respectively). In another study [bib_ref] Citrus fruits by-products as sources of bioactive compounds with antioxidant potential, Al-Juhaimi [/bib_ref] , phenolic compounds, ascorbic acid and the free radical scavenging activity of peel and pulp from Orlando orange, Kinnow mandarin and Eureka lemon fruits were assessed applying a conventional extraction method (80% aqueous ethanol at 70 - C in a shaking water bath for 3 h). These authors reported different levels of compounds in the matrices studied, demonstrating the major advantage from conventional extraction methods versus the most recent ones, namely, their versatile application in different matrices as well as their easy operation and low cost of application. The work developed by Gómez-Mejía and co-workers [bib_ref] Citrus peels waste as a source of value-added compounds: Extraction and quantification..., Gómez-Mejía [/bib_ref] is also in-line with these achievements. These authors tested the extraction of bioactive polyphenols from different citrus peels by employing magnetic stirring, evaluating the influence of extraction time (X1; 10-15 min), ethanol-water ratio (X2; from 20:80 to 40:60 v/v), and extraction temperature (X3; from 62 to 90 - C). Depending on the type of citrus peel, different extraction conditions were validated, but these authors concluded that the proposed conventional extraction method uses a lower amount of ethanol, reduced extraction times and a lower sample-to-solvent ratio than the novel techniques. Nonetheless, significant efforts have been made to replace these traditional techniques with green extraction methods, such as ultrasound-assisted extraction (UAE), subcritical water extraction (SWE), supercritical fluid extraction, microwave-assisted extraction (MAE), and enzyme-assisted extraction, not only to improve the extraction efficiency of bioactive compounds from citrus waste but also to overcome some of the most common drawbacks of traditional extraction techniques. Šafranko et al.investigated a two-step green extraction technique to recover bioactive compounds from mandarin peel (Citrus unshiu Marc. Var. Kuno). Firstly, these authors employed a supercritical CO2 (SC-CO2) extraction to recover volatile compounds, reporting limonene as the dominant component (30.65% at 300 bar), followed by farnesene, linoleic and hexadecanoic acids. Afterwards, the residue from SC-CO2 treatment was subjected to different SWE conditions to obtain bioflavonoids. Hesperidin (0.16-15.07 mg/g) was the most abundant flavanone in mandarin peel, followed by narirutin, and rutin. However, these authors reported that for extraction temperatures higher than 160 - C, the possible formation of undesirable compounds, such as chlorogenic acid and 5hidroxymethilfurfural, can represent a limitation for the large-scale exploitation of the SWE technique. Another environmentally friendly extraction technique widely used is UAE. Ordóñez-Santos et al.employed UAE for the recovery of carotenoid compounds from mandarin epicarp to be further used as a natural colorant in bakery products. These authors reported a total carotenoid amount of 140.70 ± 2.66 mg β-carotene/100 g of dry sample for an extraction performed at 60 - C for 60 min. Montero-Calderon et al. [bib_ref] Green solvents and ultrasound-assisted extraction of bioactive orange (Citrus sinensis) peel compounds, Montero-Calderon [/bib_ref] also employed a UAE technique to extract bioactive compounds from orange (Citrus sinensis) peel. At the optimal UAE conditions (50% aqueous ethanol, 30 min, 400 W of power), a total carotenoid concentration of 0.63 mg ß-carotene/100 g, vitamin C concentration of 53.78 mg AA/100 g, a total phenolic content of 105.96 mg GAE/100 g, and a hesperidin maximum concentration of 113.03 ± 0.08 mg/100 g were obtained. Despite the advances in the recovery of bioactive compounds from citrus matrices, most of the applied environmentally friendly extraction methods still cause concerns about the health and safety of the produced bioactive extracts, and the possible degradation and/or formation of undesirable compounds due to high temperatures [bib_ref] Valorization and extraction optimization of Citrus seeds for food and functional food..., Zayed [/bib_ref]. Indeed, Benassi et al. [bib_ref] Assessing green methods for pectin extraction from waste orange peels, Benassi [/bib_ref] tested three different techniques, namely, conventional hot-water extraction, rapid solid-liquid dynamic (RSLD) and MAE, to recover pectin from waste orange peel. These authors concluded that the "hot-water" extraction assisted with citric acid was the most sustainable extraction route, ensuring higher extraction yield (21%) as well as a high quality of the extracted pectin (degree of esterification of 82.5%). This fact evidences the main advantage of the traditional extraction method-namely, its simplicity of operation and/or equipment-over the novel UAE, MAE or SWE, especially at an industrial scale. Therefore, researchers need to increase their efforts to find more sustainable, economical, and rapid techniques to recover the different bioactive compounds from citrus waste and to enable their safe incorporation into food products. ## Food industrial applications ## Functional food ingredient In recent years, consumer attention is moving towards consuming dietary-fiberenriched foods. Considering that citrus fruits are excellent sources of antioxidants and dietary fibers, their inclusion in daily consumed foods such as baked goods, meat, and dairy products has become a hot topic of scientific research. Several authors have reported the potential of using citrus powder or flour in bakery and confectionery products as a functional ingredient [bib_ref] Utilization of citrus plant waste (peel) for the development of food product, Iftikhar [/bib_ref]. For example, Caggia et al.developed a low-fat bakery product (brioches) fortified with proportions (30, 50 and 70%) of debittered orange fibers, which improved the stability and nutritional properties of the developed product. The results obtained demonstrated that the addition of 50% debittered orange fiber resulted in a fat content of 4.5% in the products, in comparison to the 10% fat in the control sample. Furthermore, due to the antimicrobial properties of these natural extracts, food product safety was also ensured. In another study, the positive effects of citrus albedo addition on bread shelf life due to the high pectin and fiber content was confirmed. The authors demonstrated that the partial replacement of wheat flour with dried fruit-peel powder provided a higher ability to bind large amounts of water. However, changes in the mechanism of staling, as well as structure modification as a consequence of fortification, should be further investigated. Iftikhar et al. [bib_ref] Utilization of citrus plant waste (peel) for the development of food product, Iftikhar [/bib_ref] also demonstrated that Citrus sinensis (sweet orange) peel can be used to enhance the nutritional and functional properties of cakes due to their fiber and fat content. These authors concluded that the mixture of wheat flour with up to 3% citrus-peel flour is suitable for the development of cake with acceptable sensory attributes. ## Food additive Food additives are responsible for the flavor, color, taste, and nutritional qualities of food products. In recent years, with the increasing consumption of organic foods, the replacement of synthetic food additives with natural ones represents a great advantage in the field of food-processing industry [bib_ref] Current applications of citrus fruit processing waste: A scientific outlook, Suri [/bib_ref]. In this sense, another reported application for the bioactive compounds with antioxidant activity recovered from citrus waste is their use as food additives, especially in the preparation of candied products for confectionery/baking industry [bib_ref] Synergistic effects of nutmeg and citrus peel extracts in imparting oxidative stability..., Nishad [/bib_ref] [bib_ref] Loera-Hernández, I. Orange peel dehydration and creation of new edible products, Espinosa-Garza [/bib_ref]. For example, Romero-Lopez et al. [bib_ref] Fiber concentrate from orange (Citrus sinensis L.) bagase: Characterization and application as..., Romero-Lopez [/bib_ref] prepared muffins enriched with different proportions of dietary-fiber-rich orange bagasse and reported that the prepared muffins (with 15% extract) had a high dietary fiber (15.3%) and low fat (15%) content compared to the control muffins. Furthermore, the addition of the dietary-fiber-rich orangebagasse extract to the muffin reduced the predicted glycemic index, and no difference in sensory evaluation was observed between the control muffin and the muffin prepared with dietary-fiber-rich orange-bagasse extract. These results are of the greatest interest because the addition of dietary-fiber-rich orange-bagasse extract to bakery products may be an alternative for people who require foods with low glycemic index. In another work, Ojha and Thapa [bib_ref] Quality evaluation of biscuit incorporated with mandarin peel powder, Ojha [/bib_ref] also prepared biscuits by replacing the wheat flour with mandarin-peel powder (3, 6, and 9%). They reported that biscuits formulated with 6% of mandarinpeel powder were comparable to the control biscuits with no substitution; the content of fiber, ash, ascorbic acid, carotenoids, polyphenol and antioxidant activity improved, and the reported values were 0.85%, 1.32%, 1.5 mg/100 g, 69 µg/g, 2150 µg gallic-acid equivalents/g and 24.5%, respectively. Regarding the application of citrus extracts as food additives in another type of product, the research from Nishad et al. [bib_ref] Synergistic effects of nutmeg and citrus peel extracts in imparting oxidative stability..., Nishad [/bib_ref] should be highlighted, which investigated the potential of using citrus-peel extracts in the maintenance of oxidative stability of meat balls during frozen storage. The authors demonstrated that the natural antioxidant extracts from citrus peels can control lipid oxidation in meat products, by inhibiting enzymatic reactions responsible for oxidative damage. Moreover, the addition of citrus extract had a positive effect on the color, flavor, and overall sensory properties of the meat balls, indicating that it can be used as a natural preservative for foods rich in fatty acids. Younis et al. [bib_ref] Mosambi peel powder incorporation in meat products: Effect on physicochemical properties and..., Younis [/bib_ref] also incorporated the mosambi peel, a by-product of the juice industry, in sausages and patties and reported an enhancement in fiber content as well as in fat and moisture content. In addition, the addition of up to 6% of mosambi-peel extract improved storage stability, demonstrating the potential of using citrus waste as a food additive in meat products. ## Food colorant The peels of citrus fruit are described as an excellent source of carotenoids. Not only do they impart color to fruits, these compounds also promote health benefits, which has attracted the attention of food industry as a solution to replace harmful synthetic colorants [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref] [bib_ref] Technical and Economic feasibility of a stable yellow natural colorant production from..., Ciriminna [/bib_ref]. Barman et al. [bib_ref] Development of β-carotene loaded nanoemulsion using the industrial waste of orange (Citrus..., Barman [/bib_ref] used orange-peel waste to extract β-carotene, which was used to develop a stable nanoemulsion to be further employed as a natural colorant in food products. These authors reported that the addition of the nanoemulsion to fruit juice significantly enhances its color, thus providing an alternative to the use of synthetic colorants. Ordóñez-Santos et al.optimized the process of ultrasoundassisted extraction of total carotenoids from the mandarin epicarp and demonstrated its potential to reduce the use of tartrazine in bakery products, such as cakes and bread, and the potential of its further use as a natural coloring additive. Ciriminna et al. [bib_ref] Pectin-rich fruit wastes as biosorbents for heavy metal removal: Equilibrium and kinetics, Schiewer [/bib_ref] also investigated the technical and economic possibilities of using lemon-peel waste to produce water-soluble yellow colorant limocitrol 3-O-6"-[3-hydroxyl-3-methylglutaryl)])-dglucopyranoside as a substitute for tartrazine. The authors demonstrated that this natural colorant can be easily obtained by simple solid-liquid extraction in aqueous ethanol or via hydrodynamic cavitation of lemon-peel waste in water. Moreover, the obtained results on the chemical and physical stability of this natural colorant open the possibility to explore the industrialization of this new bioeconomy production. Despite these promising results, further research is needed to overcome the main limitations, such as the high cost of using natural biocolorants in industrial food applications [bib_ref] Citrus peel as a renewable bioresource: Transforming waste to food additives, Wedamulla [/bib_ref]. ## Flavoring agent Synthetic flavors are still widely used in the food industry; however, the use of citrus essential oils as flavoring agents is gaining increasing attention. Essential oils, mostly recovered from citrus peel, are prominent sources of terpenoids, which are widely used as flavoring agents in foods, and also have antibacterial, antifungal, and insecticidal properties [bib_ref] Citrus essential oils (CEOs) and their applications in food: An overview, Bora [/bib_ref]. Most studies have focused on determining the volatile profiles of different citrus species [bib_ref] Comparative evaluation of quality properties and volatile profiles of lemon peels subjected..., Tekgül [/bib_ref] , and only few applications of citrus in the food industry as flavoring agents have been found. Bergamot oils, a rich source of linalool and linalyl acetate with promising flavor characteristics, have been used in some flour-based confectionery in recipes to replace bergamot peels. Recently, Matsuo et al. [bib_ref] Utilization and evaluation of Citrus natsudaidai peel waste as a source of..., Matsuo [/bib_ref] studied the effects of adding Citrus natsudaidai (CN) peel extracts to aqueous solutions and reported that the solutions flavored with CN extracts exhibited preferential odor over the commercial citrus-flavored beverages, which were classified in the same group as commercial citrus juices by the electronic nose test. In addition, the solutions flavored with CN extracts exhibited sourness, bitterness, and an orange-like taste, and the overall acceptability was not significantly different from commercial citrus-flavored beverages. The use of citrus essential oils in ice cream, marmalade, and jam-like food products has also been widely described by other authors [bib_ref] Comparative evaluation of quality properties and volatile profiles of lemon peels subjected..., Tekgül [/bib_ref]. ## Thickening agent As previously reported, citrus wastes, especially citrus peel, are an excellent source of pectin, which is extensively used in jams, jellies, marmalades, milk, and confectionery products due to their gelling and stabilizing properties [bib_ref] Citrus pectins: Structural properties, extraction methods, modifications and applications in food systems-A..., Singhal [/bib_ref]. Many studies have focused on finding more environmentally friendly extraction techniques to recover pectin from citrus peels [bib_ref] Assessing green methods for pectin extraction from waste orange peels, Benassi [/bib_ref] [bib_ref] Comparisons of process intensifying methods in the extraction of pectin from pomelo..., Liew [/bib_ref] , and very few papers reported results on its incorporation in food products. For example, Mann et al. [bib_ref] Development of Phytochemical Rich Ice Cream Incorporating Kinnow Peel, Mann [/bib_ref] reported the production of ice cream using frozen Kinnow peel; both unblanched and blanched, at three levels (1, 3 and 5%). The addition of Kinnow peel improved the appearance, flavor, and overall acceptability of the ice-cream samples. The authors reported that the content of ascorbic acid and flavonoids (namely, naringin) in the ice-cream samples increased with the addition of Kinnow peel, showing that the best levels of frozen Kinnow peel, based on sensory evaluation, were unblanched-3% and blanched-5%. Mohamed et al. [bib_ref] Extraction and Characterization of Pectin from Grapefruit Peels, Mohamed [/bib_ref] reported the extraction of pectin from white and red Sudanese-grapefruit peel and confirmed that the gel-forming quality of the extracted pectin was similar to that of commercial pectin. Jellies prepared with both types of grapefruit peel pectin set within the 10-25 min, indicating them to be rapid-set pectin and demonstrating their potential to be used as a stabilizer/thickening agent in different food products. In another study [bib_ref] Extraction and characterization of pectin from grapefruit (Duncan cultivar) and its utilization..., Khan [/bib_ref] , jams were also prepared and their physicochemical and sensory properties analyzed. The authors extracted, characterized, and applied pectin recovered from grapefruit peel from Duncan cultivar to jam formulations and observed a significant effect on the texture of the final product. Despite the limited number of studies demonstrating the practical applications of extracted pectin in food products, this research has great potential, as the extracted pectin from citrus waste can replace the use of commercial pectin as a gelling agent in various foods. ## Limitations of applying citrus wastes in food industry Several studies have demonstrated the promising potential of incorporating citruswaste extracts in food-industry products. However, some parameters of citrus extracts, such as their low stability and water solubility, limit their further use at a larger scale. Since most of the bioactive compounds present in citrus extracts have poor bioavailability and increased sensitivity to different environmental conditions, such as pH, heat, and oxidation, their protection is a major challenge for the food industry in commercial applications. In addition to these limitations, the conversion of citrus wastes into value-added food products raises concerns about the safety and toxicity of the citrus-waste extracts used [bib_ref] Circular food supply chains-Impact on value addition and safety, Lavelli [/bib_ref]. In general, the potential of citrus wastes to be used as novel functional ingredients with a specific function is well-described; however, the evaluation of their safety has not yet been established. Nevertheless, the use of citrus wastes in food products must comply with current legislation and a risk assessment must be performed to assess their safeness, and very few studies have been conducted recently to address these issues [bib_ref] Biological activities and safety of Citrus spp. Essential oils, Dosoky [/bib_ref]. Therefore, a holistic research approach is needed to integrate the value-addition strategy with risk analysis and to apply forecasting and optimization studies to the whole supply chain. Furthermore, industrial-scale studies on the use of citrus food are still very limited, although they are also extremely necessary to define the barriers to a large-scale application. Therefore, collaboration between academic and industrial partners may be the key to increase the value of citrus-processing industries by converting their wastes into functional food products. ## Application in food packaging According to definition reported in the EC Regulation No 450/2009, "active materials and articles means materials and articles that are intended to extend the shelf-life or to maintain or improve the condition of packaged food; they are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food". Active packaging technology provides several advantages over the direct addition of active compounds to the packed food, such as the lower amounts of active substances required, the localization of activity at the surface, migration from the film into the food matrix, controlled release systems, and the elimination of additional steps within a standard process intended for introducing the active compounds at the industrial processing level, such as mixing, immersion, or spraying. Controlled-release systems are of industrial importance as they can prevent sensory or toxicological problems or inefficiencies of the system caused by too-high or too-low concentrations of the delivered substance [bib_ref] Advances in antioxidant active food packaging, Gómez-Estaca [/bib_ref]. In the review by Han et al. [bib_ref] Food Packaging: A Comprehensive review and future trends, Han [/bib_ref] , the problems related to development of antioxidant and antimicrobial active packaging are well-defined, making it quite difficult to set specific targets for the selection of the natural extracts to be used due to the absence of reference benchmarking products. Recent research trends have focused on the development of active food packaging by adding antioxidants into packaging materials to extend the shelf life of the product. The most commonly used synthetic antioxidants in the food industry are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate in U.S.A., especially for packaging cereals and snacks [bib_ref] Loss of antioxidants from high-density polyethylene, Miltz [/bib_ref]. However, synthetic antioxidants can also be carcinogenic and harmful to consumers. This must be considered in active packaging, as migration from food contact materials is not negligible and is, indeed, a desired phenomenon. With increasing health awareness and consumer's demand for reduced use of chemicals in food packaging, more attention has been paid to finding naturally occurring, safe substances that can act as alternative antimicrobials and antioxidants. The use of natural antioxidants derived from plant extracts in food packaging is becoming increasingly popular. Extrusion is the most popular technique to include natural extracts into the final formulation [bib_ref] Advances in antioxidant active food packaging, Gómez-Estaca [/bib_ref]. In this technique, bioactive compounds are incorporated before extrusion, so that the high temperatures of extrusion (the exact values depend on the melting temperature of the processed polymer) allow their effective and homogeneous distribution in the film, although, at the same time, they are responsible for the thermal degradation of the bioactives' activity. For example, Ha et al. [bib_ref] Multilayered antimicrobial polyethylene films applied to the packaging of ground beef, Ha [/bib_ref] used a high-temperature profile - C) to extrude an antimicrobial LLDPE-based film, which resulted in a high loss of functionality of the grapefruit-seed extract (GSE) and loss of antimicrobial activity. For this reason, heat-sensitive bioactive agents (i.e., natural extracts) should preferably be incorporated into the packaging using non-heating methods (e.g., electrospinning and surface coating). Among these methods, surface coating, in particular, is a simple process that relies on low temperatures but may suffer from poor adhesion to plastics and needs to be designed to be in direct contact with the food in cases where active packaging is the final objective of material production. Natural extracts are already produced and commercialized by different companies, mainly for direct use in food or for the cosmetic and pharmaceutical industries. There are numerous scientific articles on the incorporation of such natural extracts to extend the shelf life of food products. However, large-scale demonstration is still pending [bib_ref] Smart packaging systems for food applications: A review, Biji [/bib_ref] , especially in relation to their use as packaging materials. While synthetic antioxidants are generally added to improve the properties of the materials during processing, natural antioxidants suffer from the major drawback of thermal degradation at the typical working temperature of the extrusion processes [bib_ref] Active packaging films with natural antioxidants to be used in meat industry:..., Dominguez [/bib_ref]. Therefore, encapsulation of antioxidants can improve their thermal resistance so that they can be incorporated directly into the plastic pellets before extrusion. Green tea, rosemary extracts, essential oils and various fruit extracts are the mostused antimicrobial and antioxidant products investigated in the literature for packaging applications [bib_ref] Release kinetics of rosemary (Rosmarinus officinalis) polyphenols from polyvinyl alcohol (PVA) electrospun..., Estevez-Areco [/bib_ref] [bib_ref] Antimicrobial wrapping paper coated with a ternary blend of carbohydrates (alginate, carboxymethyl..., Shankar [/bib_ref] [bib_ref] A concise guide to active agents for active food packaging, Vilela [/bib_ref] [bib_ref] Antioxidant packaging with encapsulated green tea for fresh minced meat, Wrona [/bib_ref] [bib_ref] Use of encapsulated natural compounds as antimicrobial additives in food packaging: A..., Zanetti [/bib_ref]. In 2015, Goglio Spa (Italy) won the Packaging Oscar for its product GTea ® , an active packaging with a green-tea extract. As the importance of environmental sustainability and circular economy is increasingly recognized, it would be better to use extracts obtained from agri-food residues, such as from orange peels. ## Natural extracts requirements for incorporation into packaging material A key point in selecting the extract to be incorporated in active packaging is obviously the food-grade characteristic. It would be the best to use food-grade extracts since there will be no problem with migration restrictions, especially if active packaging is planned with the expected release of extracts into food. On the other hand, if the extract is not food-grade, it will have to be exploited to absorb substances from the packaged food or the environment surrounding the food and then incorporated only into the external coating, or into an intermediate layer (i.e., by incorporation into an adhesive if lamination is used to manufacture the multilayer film) with a barrier layer which prevents migration into the food. If the aim is to provide antioxidant and/or antimicrobial activities, these properties are crucial for selection and it is necessary to verify the maintenance of the property after incorporation and over time. Furthermore, depending on the selected target food to be packed and the coloring power of the extract, this property could be incompatible with obtaining a suitable transparent packaging. However, it should be noted that many natural extracts, such as those from citrus species, have antioxidant/antimicrobial activity due to the presence of phenolic compounds, which are often colored. At the same time, the presence of phenolic compounds and carotenoids with the ability to absorb light in the 200-800 nm range could be of interest, as UV-Vis light can catalyze many degradation reactions in food products. ## Need for encapsulation As explained earlier, natural extracts are sensitive molecules that can be denatured under harsh conditions. Encapsulation may be necessary to provide suitable solubility in the coating medium (when incorporated into the packaging material via a coating application), thermal stability at processing temperatures (when incorporated into a plastic polymer prior extrusion step), and/or light stability. The thermal stability of natural additives is the main problem in cases of the direct incorporation of the extract for compounding a functionalized polymer masterbatch. The working temperature during extrusion to form the plastic film is the most challenging point, since it can exceed 150-200 - C depending on the processed polymer. For this reason, direct incorporation prior to film extrusion is often discarded. Spray-drying encapsulation can be applied to increase the thermal stability of the extracts. Thermal stability may also be required for some specific uses of packaging in the food industry, such as hot filling and thermal treatment after packing. In addition, other encapsulation technologies may be considered: extrusion with vibrating nozzles, jet cutter, coacervation and others. Information on the maximum temperature that could be reached during the coating preparation or during the melting/extrusion process of the plastic material (in the case of direct incorporation of the extract into the polymer), or, eventually, by the final food industry end user, is, therefore, key to defining thermal-stability requirements. Solubility in water or in another solvent is necessary if the extracts are to be incorporated via a coating application, depending on the solvent on which the coating is based. Furthermore, in Europe, the legislation for plastic food-contact materials (FCM) reports the use of different simulants to simulate the different ranges of food products (ethanol 10%, 20%, 50%; acetic acid 3%; vegetable oil with less than 1% unsaponifiable content, and simulant E for dry foods). Evaluation of solubility in these simulants is important in terms of desired or undesired release in the packaged food. Encapsulation may affect and eventually improve thermal stability and solvent solubility, depending on the carrier materials used, but it is also important to check the potential effect of the encapsulation process on the antioxidant and antimicrobial properties of the original extract. ## Literature examples of citrus extracts use to develop antimicrobial/antioxidant packaging Plant/fruit extracts or essential oils are known for their potential antioxidant and antimicrobial properties and have been widely investigated in the literature for these properties as well as for their use in active food packaging [bib_ref] Trends in the use of natural antioxidants in active food packaging: A..., Sanches-Silva [/bib_ref]. Many of the extracts studied are obtained from fresh plants, fruits and herbs, but not from processing residues. The antibacterial capacity of 32 essential oils against five foodborne (L. monocytogenes, S. aureus, E. coli, S. Typhimurium, P. aeruginosa) and spoilage bacteria in liquid phase (as minimum inhibitory concentration, MIC, values) was evaluated by Ghabraie et al. [bib_ref] Antimicrobial effect of essential oils in combinations against five bacteria and their..., Ghabraie [/bib_ref]. Among the oils tested, Chinese cinnamon, cinnamon bark and wild-bergamot essential oils were the only ones that exhibited inhibitory activity against all five pathogenic microorganisms tested. However, these essential oils were not produced from residues and by-products, like in the case of essential oils from fruit peels. In addition, essential oils have a typical strong flavor that may interfere with their use in food packaging, as they could have a strong impact on the sensory profile of the packed foods. Grapefruit-seed extract is made from the seeds and pulp of grapefruit and it contains tocopherol, citric and ascorbic acids [bib_ref] Preparation of a barley bran protein-gelatin composite film containing grapefruit seed extract..., Song [/bib_ref]. The antioxidant and antimicrobial effects of this extract have been reported in different products such as ground beef [bib_ref] Preparation of a barley bran protein-gelatin composite film containing grapefruit seed extract..., Song [/bib_ref]. There are several studies in the literature in which grapefruit-seed extracts were incorporated into bio-packaging [bib_ref] Antimicrobial wrapping paper coated with a ternary blend of carbohydrates (alginate, carboxymethyl..., Shankar [/bib_ref] , which showed good antimicrobial activity against L. monocytogenes and E. coli. Kanmani and Rhim [bib_ref] Antimicrobial and physical-mechanical properties of agar-based films incorporated with grapefruit seed extract, Kanmani [/bib_ref] incorporated a GSE at different concentrations (from 0.6 to 13.3 µg/mL) into an agar-based film through a casting technique and the obtained films were evaluated for their antimicrobial activity against L. monocytogenes, Bacillus cereus, and E. coli. Only the enriched films showed antimicrobial activity, with an extract-dose correlation, and better performance against L. monocytogenes (Gram +) than against E. coli (Gram -), probably due to the specific outer membrane which inhibits the diffusion of the active compounds through the lipopolysaccharide layer. Extracts can be obtained from citrus peel, which is a residue of citrus-juice production. Jridi et al.incorporated phenolic extracts from red-orange peels (C. sinensis) in both dried and fresh forms at concentrations of 5 and 10 mg/mL into a fish gelatin-based film using the casting technique. The films were tested for their antimicrobial activity against Micrococcus luteus, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Salmonella enterica, Listeria monocytogenes and Enterobacter sp. Some antimicrobial activity was exhibited against all the tested microorganisms, with S. aureus being the most sensitive. In general, the fresh extract was more effective than the dry extracts, showing a reduction in activity after drying. The focus of the study of Bassani et al. [bib_ref] Model of spray-drying for encapsulation of natural extracts, Bassani [/bib_ref] was the development of an innovative, biodegradable, and sustainable PLA-based active-packaging solution incorporating an antioxidant extract from orange peel. The extract was first obtained using hydro-alcoholic extraction and then purified by a resin absorption process (which was required to remove sugars and organic acids and increase antimicrobial activity). The extract contained up to 50% total phenols on dry matter and it was either freeze-dried or spray-dried with pectin or ß-cyclodextrins as a carrier material to obtain powder formulations. Encapsulation could improve thermal stability compared to the freeze-dried extract, particularly when cyclodextrins were used. The three powder extracts were incorporated into commercial PLA at different concentrations (0.25, 0.50, 1.50, 2.0 wt %.) and film samples were obtained by hot pressing. The films were assayed to evaluate the influence of the extract addition on the thermal stability of the polymer, color, and mechanical properties of the films under accelerated light storage conditions (in a Suntest XXL+ aging chamber, for 500 h). Positively, extract addition preserved the transparency of the bioplastic and did not modify the degradation temperature profile of the PLA film. However, the extracts resulted in a yellowish coloration that increased with the amount added and achieved an unacceptable browning at a dosage of 2%. The accelerated light storage test highlighted that encapsulation improved color stability and that the film performed worse in terms of mechanical properties (Young module, tensile strength and elongation at break) when it was enriched with the extracts (in this case, the freeze-dried formulation was preferable to the encapsulated ones). In the work of Fiorentini et al., different commercial citrus-peel extracts were investigated for their thermal stability, which was then improved by a spray-drying encapsulation process with beta-cyclodextrins. The study revealed that the antioxidant capacity was retained after the encapsulation process, with an apparent 20-25% reduction in the total phenolic content of the original extract. In addition, the antimicrobial activity against S. aureus was almost unaffected by spray drying, with MICs ranging from 5-0.625 mg/mL to 5-1.25 mg/mL. The encapsulated extract with the best antioxidant and antimicrobial activity was incorporated into a polylactic acid/polyhydroxy butyrate (PLA/PHB) film produced on an industrial scale by cast extrusion. The obtained extract-enriched film was proven to be compliant with European regulations for food-contact materials in relation to overall migration in contact with acidic, hydrophilic and fat-containing food categories. The authors also evaluated the migration of active compounds and observed a potential release of 13.41% in hydrophilic food products and 11.02% in acidic products (pH < 4.5). The film showed growth inhibition by 30 and 60% against E. coli and S. aureus, respectively. The main steps applied in the works [bib_ref] Model of spray-drying for encapsulation of natural extracts, Bassani [/bib_ref] for the production of antioxidant/antimicrobial extracts and their incorporation into biobased plastic materials, are summarized in . It is clear how different encapsulation processes and percentages of addition may lead to more or less colored final materials. The main steps applied in the works [bib_ref] Model of spray-drying for encapsulation of natural extracts, Bassani [/bib_ref] for the production of antioxidant/antimicrobial extracts and their incorporation into biobased plastic materials, are summarized in . It is clear how different encapsulation processes and percentages of addition may lead to more or less colored final materials. . Schematization of the process from waste citrus peels to purified spray-dried extracts (powders obtained with β-cyclodextrins on the right or pectins on the left, at different extract-tocarrier ratios, and example of a SEM observation), and to their incorporation into PLA (on the right) or PLA/PHB (on the left) films. and Nerin [bib_ref] Role of catechins in the antioxidant capacity of an active film containing..., Colon [/bib_ref] tested a grapefruit dried hydroalcoholic extract for incorporation into a coating applied on a PET film. The system was prepared according to EU patent EP1477519-A1.41 and the coating was applied at room temperature reaching a maximum of 40 °C using hot air to eliminate the solvent. Active films were prepared with different weight percentages of the active agent/active layer, which varied from 0.7 g active/m 2 film to 3.0 g active/m 2 film. The obtained films were subjected to a free radical gas stream to increase oxidation and the antioxidant capacity was then determined based on the oxygen radical absorbance capacity (ORAC) assay. The grapefruit extracts revealed a lower antioxidant activity (based on ORAC assay) compared to green-tea or green-coffee extracts. A commercial grapefruit-seed extract (GSE) was also used by Wang et al.to develop active films based on poly(lactide) (PLA) or antimicrobial low-density polyethylene (LDPE). In this study, the natural extract was added before the extrusion step. Thermo- . Schematization of the process from waste citrus peels to purified spray-dried extracts (powders obtained with β-cyclodextrins on the right or pectins on the left, at different extract-tocarrier ratios, and example of a SEM observation), and to their incorporation into PLA (on the right) or PLA/PHB (on the left) films. and Nerin [bib_ref] Role of catechins in the antioxidant capacity of an active film containing..., Colon [/bib_ref] tested a grapefruit dried hydroalcoholic extract for incorporation into a coating applied on a PET film. The system was prepared according to EU patent EP1477519-A1.41 and the coating was applied at room temperature reaching a maximum of 40 - C using hot air to eliminate the solvent. Active films were prepared with different weight percentages of the active agent/active layer, which varied from 0.7 g active/m 2 film to 3.0 g active/m 2 film. The obtained films were subjected to a free radical gas stream to increase oxidation and the antioxidant capacity was then determined based on the oxygen radical absorbance capacity (ORAC) assay. The grapefruit extracts revealed a lower antioxidant activity (based on ORAC assay) compared to green-tea or green-coffee extracts. A commercial grapefruit-seed extract (GSE) was also used by Wang et al.to develop active films based on poly(lactide) (PLA) or antimicrobial low-density polyethylene (LDPE). In this study, the natural extract was added before the extrusion step. Thermoplasticized starch was used as a carrier to introduce the extract into the polymer. It was prepared by mixing corn starch with 20% wt glycerol (as plasticizer) and 40% of GSE and heating at 120 - C for 30 min in an autoclave. The plasticized starch mixture was then cooled to room temperature and pulverized using a blender. The obtained powder was mixed with the plastic resins (PLA and LDPE) at the ratio of 1:10 w/w to obtain the masterbatches, which were cast and blow extruded, respectively. The addition of the extract decreased the lightness of the films and increased the color (increase in the chromatic coordinates a* and b*) due to both the dextrinization of the starch and the phenolic content of GSE. The starch blend addition increased the LDPE thickness but not the PLA film. This is due to the fact that the hydrophilic thermoplastic starch is more compatible with more hydrophilic polymers, such as PLA. The antimicrobial activity of the films was tested against L. monocytogenes and E. coli and was high for the enriched PLA film, but not for the LDPE. Finally, the capacity of the films to act as active food packaging was tested on a fish paste, where the activity of the GSE-PLA film was confirmed, while the GSE-LDPE showed inhibitory activity only after 6 days of contact. This was consistent with the results of the migration tests, which showed a slower release of the GSE components from LDPE than from PLA. ## The use of citruses in cosmetics The global growth and development of the cosmetics market is a well-recognized trend worldwide, accounting for 41% in 2021. It is estimated that at an annual growth by 6%, the value of the cosmetics market will reach USD 675 billion by 2026 (Expert Market Research). The cosmetics industry follows a continuous research and development (R&D) program, not only studying consumer behaviors and changes in beauty preferences, but also focusing on new technologies and sustainable development. Among all cosmetics categories (facial care, body care, hair care, sun care, decorative/beauty cosmetics, fragrances), skin-care products account for the largest market share, with trending demands in anti-aging and organic products with natural ingredients. It is estimated that the total expenditure on R&D in the cosmetics industry is EUR 2.35 billion, with an average of 5 years spent on innovative research and formulation to bring a new product to market (expert market research). Before launching a new cosmetics product on the market, the safety of the product and compliance with regulatory requirements must be confirmed. This is most directly linked to the specific ingredients of the final formulation, as well as the packaging material. Before a new product is launched, the microbiological safety and efficacy of the new cosmetic formulation must be evaluated and confirmed through standardized testing. It is imperative to demonstrate in controlled studies that the new cosmetic product provides the claimed benefits to consumers. Subsequent stability tests provide information on the compatibility of the product and specific packaging, defining the products' shelf-life. After all laboratory testing is complete, the process development scales up the technology, assuring the maintenance of the product quality. Finally, it is important to establish a supply chain which includes raw materials, packaging, and labeling to assure sustainability in the production, independently of the market demand. Clear trends that can be recognized in the cosmetics industry are related to the inclusion of safe and environmentally friendly ingredients and technologies that respond to ever-changing consumer expectations. Increasing consumer awareness of the side effects of synthetic cosmetic ingredients and the advantages of natural and organic cosmetics, which generally produce fewer allergic responses and unwanted or unexpected effects, are leading to a shift in consumers' demands for products in which the key active ingredients are of natural origin, most often plant-derived. The increasing demand for natural and organic products and attractive marketing strategies in this field has propelled the expansion of a wide range of new products containing various natural extracts and phytochemicals. In this respect, citrus fruits, as well as the by-products of their processing, represent very attractive alternatives for the reformulation of cosmetic products, or the design of completely new products with additional benefits. Citrus fruits are rich in numerous phytochemicals and bioactive components, representing excellent sources of antioxidants, polyphenols (mainly flavonoids), carotenoids, vitamin C, folic acid, minerals, and pectin [bib_ref] Citrus peel as a source of functional ingredient: A review, Rafiq [/bib_ref]. Among flavanones, naringin and hesperidin have demonstrated numerous bioactivities that may be useful for the cosmetics industry, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic properties, and they are mainly found in citrus peel and albedo. In addition to the use of citrus processing by-products to extracts' added-value compounds, citrus essential oils are often incorporated as fragrance components due to their specific, unique fragrance and compatibility with cosmetic products [bib_ref] Cosmetics-Food waste recovery, Pinto [/bib_ref]. Considering the worldwide mass production and processing of citrus, and the global industrial framework oriented towards circular economy and a sustainable approach in biowaste valorization, the recovery of bioactive ingredients and essential oils from citrus bio-waste is a current trend in the R&D sector of the cosmetic industry [fig_ref] Figure 2: By-products created as a result of industrial processing of citrus-fruit wastes [/fig_ref]. ## The extraction of cosmeceuticals from citrus biowaste Citrus fruits are cultivated mostly in tropical and subtropical regions, with multimillion tons of annual production, generating approximately 40 million tons of citruses waste, which can be used to produce valuable compounds with potential uses in the food, nutraceutical, pharmaceutical and cosmetic sectors. In the food sector, citruses are mostly processed into juices and marmalades, generating large amounts of waste consisting of peels, pomace, seeds, and membrane residues. Citrus-peel waste accounts for 50-70% of processing waste and can be used for the production of flavoring agents, flavonoids, and citric acid for the cosmetic and pharmaceutical industries. Strict legislative requirements for cosmetic products in terms of their safe use and residues of unwanted contaminants, such as traces of organic solvents or heavy metals, have made "green" techniques for the isolation of cosmeceuticals from citrus-fruit waste mandatory. An array of bioactive compounds with beneficial properties for skin care (such as anti-radical, antioxidant, whitening, and anti-inflammatory, etc.), and belonging to different chemical classes, can be isolated using modern extraction techniques with improved performance, lower energy and time consumption and reduced use of organic solvents. For example, carotenoids, which are concentrated in citrus peels and are responsible for the orange, yellow and red color of the fruits, have been extracted using ultrasound-assisted extraction, using different solvents, such as ionic liquids [bib_ref] Ionic liquid associated with ultrasonic-assisted extraction: A new approach to obtain carotenoids..., Murador [/bib_ref] , ethanol [bib_ref] Green solvents and ultrasound-assisted extraction of bioactive orange (Citrus sinensis) peel compounds, Montero-Calderon [/bib_ref] or limonene [bib_ref] Development of a green procedure of citrus fruits waste processing to recover..., Boukroufa [/bib_ref]. In addition, carotenoids can also be extracted using supercritical carbon-dioxide [bib_ref] Sequential extraction of bioactive compounds from tangerine (Citrus Unshiu) peel, Tsitsagi [/bib_ref] , or, as an advanced approach, high-voltage electric discharge (HVED) technology [bib_ref] Bioaccessibility of bioactive compounds and antioxidant capacity from orange peel after pulsed..., Buniowska [/bib_ref]. On the other hand, the essential oils of different citrus fruits (e.g., mandarin, orange, grapefruit, lemon, lime, kinnow mandarin) can be extracted from different parts of the plant, representing valuable natural cosmetic fragrances with distinctive characteristics and beneficial properties. The essential-oil yield obtained by microwave-assisted hydro-distillation from the peels of different citrus varieties can vary from 0.42% (Citrus sinensis L., orange) [bib_ref] An improved microwave Clevenger apparatus for distillation of essential oils from orange..., Ferhat [/bib_ref] to 2.73% (C. sinensis var. Valencia, mandarin) [bib_ref] Estimation of mass transfer coefficients of the extraction process of essential oil..., Franco-Vega [/bib_ref]. The yields obtained by supercritical carbon-dioxide extraction are much higher, reaching~28% in citron (C. medica) peel [bib_ref] Chemical composition and bioactivity of Citrus medica L. cv. Diamante essential oil..., Menichini [/bib_ref]. Garrido et al. [bib_ref] Influence of extraction methods on fatty acid composition, total phenolic content and..., Garrido [/bib_ref] isolated essential oils from lime and orange seeds with hexane assisted by ultrasound and achieved yields of 22%. Other classes of compounds of interest to the cosmetic industry from an addedproperty point of view include phenolics, which can be extracted from virtually all citrus wastes, and all the green technologies listed. For example, total phenolics extracted with 20% glycerol from grapefruit peels by high-voltage electrical discharge was 1880 mg GAE/100 g [bib_ref] Green extraction of polyphenols from grapefruit peels using high voltage electrical discharges,..., El Kantar [/bib_ref] , while with high hydrostatic-pressure technology applying 300 MPa, 266.23 mg GAE/100 g was extracted from lemon peel, 397.21 mg GAE/100 g from lime peel, 587.28 mg GAE/100 g from tangerine peel, and 288.16 mg GAE/100 g from orange peel, respectively [bib_ref] Evaluation of the effect of high pressure on total phenolic content, antioxidant..., Casquete [/bib_ref]. The extraction of mandarin peels (Citrus inshiu) by microwaveassisted extraction with 70% ethanol resulted in the recovery of 5860 mg/100 g of hesperidin and 1310 mg/100 g of narirutin [bib_ref] Isolation of hesperidin from peels of thinned Citrus unshiu fruits by microwave-assisted..., Inoue [/bib_ref]. Depending on the solvent used, ultrasound-assisted extraction leads to similar yields of phenolic compounds as microwave-assisted extraction. On the other hand, supercritical carbon-dioxide extraction, even at a high concentration of co-solvent (40% ethanol), generally produce lower yields (0.67 mg GAE/100 g in grapefruit peels, 0.66 mg GAE/100 g in lemon peels, 0.45 mg GAE/100 g in orange peels, and 0.38 mg GAE/100 g in tangerine peels) [bib_ref] Optimization of focused ultrasound extraction (FUSE) and supercritical fluid extraction (SFE) of..., Omar [/bib_ref]. ## The use of citrus biowaste extracts in cosmetics The bioactivity of different citrus-waste extracts has been undoubtedly confirmed by numerous studies, but real applications in the cosmetic industry are still scarce; thus, in-depth research in this field is needed. The next steps in the implementation of citrus waste in the cosmetic industry must be oriented towards the development of formulations integrating safe doses of extracts as well as studies of the quality parameters of such products, including basic physico-chemical quality parameters, efficacy, stability tests, and, no less important, consumer acceptance. Kim et al. [bib_ref] Citrus peel wastes as functional materials for cosmeceuticals, Kim [/bib_ref] confirmed several beneficial activities of ethanolic extracts of Citrus unshiu waste. In addition to antioxidant effects and dose-dependent inhibition of melanin synthesis, the extracts also showed moderate antibacterial effects against bacteria associated with acne, namely, Propionibacterium acnes, while they were ineffective against normal skin microflora Staphylococcus epidermidis. The absence of toxicity in human keratocytes was confirmed at concentrations below 10 µg/mL, resulting in 100% viability, while increasing the concentration to 100 µg/mL reduced the viability to 80%. The extracts of obtained from C. reticulata Blanco peel showed promising anti-collagenase and anti-elastase activities which could be exploited for the formulation of anti-aging cosmetic products [bib_ref] Evaluation of Skin Anti-aging potential of Citrus reticulata Blanco peel, Apraj [/bib_ref]. The authors compared the activity of extracts obtained using Soxhlet extraction and maceration and found better activities for hot extraction. The EC 50 values calculated for inhibition of collagenase and elastase, for hot extraction and maceration were 329.33 µg/mL, 466.93 µg/mL, 3.22 mg/mL and 5.09 mg/mL, respectively. The authors also confirmed high anti-radical activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, and 2, 2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. Skin-whitening cosmetic formulations have significantly increased in popularity in some countries due to different beauty perceptions. In some cultures, and modern societies, pale skin is considered aesthetic, giving a rise to numerous skin-whitening products, such as soaps, tonics, and creams, etc. Some of these products contain dangerous synthetic chemicals, such as hydroquinone, or pure natural isolated compounds with anti-tyrosinase activity, such as kojic acid, alpha and beta arbutin, or the isoflavone glabridin, isolated from licorice root. Although they are all of natural origin, some of these phytochemicals have some safety concerns, such as kojic acid, which can trigger contact dermatitis [bib_ref] Contact allergy to kojic acid in skin care products, Nakagawa [/bib_ref]. ## The use of citrus essential oils in cosmetics The essential oils from citruses can be produced by steam distillation from different parts of the plant, mostly from peels, but also from flowers, shoots, buds, and leaves. Due to their very pleasant fragrance and antibacterial effects, citrus essential oils have been used in folk medicine and perfumery for centuries and are now popular ingredients in aromatherapy, air fresheners, house cleaning products, and cosmetics. Although citrus essential oils have a number of beneficial effects in skin care, such as degreasing, antiseptic, brightening and astringent properties, the contents of particular essential-oil components are regulated by legislation for cosmetic products due to potential adverse effects they might exhibit, such as phototoxicity or allergenicity. The phototoxicity of citrus essential oils and extracts may be related to furocoumarins, which strongly absorb UV light and carry a risk of skin burns, inflammation and swelling. Identified major phototoxins in citruses include bergamottin, citropten, herniarin or oxypeucedanin, bergapten and other derivatives [bib_ref] Current and potential use of Citrus essential oils, Palazzolo [/bib_ref]. Due to its recognized phototoxicity, the Scientific Committee on Cosmetic Products (SCCS) has implemented a restriction on furocoumarin-like substances in cosmetics, limiting their content to 1 ppm (SCCNFP, 2001). However, furocoumarins may have other medicinal applications in different skin conditions. The intense UV-light absorption of furocoumarins is the underlying mechanism for psoralen UV A (PUVA) treatment of psoriasis or vitiligo. In topical psoriatic plaques, psoralens form adducts with cellular DNA that induce apoptosis upon exposure to UV light. In this treatment, furocoumarins are applied either orally or topically before UV treatment. In topical application, the limiting step is weak percutaneous permeability, and in both cases, there is a risk for the development of cancer with long-term therapy [bib_ref] A clinical review of phototherapy for psoriasis, Zhang [/bib_ref]. The antimicrobial effects of citrus essential oils are beneficial in different skin inflammation conditions, such as acne, which is attributed to different mono-and sesquiterpenoids, aldehydes, ketones, acids, alcohols, and esters, such as p-cymene, limonene, carvacrol, geraniol, eugenol, and others, which can be well dermally absorbed due to their small size and hydrophobic characteristics. The peel of citrus fruits yields about 0.5-5% (w/v) essential oil, which is mainly composed of volatiles (85-95%) and non-volatile fractions consisting of coumarins, fatty acids, sterols, carotenoids and polymethoxylated flavonoids [bib_ref] Current and potential use of Citrus essential oils, Palazzolo [/bib_ref]. Many components of citrus essential oils act as efficient free radical scavengers, important for anti-ageing processes but also involved in melanogenesis via the activation of a melanin-producing enzyme-tyrosinase. It has been confirmed that essential oils of different citruses act as efficient tyrosinase inhibitors [bib_ref] Tyrosinase inhibitory activity of citrus essential oils, Matsuura [/bib_ref] , and are, therefore, frequently added to cosmetic products as whitening compounds. ## Cosmetic formulations with components isolated from citruses Data on cosmetic formulations containing citrus extracts or oils are rather scarce in the literature; however, several formulations based on peels' extracts were reported. The authors mostly emphasized the antiradical activity of peels extracts, confirming them using standardized spectrophotometric assays, such as the DPPH test. Kamalambigeswari et al. [bib_ref] Formulation of face cream using methanolic extract of Citrus limon, Kamalambigeswari [/bib_ref] , for example, used the methanolic extract of lemon peel, while Riski et al. [bib_ref] Activity test and formulation of antioxidant cream from ethanol extract combination of..., Riski [/bib_ref] prepared individual and combined 70% ethanolic extract from the peel of several citrus species, namely, lime fruit (C. aurantifolia Swingle.), kaffir lime (C. hystrix DC), and sweet orange (C. sinensis L.), to formulate creams with different extract concentrations (1-3%). The formulations met all quality and stability criteria for this product. It is even rarer to find scientific publications on cosmetic formulations incorporating citrus essential oils, probably due to their photosensitivity and allergenicity. However, Nareswari and Kuncoro [bib_ref] Preparation of essential oil ointment of lime leaves (Citrus amblycarpa) and stability..., Nareswari [/bib_ref] extracted essential oil from lime (Citrus amblycarpa) leaves yielding 0.47%, and prepared an ointment with antibacterial properties. The principal stability parameters (pH, dispersiveness, density, homogeneity, sensory, viscosity, and adhesion) were evaluated after 8 weeks and found to be satisfactory. Another example of essential-oil addition for the purpose of additional activity was the formulation of a cream with mosquito-repellent activity with the addition of a lemon-grass (Cymbopogon citrus) essential oil which resembles citrus in fragrance [bib_ref] Formulation of herbal cream using essential oils of Cymbopogon citrus (Lemon grass)..., Jayarathna [/bib_ref]. The mosquito-repellent activity was confirmed in vivo in volunteers exposed to three mosquito species, namely, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, for a duration of 240 min, in comparison to commercial herbal creams that were used as control. Recognizing that citrus essential oils are prone to photo-sensitive degradation and volatility, Kaur et al. [bib_ref] Synthesis and characterization of Citrus limonum essential oil based nanoemulsion and its..., Kaur [/bib_ref] formulated a nanoemulsion of C. limonum essential oil for further use in cosmetics. The authors used Tween 80 and ethanol as surfactant and co-surfactant, respectively, to form an oil-in-water emulsion with a particle size of 60 nm, a polydispersity index of 0.125 and a zeta potential of −14.9 mV. The antioxidant potential, chemical profile, and thermodynamic stability of the plain essential oil and the nanoemulsion-protected essential oil were monitored over a period of 6 months. The developed nanoemulsion proved to be much more stable; thus, its use in cosmetic formulations is recommended. For recognized anti-inflammatory and antibacterial properties, some authors reported that bergamot essential oil (C. bergamia) can be useful in antidandruff and hair-growth products [bib_ref] Potential use of essential oils in cosmetic and dermatological hair products: A..., Abelan [/bib_ref]. The California Navel orange-peel extract (C. sinesis L.), due to its high anti-tyrosinase activity (IC 50 = 255.10 µg/mL), was used for facial-cream formulation [bib_ref] Matrix metalloproteinase-2 inhibition activity of Plukenetia volubilis L. leaves extract for anti-aging..., Wuttisin [/bib_ref]. The facial cream was formulated with 2% (w/w) orange-peel extract and was evaluated as pleasant by a sensory analysis panel. The formulated product reduced melanin pigment by 17.33% after one month of use, without a single case of skin irritation reported in 20 volunteers [bib_ref] Matrix metalloproteinase-2 inhibition activity of Plukenetia volubilis L. leaves extract for anti-aging..., Wuttisin [/bib_ref]. # Conclusions This review provided an overview of the chemical constituents of the main citrus species and a comprehensive assessment of the health benefits of their consumption, with reference to various secondary metabolites such as flavonoids and terpenoids. The wellknown nutraceutical and medicinal value of citrus fruits guarantees a protective effect against a series of chronic diseases, partly linked to the high content of nutrients with antioxidant capacity. Therefore, thanks to their innumerable beneficial properties, due to the synergistic action of their bioactive constituents, citrus fruits can be classified as functional foods. Furthermore, in this review, aspects related to the use of citrus fruits and their extraction products such as essential oils, in the food and cosmetic industry sectors, also highlighting the latest technological developments, were taken into consideration, thus showing the high potential of citrus fruits as a source of multifunctional natural agents. ## Conflicts of interest: The authors declare no conflict of interest. [fig] Table 1: Overview of the studies (from 2015 to present) on the chemical constituents in major citrus species. [/fig] [fig] Figure 1: Citrus-fruit wastes: seeds, pulp, pomace and peels. [/fig] [fig] Figure 2: By-products created as a result of industrial processing of citrus-fruit wastes. [/fig] [fig] Author: Contributions: Conceptualization, A.B.H., R.B.S. and W.M.; writing-original draft preparation, A.B.H., C.S., I.G.M., S.G., J.Š.-G., F.R., S.M., M.M.M., E.F., G.S., T.B.-B., B.B.A., R.B.S. and C.D.-M.; writing-review and editing, I.G.M., S.G., J.Š.-G., F.R., G.S., T.B.-B., C.D.-M. and W.M.; visualization, C.S., S.M., M.M.M., E.F., B.B.A. and R.B.S.; supervision, J.Š.-G., F.R., G.S., C.D.-M. and W.M.; project administration, A.B.H. and W.M.; funding acquisition, W.M. All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This research was co-funded by the projects UIDB/50006/2020, UIDP/50006/2020, and LA/P/0008/2020, all supported by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). The authors extend their appreciation to the Deanship of Scientific Research at University of Bisha for funding this research through the general research project under grant number (UB-GRP-66-1444). Part of the research has received funding from Ecosystem for Sustainable Transition in Emilia-Romagna", project funded by European Union under the National Recovery and Resilience Plan (NRRP), Mission 04 Component 2 Investment 1.5-NextGenerationEU, Call for tender n. 3277 dated 30/12/2021, Award Number: 0001052 dated 23/06/2022. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at University of Bisha for funding this research through the general research project under grant number (UB-GRP-66-1444). Manuela M. Moreira (CEECIND/02702/2017) and Francisca Rodrigues (CEECIND/01886/2020) are thankful for their contracts financed by FCT/MCTES-CEEC Individual Program Contract. Jaroslava Švarc-Gajić and Tanja Brezo-Borjan are grateful to The Science Fund of the Republic of Serbia (CleanNanoCatalyze, Grant No 7747845 "In situ pollutants removal from waters by sustainable green nanotechnologies"-CleanNanoCatalyze) and the Ministry of education, science and technological development of the Republic of Serbia (Grant No 451-03-68/2020-14/200134). [/fig]

Reviewing Chitin/Chitosan Nanofibers and Associated Nanocomposites and Their Attained Medical Milestones Citation: Sivanesan, I.; Gopal, J.; Muthu, M.; Shin, J.; Oh, J.-W. # Introduction Miniaturization has become the recent state-of-the-art technology, resulting in modifying material shapes, surfaces, characteristics, and functions. Nanotechnology leads the forefront in the arena of miniaturization strategies. The impressive potential applications of nanotechnology have transformed this field and opened it for many researchers and scientists to work on. Nanotechnology today has expanded into different scientific areas, extending from electronics to industrials to cosmetics. Nanotechnology has also strongly impacted core areas of human health and welfare. Polymers of natural origins have been extensively employed not only in the food industry but also in pharmaceuticals. Chitin, chitosan, alginate, and carrageenan are the most commonly used polysaccharide polymers in various pharmaceutical applicationsand food quality, safety, and preservation. Polysaccharide polymers are sought after owing to their non-toxicity, biocompatibility, and biodegradability. Chitin (Ch) consists of repeated N acetyl-D-glucosamine (GlcNAc) units linked by β-(1,4) glycosidic bonds. Naturally, chitin exists in three crystalline polymorphic forms. Within each form, there are different orientations of the microfibrils: α-chitin has parallel chains, βchitin has antiparallel chains, and γ-chitin has a mixture of parallel and anti-parallel chains.displays the representative organisms for each of these polymorphs. Chitin is insoluble in water; however, water-soluble chitosan (ChS) has been produced after a chitin deacetylation process. A ChS molecule contains an -NH2 group and two -OH radicals in each glycoside residue. Although chitin is naturally produced at a rate of around 1010 tons per year, most chitin is thrown away as commercial waste. Chitin is disposed of as marine shell wastes or industrial wastes in the magnitude of multiple tons. ChS, a polymer of interest, is used to deliver different therapeutic agents, increasingly known for its use in pharmaceuticals. This is attributed to its biocompatibility, ability to bind few organic compounds, susceptibility to enzymatic hydrolysis, and intrinsic physiological activity combined with nontoxicity. These properties make ChS amenable to a wide variety of biomedical applications, including drug delivery and targeting, wound healing, tissue engineering, and similar allied fields. Moreover, ChS is also known for its antimicrobial properties, as well as color stabilization, emulsification, antioxidant activities, and dietary fiber-like properties, water-retention, and fat entrapment. Chitosan has attracted great attention as a material for drug delivery biomedical applications as a promising material specifically for delivering macromolecules. Therapeutic agents have been incorporated into such polymeric matrix to protect the biologically active compound from degradation, promote controlled drug release and improve absorption and therapeutic effect. Miniaturization of chitosan has also been attempted, with ChS-based nanomaterials ranging from microparticles to NP composites and nanofilms, showing enhanced biomedical properties. A nanofiber (NF) is generally defined as a fiber less than 100 nm diameter and an aspect ratio greater than 100:1. Properties of NFs are distinct from that of microfibers, because NFs have a characteristic morphology, an extremely high surface-to-volume ratio, and opticaland mechanical properties. In addition to these, they are biodegradable, biocompatible, renewable, and sustainable. The electro-spinning process is well known for producing artificial NFs from a wide range of polymers. Owing to Chitin is insoluble in water; however, water-soluble chitosan (ChS) has been produced after a chitin deacetylation process. A ChS molecule contains an -NH 2 group and two -OH radicals in each glycoside residue. Although chitin is naturally produced at a rate of around 1010 tons per year, most chitin is thrown away as commercial waste. Chitin is disposed of as marine shell wastes or industrial wastes in the magnitude of multiple tons. ChS, a polymer of interest, is used to deliver different therapeutic agents, increasingly known for its use in pharmaceuticals. This is attributed to its biocompatibility, ability to bind few organic compounds, susceptibility to enzymatic hydrolysis, and intrinsic physiological activity combined with nontoxicity. These properties make ChS amenable to a wide variety of biomedical applications, including drug delivery and targeting, wound healing, tissue engineering, and similar allied fields. Moreover, ChS is also known for its antimicrobial properties, as well as color stabilization, emulsification, antioxidant activities, and dietary fiber-like properties, water-retention, and fat entrapment. Chitosan has attracted great attention as a material for drug delivery biomedical applications as a promising material specifically for delivering macromolecules. Therapeutic agents have been incorporated into such polymeric matrix to protect the biologically active compound from degradation, promote controlled drug release and improve absorption and therapeutic effect. Miniaturization of chitosan has also been attempted, with ChS-based nanomaterials ranging from microparticles to NP composites and nanofilms, showing enhanced biomedical properties. A nanofiber (NF) is generally defined as a fiber less than 100 nm diameter and an aspect ratio greater than 100:1. Properties of NFs are distinct from that of microfibers, because NFs have a characteristic morphology, an extremely high surface-to-volume ratio, and opticaland mechanical properties. In addition to these, they are biodegradable, biocompatible, renewable, and sustainable. The electro-spinning process is well known for producing artificial NFs from a wide range of polymers. Owing to its linear structure, chitin has high crystallinity and is arranged as NFs; these NFs are embedded in a protein matrix. Crab and prawn shells also have a complex hierarchical structure consisting of NFs. In this current review, we briefly run through the existing Ch and ChS based nanomaterial synthesis and their applications. The biomedical applications of Ch and ChS nanofibers have been dealt with elaborately in this review. The future perspective of Ch and ChS NFs is discussed and the reason why Ch NFs are less applied for biomedical applications than ChS NFs speculated. ## Chitin and chitosan nanomaterials ## Overview of ch/chs nanomaterial preparation methods Ch is recovered from natural sources by a two-step process. The first step is the extraction of Ch and removal of calcium carbonate (CaCO 3 ) from crustacean shells using dilute hydrochloric acid, followed by deproteination using dilute aqueous NaOH. During the second step, 40-50% aqueous NaOH at 110-115 - C is used for the deacetylation of Ch. When the degree of deacetylation exceeds 50%, then ChS is produced. Ch with a degree of deacetylation of ≥75% is also recognized as ChS. Elgadir et al., 2015 have elaborately reviewed these preparation methods. ChS NPs were first characterized byfor the delivery of 5-fluorouracil, a chemotherapy medication. The basic techniques demonstrated for the preparation of ChS NPs revolve around emulsification, precipitation, ionic or covalent crosslinking, or combinations thereof. Emulsification and crosslinking are the pioneering methods demonstrated in literature that are based on the amino group of ChS and the aldehyde group of a crosslinking agent. The reversed micelles (microemulsion) method based on covalent crosslinking, has also been applied for the effective preparation of ChS NPs. ChS NPs can also be produced by precipitation-based methods. The phase inversion precipitation method combines emulsification and precipitation techniques. Alternatively, the emulsion-droplet coalescence method, also called desolvation, has been described, based on the coalescence of two water-in-oil emulsions which induce precipitation of ChS NPs. Ionic gelation was first described by; this method can also be used in combination with radical polymerization, which induces gelation of chitosan simultaneously with polymerization of acrylic or methacrylic acid. ChS NPs can also be produced via self-assembly, which is a widely used method based on multiple electrostatic, or hydrophobic and/or van der Waals force based interactions that may be between ChS and other molecules. Recently, green preparation routes and the use of "mild" compounds have become popular. Spray drying is one such method; ChS in aqueous acetic acid is passed through a nozzle exposed to air temperatures from 120-150 - C, resulting in ChS NPs. Magnetic ChS NPs have been produced using this method. Supercritical-CO 2 -assisted solubilization and atomization (SCASA) is one of the pioneering green methods as it is a process that is free of acid-or harmful solvent and uses only water and CO 2 . The dissolution of ChS in water occurs through the acidifying effect of pressurized CO 2 under high pressure. After a 48 h dissolution step, the ChS solution is fed in for atomization. NPs form due to the drying process and are collected by a filter. The long processing time and the size of the particles and their distribution are the disadvantages of this method. There are few other green preparation methods reported for ChS NP synthesis, based on ultrasonication, microwave synthesis and microbe-based methods. There are almost no reports on the preparation of Ch NP, except for the report by Lu et al., who demonstrated a route for preparing suspensions of Ch crystallite particles. However, Ch nanowhiskers have been reported by several authors. Ch was treated with strong HCl, followed by controlled hydrolysis to obtain Ch nanowhiskers. Another method used for Ch nanowhiskers' isolation is TEMPO mediated oxidation (2,2,6,6tetramethylpiperidine-1-oxyl radical mediated oxidation). Whiskers have recently been prepared from Ch from different sources, such as crab, shrimp, and lobster shells and squid pens. Morin and Dufresneprepared nanochitin whiskers from Riftia (marine invertebrates). In another study, Gopalan and Dufresne obtained nanochitin whiskers from crab shells. Rujiravanit et al. reported the preparation of Ch whiskers via acid hydrolysis of shrimp shells. Revol et al. reported the preparation of a suspension of chitin crystallites of rod-like particles through acid hydrolysis, with average sizes of 200 ± 20 nm in length and 8 ± 1 nm in width. Salaberria et al. extracted chitin from yellow lobster shell wastes followed by the isolation of nanocrystals by acid hydrolysis. Zeng et al.and Morin et al.have covered more details on this topic in their reviews. ## Biomedical applications of ch/chs nanomaterials Compared to Ch, ChS NPs have been extensively studied for their varied applications in medicine and pharmaceutics. Although the reason why exactly ChS is preferred over Ch is not clearly laid out, there is definitely a clear edge that ChS holds over Ch when it comes to publications reporting medical applications. The insoluble feature of Ch in common solvents greatly restricts its application into biomedical of different fields, this being the case, two other derivatives of chitin (carboxymethyl chitin (CMC) and chito oligosaccharide (COS)), are those that are reported with respect to biomedical applications. ChS being biocompatible, allow encapsulation and chain grafting of drugs and active ingredients. In addition, ChS NPs possess interesting medical applications. ChS NPs are known to reduce enzymatic degradation of drugs, as well as reduce the damage of non-targeted tissues. This comes very handy when it comes to their usefulness for drug delivery, cancer treatment, and biological imaging and diagnosis. The highly positive surface charges of ChS NP surfaces, aid in carrying substances into the human system. Studies on nanoencapsulation with ChS NPs are mostly related to the delivery of therapeutic peptides such as insulinand Cyclosporin A, and DNAfor gene therapy. ChS NPs have been demonstrated as carriers for the controlled delivery of doxorubicin (DOX), an anticancer drug used for the treatment of several tumors. DOX treat several cancers such as lymphomas, acute leukemia, osteogenic sarcomas, pediatric malignancies, breast and lung carcinomas. The challenge here is that only small amounts of DOX reach the tumor target site. This is because~40% is excreted via liver metabolism. In addition to this, DOX also induces cardiac toxicity. ChS NPs conjugated with DOX have been proved to overcome all these above-said limitations. ChS NPs have also been used for drug delivery of 5-fluorouracil (5-FU) and leucovorin (LV), both of these that are used for the treatment of colon cancer, as well as for delivery of avidin and biotin, which are used for hepatic carcinoma treatment. ChS NPs have also been demonstrated for their gene delivery applications, small interfering RNA (siRNA) delivery, vitamin C release, plasmid DNA (pDNA) delivery against hepatitis B, and insulinor bovine serum albumin (BSA) deliverydelivery of polyphenolic antioxidants called catechins to the intestine. Other researchers showed that ChS NPs loaded with insulin improved the systemic delivery of insulin through the nasal passage. To protect metronidazole (MZ) from dissolution in saliva, the drug was loaded into ChS NPs for controlled release of the drug over 12 h, to dampen their side effects. As a therapeutic delivery entity, ChS NPs attract great attention due to their approach via different pathways of intake such as oral, nasal, and intravenous. The positive charge of ChS NPs, gives them the advantage of high affinity for negatively charged cell membranes. The hydrophobic nature of ChS NPs influences the efficient encapsulation of hydrophilic therapeutics. ChS NPs can increase drug permeability across absorptive epithelia. Since ChS NPs are internalized via endocytosis, they are able to deliver biologically active materials into cells without compromising the integrity of the cargo or the cell. Yu et al. synthesized a copolymer of poly (l-lysine) with ChS and studied its efficiency in relation to plasmid DNA adherence capability and gene transfection effect. Rizeq et al.and Joseph et al.have extensively reviewed the various biomedical applications of ChS NPs. ## Chitin/chitosan nfs ## Overview of ch/chs nfs fabrication methods The usual techniques that are validated for producing NFs include: electrospinning, printing, self-assembly, phase separation, and template synthesis. With respect to Ch/ChS NFs fabrication: electrospinning, self-assembly, and phase separation are the more predominantly used methods. Few other add-on techniques have also been applied in the case of Ch/ChS NFs. These include microcontact printing, simple mechanical treatment, ultrasonication, and 2,2,6,6-tetramethylpiperidinooxy (TEMPO) mediated oxidization. Electrospinning, uses electrostatic forces to generate polymer NFs, and is an age-old technique, with an experience of 60 years. In case of Ch/ChS NFs, electrospinning has been the most frequently used and published method. The electro-spinning process is the typical method of artificially producing NFs from a polymer solution. If the characteristic NF structure of chitin is maintained after deacetylation, a downsizing process may be useful for the production of chitosan NFs. Dry chitosan powder was treated using the high-pressure waterjet system and disintegrated into NFs. An electrospun nanofibrous mat of pure chitosan was successfully prepared by Ohkawa and coworkersusing trifluoroacetic acid (TFA) as electrospinning solvent. Under optimized conditions, homogenous chitosan fibers with a mean diameter of 330 nm were prepared. Sangsanoh et al.developed electrospun chitosan nanofibers (average diameters of 126 ± 20 nm) using TFA/DCM (70:30 v/v) as electrospinning solvent. Haider and Parkprepared similar chitosan nanofiber mats by electrospinning. Vrieze et al.attempted a range of acid aqueous solutions for developing chitosan nanofibers by electrospinning. Molecular self-assembly is another powerful approach, mediated by notable hydrogen bonds, weak non-covalent bonds, ionic bonds, van der Waals interactions and hydrophobic interactions. Ch NFs with diameters of 3 nm have been fabricated in hexafluoroisopropanol (HFIP) using facile self-assembly. Phase separation technology works on the thermodynamic demixing of a homogeneous polymer-solvent solution into a polymerrich phase and polymer-poor phase; this has been used to prepare ChS NFs. Microcontact printing is a powerful technique successfully applied to the fabrication of nanostructured dendrimers, peptides, and conducting polymers. Ch NFs have been obtained using the microcontact printing method by dissolving Ch in HFIP. Ultrasonication has also been widely used to obtain individualized Ch/ChS NFs. Using this simple technique, Ch NFs that are 3-4 nm wide and few microns in length were prepared. The TEMPO-mediated oxidation was originally devised for cellulose NFs, Fan et al. successfully prepared Ch NFs by TEMPO-mediated oxidation. Ifuku and his research group have elaborately worked on the fabrication of Ch NFs from various sources using a simple mechanical technique of grinding. They have successfully demonstrated the preparation of Ch NFs from crab shells by a disintegration process. Crab shells have a hierarchical organization with various structural levels.displays the representatives of chitin organization in various chitin sources. They (Ch NFs) are enveloped within protein sheaths and occur as such in the exoskeletons of crabs. The shells were first purified by a series of conventional chemical treatments and then subjected to mechanical treatment. Proteins and minerals were removed using NaOH and HCl treatmentsand then a grinder was used. A pair of grinding stones disintegrated the hierarchical organization of Ch in the crab shells. Highly uniform Ch NFs with a width of approximately 10 nm were obtained. This simple but powerful method obtains homogeneous chitin NFs from waste crab shells in large quantities. The same group also successfully prepared Ch NFs from prawn shellsusing a method similar to that of the crab shells, since prawn shells are also made up of a hierarchical organization. Penaeus monodon (black tiger prawn), Marsupenaeus japonicas (Japanese tiger prawn), and Pandalus eous Makarov (Alaskan pink shrimp) shells were demonstrated for extraction of Ch NFs. These species serve as seafood delicacies and their shells are thrown away as food waste. These authors also reported the preparation of Ch NFs from mushroom. The cell walls of mushrooms consist of Ch NFs that occur complexed with glucans. Five widely consumed species of mushrooms: Pleuotus eryngii (king trumpet mushroom), Agaricus bisporus (common mushroom), Lentinula edodes (shiitake), Grifola frondosa (maitake), and Hypsizygus marmoreus (bunashimeji) were used to extract Ch NFs. Because the organization of chitin in the mushroom cell walls is different from those of crab and prawn shells (which have a hierarchical organization of chitin), the extraction method was modified. Ch NFs were also extracted from squid pens. β-type chitin is present in squid pens. Fan and Isogai et al. prepared Ch NFs from squid pen β-chitin. Mechanical treatments under acidic conditions lead to extraction of Ch NFs. Cationic charges accelerate NF conversion and lower crystallinity, parallel chainpacking mode and relatively weak intermolecular forces of squid pen β-chitin play a crucial role in the preparation of chitin NFs. Ch NFs have also been prepared from commercial chitin. Dry chitin was dispersed in acidic water and passed through a grinder. Acquiring NFs from commercial chitin is advantageous because a large amount of chitin can be obtained within a few hours. A high-pressure waterjet system called the Star Burst instrument was used successfully for the nanofibrillation of dry chitin. Ifuku and group have extensively worked on and published on Ch NFs and their preparations and modifications.gives the overall work flow towards procuring Ch/ChS NFs. The shells were first purified by a series of conventional chemical treatments and then subjected to mechanical treatment. Proteins and minerals were removed using NaOH and HCl treatmentsand then a grinder was used. A pair of grinding stones disintegrated the hierarchical organization of Ch in the crab shells. Highly uniform Ch NFs with a width of approximately 10 nm were obtained. This simple but powerful method obtains homogeneous chitin NFs from waste crab shells in large quantities. The same group also successfully prepared Ch NFs from prawn shellsusing a method similar to that of the crab shells, since prawn shells are also made up of a hierarchical organization. Penaeus monodon (black tiger prawn), Marsupenaeus japonicas (Japanese tiger prawn), and Pandalus eous Makarov (Alaskan pink shrimp) shells were demonstrated for extraction of Ch NFs. These species serve as seafood delicacies and their shells are thrown away as food waste. These authors also reported the preparation of Ch NFs from mushroom. The cell walls of mushrooms consist of Ch NFs that occur complexed with glucans. Five widely consumed species of mushrooms: Pleuotus ## Medical milestones of ch nfs When it comes to nanofibers, Ch NFs and their preparation methods have been well documented. However, the medical applications of Ch NFs include scattered reports supported by few publications. The available scanty information are summarized below. Ch possesses nonspecific antiviral and antitumor activitiesand nanosized chitin influences its effects on immune cells. Ch NFs have been proposed for tissue engineering scaffolds, drug delivery and wound dressing application. There are a few reports on the successful use of Ch/ChS NFs in the area of tissue engineering because the structure is similar to glycosaminoglycans. Ch/ChS NFs attach and proliferate osteoblast cells, support the development of mineralized bone matrix. They also are easy to mold into various geometries and are biocompatible, biodegradable, and exhibit strong antibacterial activity. Bone tissue engineering procedures usually combine hydroxyapatite (HA) with the chitin and chitosan scaffolds to improve the activity and viability of cells, as well as enhance mechanical and cell attachment properties of scaffolds..gives the overall work flow towards procuring Ch/ChS NFs. ## Medical milestones of ch nfs When it comes to nanofibers, Ch NFs and their preparation methods have been well documented. However, the medical applications of Ch NFs include scattered reports supported by few publications. The available scanty information are summarized below. Ch possesses nonspecific antiviral and antitumor activitiesand nanosized chitin influences its effects on immune cells. Ch NFs have been proposed for tissue engineering scaffolds, drug delivery and wound dressing application. There are a few reports on the successful use of Ch/ChS NFs in the area of tissue engineering because the structure is similar to glycosaminoglycans. Ch/ChS NFs attach and proliferate osteoblast cells, support the development of mineralized bone matrix. They also are easy to mold into various geometries and are biocompatible, biodegradable, and exhibit strong antibacterial activity. Bone tissue engineering procedures usually combine hydroxyapatite (HA) with the chitin and chitosan scaffolds to improve the activity and viability of cells, as well as enhance mechanical and cell attachment properties of scaffolds. The in vivo effects of Ch NFs after oral administration are undocumented. The preventive effects of Ch NFs in a mouse model of DSS-induced acute ulcerative colitis (UC) The in vivo effects of Ch NFs after oral administration are undocumented. The preventive effects of Ch NFs in a mouse model of DSS-induced acute ulcerative colitis (UC) was studied. They lead to decreased colon inflammation and histological tissue injury in mouse models. Ch NFs have anti-inflammatory properties and are able to suppress NF-κB and MCP-1 activation and fibrosis in acute UC mouse models. This confirms that ChNF can be prescribed as a potentially novel medicine or functional food for patients with inflammatory bowel disease. Furthermore, the application of Ch NFs to skin improved the epithelial granular layer, increased granular density and resulted in lower production of TGF-β. This implies that Ch NFs can be incorporated into cosmetics or textiles. Ch NFs are reported to possess other powerful biological activities and few applications have been proposed with respect to biomedical applications. However, as of now, their (Ch NFs) biomedical accomplishments still are restricted to limited research validations. ## Medical milestones of chs nfs ChS NF are reported for their excellent biological properties related to biocompatibility, biodegradability, cellular binding capability, wound healing, antitumor, and antibacterial applications. ChS NFs incorporated with hydroxyapatite have been used in bone tissue engineering because of their structural similarity to the extracellular matrix of the native bone tissue. ChS NFs containing hydroxyapatite were electrospun to test its ability to regenerate bones. Hydroxyapatite blended ChS NFs crosslinked with genipin facilitated the formation of bones. ChS NFs were electrospun and used as scaffolds in vascular tissue engineering. A 3D gradient heparinized chitosan/poly-3-caprolactone NF scaffold was used. Cooper et al. have fabricated randomly oriented and aligned chitosan/poly-3-caprolactone (chitosan/PCL) fiber scaffolds and investigated their potential use in nerve regeneration. Chitosan NFs with diameters of 4 nm and 12 nm were coupled with poly-D-lysine (PDL) and employed in mouse cortical neuron cultures to examine their capability to support cell attachment, neurite coverage and survival, suggesting significant improvement in long-term cell viability. PLGA/Ch NFs were tested for their potential as drug release systems. ChS blended with ethylenediaminetetraacetic acid (EDTA) and polyvinyl alcohol (PVA) (ChS-EDTA/PVA) as NF scaffolds have been reported. Garcinia mangostana fruit hull extracts and lysozyme were incorporated into ChS-EDTA/PVA NFand used as wound dressings. Chitosan is endowed with natural antimicrobial activity, Ch NF antibacterial activity has come in handy for wound dressing, antibacterial filtration and tissue engineering applications. ChS NF biosensors have also been developed. ChS/PVA NF were electrospun for lipase immobilization. A biosensor based on ChS NFs incorporated with cholesterol oxidase and Au NPs was developed to detect cholesterol. The clinical use of chitin and chitosan-based NFs still remains challenging and needs further insights. Not many supportive clinical trials have been achieved. Kossovich et al. developed ChS/PEO NFs towards wound dressings for IIIa and IIIb degree burns. The results were highly promising. However, nothing much has been progressing in this direction of putting ChS NFs to clinical use. Bazhaban and her colleagues in 2013 fabricated a biodegradable nanofibrous controlled release drug delivery system using chitosan and Beta cyclodextrin (β-CD); salicylic acid was the model drug. The results were promising, and suggested that the electrospun nanofibers of PVA/ChS NFs-g-β-CD could be of high potential for biomedical applications. Its almost a decade this report has come out and yet, once again no progress has been made in this direction. Nawzat et al., 2019, have presented an overview of ChS NFs and their drug delivery applications. ## Ch/chs nf modifications and nanocomposites Chitin nanomaterials have a reactive surface covered with hydroxyl groups, opening up the possibility for extensive chemical modification. Using surfactants or by chemical grafting/modifications, the mechanical performance of the Ch nanocomposites can be enhanced. Reinforced Ch/ChS based nanocomposites blended with poly (methyl methacrylate), epoxy, polystyrene, polyaniline, polysulfone, polycarbonate, and thermoplastic polyurethanehave been reported. Apart from these, Ch/ChS composites with hydrophilic and bio-based polymeric matrices, namely, chitosan, starch, PLA, and cellulose, have also been fabricated. Several chemical modifications of chitin NF surfaces have been achieved: acetylation, deacetylation, phthaloylation, naphthaloylation, maleylation, chlorination, TEMPO-mediated oxidation, and graft polymerization. Modification is a promising and effective way to design functional materials and enhance their functionality. The blending in of two to three different materials, allows the harnessing of their individual properties in one material. ChS nanofibers have been developed using chitosan and synthetic polymers such as poly(vinyl alcohol) (PVA), poly(ethylene oxide) (PEO), poly(ethylene terpthalate) (PET), polycaprolactone (PCL), poly(lactic acid) (PLA), and nylon-6. These composite nanofibers are more advantageous than pure chitosan, because their mechanical, biocompatible, and antibacterial properties were drastically enhanced by the addition of these polymers. Ding et al., 2014 have published a comprehensive summary of the various Ch/ChS NF materials and their medical applications. Chitin based binary blend NFs were fabricated using HFIP as solvent. Park et al., reported Ch/PGA blend NFs using HFIP as the solvent via electrospinning method. Using the same solvent, Ch/silk blend NFs were fabricated as well. Synthetic polymers, such as PVA, PLGA, PEO, and PGA have been widely used with ChS NFs. These are expected to deliver much towards medical applications. Biopolymers such as silk, cellulose, collagen, hyaluronic acid, alginate, and zein have been electrospun with ChS. Native cellulose/chitosan NF composites have also been successfully produced. Hydroxyapatite/ChS NF scaffolds have been prepared for tissue engineering. A further development in chitin/chitosan based nanocomposites, is the production of ternary composite NFs blended with synthetic polymers, biopolymers and inorganic substances. These composite scaffolds are expected to show better mechanical properties and biocompatibility. Other chitosan/synthetic polymer/natural polymer ternary blend NFs include, carboxymethyl chitosan/poly(vinyl alcohol)/silk fibroin, chitosan/lysozyme/poly(vinyl alcohol), poly (lactic acid)/chitosan/collagen, and collagen/chitosan/hermoplastic polyurethane. Some inorganic substances such as silica, Au nanoparticles, Ag nanoparticles, multiwalled carbon nanotubes, and hydroxyapatite were incorporated into chitosan/synthetic polymer (PVA and PEO) NFs to enhance the mechanical properties and confer antibacterial activity, protein adsorption ability, cell attachment and proliferation. Carboxymethyl chitosan is one of the most frequently used chitosan derivatives to obtain NFs using the electrospinning method. In order to enhance the spinning capacity, some synthetic polymers such as PVA and PEO were blended with carboxymethyl chitosan to form NFs, their medical applicability has also been demonstrated. Quaternized chitosan was another chitosan derivative which was chosen to form NFs owing to its excellent antibacterial activity and biocompatibility.summarizes the Ch/ChS NF based nanocomposites that are currently established. ## Future outlook and conclusions The current status with respect to Ch/ChS NF synthesis, methods, medical deliverables, and consolidation of available nanocomposites for future endeavors have been comprehensively presented in this review. During the process of reviewing the existing scenario with respect to Ch/ChS NFs, staggering ends and grey areas and lacunae have been identified. For instance, there is almost no information or research findings on Ch based NPs, when ChS based NPs have been extensively prepared and applied towards biomedical applications. The one straightforward existing reportconfirms the preparation and usage of Ch NPs for wound healing applications. This being true, then there could ## Future outlook and conclusions The current status with respect to Ch/ChS NF synthesis, methods, medical deliverables, and consolidation of available nanocomposites for future endeavors have been comprehensively presented in this review. During the process of reviewing the existing scenario with respect to Ch/ChS NFs, staggering ends and grey areas and lacunae have been identified. For instance, there is almost no information or research findings on Ch based NPs, when ChS based NPs have been extensively prepared and applied towards biomedical applications. The one straightforward existing reportconfirms the preparation and usage of Ch NPs for wound healing applications. This being true, then there could definitely be more to harness from Ch NPs. This is somewhere research needs to be focused, and if there is a challenge, it needs to be exposed and addressed. This review interestingly also found that there was a strong bias in the usage of Ch and ChS NFs for biomedical applications. Ch NFs are very less represented when it comes to medical applications. The application aspect of Ch NFs is significantly lagging. There are few reasons, that of course indicate that chitosan is better than chitin, owing to its solubility and chitosan's degree of deacetylation which is proportional to its biological activity. However, there is no information on the continuing retardation in the bio applicability of Ch NFs versus ChS NFs. Chitin as such, facing inadequacy is understandable, but with the introduction of nano inputs and preparation of advanced NFs, there is always plenty of room for reversal of the inherent material limitations. This being so, this review provokes researchers to apply Ch NFs for biomedical aspects where previously chitin materials have failed. Drug delivery is another aspect that ChS NFs have been luxuriously used and Ch NFs have been seldom used. This is another research direction. ChS-based composites, binary and ternary composites have been well documented, but their medical applications remain restricted to scattered scanty publications; this is one area that needs development. Ch based nanocomposites are once again relatively fewer compared to ChS NF nanocomposites; this is another area that can be built on. Ch NF nanocomposites could be a real launching factor, breaking limitations and bringing Ch based biomedical applications to the forefront. Meanwhile ChS NFs also need expansion laterally blending with composites. Synergistic composites combining multiple polymeric and inorganic metal nanoparticle systems, could extend their (ChS NFs) utility for medical applications further. This review expects to draw the attention of workers to expand on these grey areas.

Patient-reported outcome measures for cancer caregivers: a systematic review Purpose Informal caregivers provide invaluable help and support to people with cancer. As treatments extend survival and the potential burdens on carers increase, there is a need to assess the impact of the role. This systematic review identified instruments that measure the impact of caregiving, evaluated their psychometric performance specifically in cancer and appraised the content. Methods A two-stage search strategy was employed to:(1) identify instruments that measure the impact of caregiving, and (2) run individual searches on each measure to identify publications evaluating psychometric performance in the target population. Searches were conducted in MEDLINE, EMBASE, CINAHL and PsycINFO and restricted to English for instrument used and article language. Psychometric performance was evaluated for content and construct validity, internal consistency, test-retest reliability, precision, responsiveness and acceptability. Individual scale items were extracted and systematically categorised into conceptual domains. Results Ten papers were included reporting on the psychometric properties of eight measures. Although construct validity and internal consistency were most frequently evaluated, no study comprehensively evaluated all relevant properties. Few studies met our inclusion criteria so it was not possible to consider the psychometric performance of the measures across a group of studies. Content analysis resulted in 16 domains with 5 overarching themes: lifestyle disruption; well-being; health of the caregiver; managing the situation and relationships. Conclusions Few measures of caregiver impact have been subject to psychometric evaluation in cancer caregivers. Those that have do not capture well changes in roles and responsibilities within the family and career, indicating the need for a new instrument. # Background Informal caregivers, whether they are spouse, family member or friend, often provide a significant amount of help and support for people with cancer. Informal caregiving is pivotal to the overall outcome of a patient's treatment, and thus, maintaining the health and satisfaction of caregivers is essential to maximise the well-being of both parties [bib_ref] Interventions with family caregivers of cancer patients: Meta-analysis of randomized trials, Northouse [/bib_ref]. Caregiving can undoubtedly place a strain on the caregiver [bib_ref] Psychological adjustment of cancer caregivers with multiple roles, Kim [/bib_ref] , but the role can also provide a source of happiness and boost self-efficacy and a sense of worth . An increasing number of patients are living a longer life with cancer. As such, there is a growing recognition that broader aspects of their lives and those of the family are affected across the disease trajectory [bib_ref] Advanced cancer: Emergence of a new survivor population, Haylock [/bib_ref]. Informal caregivers could be viewed as ''second-order patients in their own right'' [bib_ref] Lifestyle interference and emotional distress in family caregivers of advanced cancer patients, Cameron [/bib_ref] ; consequently, a well-validated measure to assess the impact of disease and treatment on their lives and overall well-being is crucial. There is a raft of measures designed to assess caregiver impact so the choice for researchers may be unclear. The instruments currently used focus on three areas: caregiver burden, caregiver need and quality of life. Some measures are not well validated, and many have been developed for use with caregivers in very different circumstances, for example the elderly with cognitive impairment [bib_ref] Care for the caregivers: A review of self-report instruments developed to measure..., Deeken [/bib_ref]. In order to better inform researchers on the content and evaluation of commonly used instruments, we identified and evaluated the psychometric performance of measures used in the cancer caregiver population and appraised their content, what is and what is not captured, with particular regard to broader areas of life experience such as the impact on career and family. # Methods The review involved a two-stage search: to identify generic and cancer-specific self-report instruments used to measure the impact of caregiving on informal caregivers, and [bib_ref] Interventions with family caregivers of cancer patients: Meta-analysis of randomized trials, Northouse [/bib_ref] to identify evidence about psychometric properties and performance of these instruments in the specific context of cancer. ## Search stage 1: identifying candidate instruments A combination of controlled syntax (MeSH) and free-text terms were used. Four groups of terms were generated: (1) generic names for measures; (2) impact on caregiver; (3) describing the population; and (4) psychometric performance. OvidSP was used for MEDLINE [MEDLINE(R) In-Process & Other Non-Indexed Citations and MEDLI-NE(R) 1946 to Present] and EMBASE (1947-current) searches. EBSCOhost was used for CINAHL (1937-present) and PsycINFO (from 1800s to present) searches. Terms were modified as appropriate for each database and limited to English language only. Searches were run on 20 November 2014 (see ''Appendix 1'' for the search strategy used for MEDLINE, adapted for other databases). ## Study selection criteria Inclusion criteria for stage 1 were self-report instruments of the impact of caring for patients with cancer or any other condition on the caregiver. Searches were not limited by study design or date, but were restricted to articles in the English language. Instruments were excluded if they were developed: (1) to address a broad population not specifically for caregivers; [bib_ref] Interventions with family caregivers of cancer patients: Meta-analysis of randomized trials, Northouse [/bib_ref] to focus on caregivers of children or children who are themselves caregivers; [bib_ref] Psychological adjustment of cancer caregivers with multiple roles, Kim [/bib_ref] to be administered only by an interviewer or clinician; (4) to measure unmet needs or objective aspects of caregiving, e.g. the amount of time or nature of tasks fulfilled; [bib_ref] Advanced cancer: Emergence of a new survivor population, Haylock [/bib_ref] to evaluate caregivers' assessment or beliefs about their caregiving skills or performance; [bib_ref] Lifestyle interference and emotional distress in family caregivers of advanced cancer patients, Cameron [/bib_ref] for use in a non-English-speaking population and for which an English version was not available; [bib_ref] Care for the caregivers: A review of self-report instruments developed to measure..., Deeken [/bib_ref] for use by patients rather than caregivers (e.g. patient estimates of the impact of their illness on the caregiver); and/or to measure caregiver bereavement. Titles and abstracts were screened independently by two reviewers (VS/LM) for names of instruments that met the inclusion criteria, resulting in a list of eligible candidate instruments . Search stage 2: Identifying evidence of the psychometric properties of candidate instruments in the cancer caregiver population Separate searches were conducted for each of the candidate instruments for studies designed to evaluate their psychometric performance in caregivers of cancer patients. Search terms are grouped as follows: (1) names and acronyms of the candidate instruments identified in stage 1; (2) target population; (3) psychometric terms; and (4) cancer terms (see ''Appendix 2'' for the search strategy used for MED-LINE and adapted for other databases). Searches were run on 16 January 2015 (CINAHL and PsycINFO) and 5 February 2015 (MEDLINE and EMBASE). ## Study selection criteria Studies that reported the reliability, validity, responsiveness, precision and/or acceptability of the caregiver impact measure and met the inclusion and exclusion criteria listed above were selected for this review. Cross-cultural studies were included only if referencing an English language version of the instrument. Searches were not limited by study design or date, but were limited to articles and instrument use in the English language. In addition to the exclusion criteria from stage 1, papers were excluded if: the instrument was used as a ''gold standard'' to test other measures; psychometric evidence was reported incidentally in studies not designed to evaluate those properties; studies addressing preference weighting or scaling issues for preference-based measures; editorials, opinions, letters and meeting abstracts. Titles and abstracts were screened independently by two reviewers (VS/LM, [fig_ref] Figure 2: Flow chart showing study selection in search stage 2 [/fig_ref]. ## Citation chasing Backwards citation chasing (one generation) using reference lists of all studies included in this stage of the review and forwards citation chasing (one generation) using Science Citation Index Expanded and Social Science Citation Index Expanded via Web of Science identified no additional eligible studies. ## Data extraction For each included measure, we extracted: name of measure and acronym, key reference/development paper, purpose of measurement, number of items, completion time, response options, recall period, population originally developed with and types of domains/dimensions assessed. For each included paper, the following descriptive data were extracted: instrument version, first author name, publication year, study aim, study population, number of participants and setting/country where the study was conducted. Any data on evidence of the psychometric properties or performance of instruments were extracted including content validity (theoretical framework and/or qualitative research), construct validity (structural validity and hypothesis testing), internal consistency, test-retest reliability, precision, responsiveness and acceptability. Data were extracted by one reviewer (LM/VS) and checked by a second reviewer (LM/VS). ## Evidence for psychometric performance Evidence of psychometric performance was compared to reference criteria for (1) content validity (qualitative research with potential respondents and involvement in development stage and item generation, clear conceptual framework); (2) construct validity assessed through convergent and divergent validity demonstrated by the ability to differentiate known groups, and/or a pattern of correlation between the scale and other measures; (3) structural validity from factor analysis; (4) criterion validity (concurrent validity assessed through correlation with a gold standard and/or predictive validity where the predicted strength and direction of correlations/direction of group differences should be identified a priori); (5) reproducibility/test-retest reliability (intraclass correlation coefficient [0.7 adequate, [0.9, excellent); (6) internal consistency (Cronbach's alpha coefficient 0.7 B a C 0.9, item total correlations [0.2); (7) responsiveness (change pre-post intervention statistically significant and/or difference of expected magnitude); (8) precision (assessment of measurement error, floor or ceiling effects \15 %; evidence from Rasch analysis); and (9) acceptability (nonresponse/non-completion of questionnaires, proportion of missing data) . For each property, the paper was given a rating of 0 if it did not evaluate or report the property, * if the property was evaluated and met the criteria partially (e.g. not for all domains), ? if the property was evaluated and met the criteria and -if the finding went against the prediction. Judgements on whether criteria were met were made by two reviewers (VS/LM) with disagreement resolved in discussion with another reviewer (LJF/VJ) where necessary. Content validity is only appraised for papers reporting measure development. ## Examination of instrument content and categorisation into related domains Individual scale items from all included measures were systematically categorised by the authors into conceptual domains. Initial domains were identified from the literature, and additional domains were defined until all individual items had been mapped. The content of each was then reviewed by the team to ensure that the concepts were consistently applied and had face validity . # Results The purpose of stage 1 was to generate a list of eligible candidate instruments. Thirty-two were identified , and in stage 2, individual searches were conducted for each. Ten measures were excluded as no candidate papers After deduplication, 186 unique records were screened. One hundred and seventy-six were excluded because they did not meet inclusion criteria resulting in the inclusion of 10 papers that reported on the psychometric properties of 8 eligible measures in the cancer caregiver population (see [fig_ref] Figure 2: Flow chart showing study selection in search stage 2 [/fig_ref] for full details of reasons for exclusion). [fig_ref] Table 1: General characteristics of identified measures [/fig_ref] details the general characteristics of the 8 included measures, including full name and acronym. We refer to measures by the acronym. Of the 8 included measures, 5 were initially developed for cancer caregivers, 2 of which were specifically developed for use in the palliative setting [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref]. Some measures were developed relatively recently [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref] , five between 1980 and 1999 [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] [bib_ref] Family caregiving in cancer pain management, Ferrell [/bib_ref] [bib_ref] Relatives of the impaired elderly: Correlates of feelings of burden, Zarit [/bib_ref]. Two [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] measure caregiver appraisal specifically, with a theoretical underpinning from the stress and coping model of Lazarus and Folkman; two were designed to measure subjective burden ± distress [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref] [bib_ref] Relatives of the impaired elderly: Correlates of feelings of burden, Zarit [/bib_ref] (the Zarit Burden Interview was later revised; three were multidimensional quality of life measures [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] Family caregiving in cancer pain management, Ferrell [/bib_ref] and one a multidimensional measure of caregivers' reactions to caring for a family member [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref]. [fig_ref] Table 2: Studies evaluating psychometric performance of measures in the cancer caregiver population [/fig_ref] describes the ten studies reporting on the psychometric properties of the measures in the cancer caregiver population in terms of the instrument and version, study aim, population, setting country and number of participants. ## Psychometric performance Appraisal of the psychometric performance reported in each paper is given in [fig_ref] Table 3: Appraisal of measure performance and characteristics in the cancer caregiver population [/fig_ref]. ## Content validity Of the six studies describing measure development, content validity was generally well described and acceptable. Four (BASC, CQOLC, CRA, QOLLTI-F) describe qualitative work with potential respondents for item development and reduction [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] ; two (ACS, FACQ-PC) describe a clear underpinning conceptual framework but no involvement of potential respondents [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref]. ## Criterion validity Concurrent validity: the 6 short forms of the ZBI were validated against the 22-item version as gold standard. Spearman rank order correlations ranged from 0.63 for the one-item version to 0.95 for the 12-item scale [bib_ref] Short-form Zarit Caregiver Burden Interviews were valid in advanced conditions, Higginson [/bib_ref]. Concurrent validity of the ACS Benefit subscale only was assessed against the Benefit Finding Scale as the gold standard (r = 0.56) [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref]. Predictive validity: predictive validity of the ACS was assessed against hopelessness and depression scores at time 2 [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref]. Although overall a significant amount of variance in hopelessness (33.3 %) and depression (27.8 %) was explained by ACS scores at time 1, only half of the predictive validity hypotheses were supported. Criterion validity of the QOLLTI-F was assessed using a 2-item measure of global quality of life. QOLLTI-F was predicted between 43 and 55 % of the variance depending on whether individual items (55 %), subscale scores (53 %) or total score (43 %) was regressed. ## Structural validity Structural validity using factor analysis was described in five of the studies. For the CRA [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] , exploratory factor analysis supported the five-subscale solution accounting for 65.1 % of variance. Confirmatory factor analysis demonstrated factorial invariance across disease (cancer vs dementia), caregiver type (spouse vs non-spouse) and over time. For the QOLLTI-F [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] , the authors describe an acceptable seven-factor solution with exploratory factor analysis (although the total amount of variance explained is not reported) with factor loadings from 0.39 to 0.88. For the FACQ-PC [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] , principal axis factor analysis supports a four-factor solution with factor loadings ranging from 0.33 to 0.92. Although all items load highest on the predicted factor, two items cross load ([0.3). Lambert and colleagues [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref] report a three-factor solution for the ACS which supports the original subscales, had minimal cross-loadings and factor loadings ranging from 0.405 to 0.726. Glajchen et al. [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref] report a five-factor solution for the BASC while noting that one item cross loads. The authors do not report their methods or the factor loadings. ## Construct validity: hypothesis testing Six studies assessed construct validity through convergent and divergent validity. For the ACS, only 5/12 correlations between subscales of the ACS and other measures exceeded the authors' criterion of ±0.3 to demonstrate construct validity [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref]. Both papers assessing the CQOLC report moderate-to-high correlations with measures completed at the same time [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) Scale: Revalidation in a home..., Weitzner [/bib_ref]. Only the initial validation study assessed divergent validity using dissimilar measures and found that these gave low correlations with CQOLC scores as expected [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref]. Construct validity of the BASC was supported by an appropriate pattern of moderate-strong correlations with similar measures [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref]. Strong correlations were found between subscales of the FACQ-PC [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] and measures used to test convergent validity; however, positive caregiving appraisals were only weak-moderately associated with positive affect (r = 0.3). The data on confirmatory factor analysis are also reported by Stommel and colleagues which was identified in forward citation searches. This paper is excluded as it concerns the same data b Authors have now produced a second version of this questionnaire with minor changes and an additional question; however, the validation studies are as yet unpublished Similarly, to demonstrate divergent validity, four correlations were calculated between subscales and other measures which should yield low, negative correlations. While all were negative, two correlations were moderate in magnitude (r = -0.4 and r = -0.38). Construct validity for the CRA [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] was assessed by correlating subscale scores with caregiver depression and patient dependencies in activities of daily living (ADL). The five subscales were, as predicted, weakly correlated with patient dependencies in ADL. Correlations with depression were in the appropriate direction and ranged from -0.23 to 0.57 in magnitude. Three studies conducted hypothesis testing by assessing ''known-group'' differences. Group differences analysis for the ACS was only partially supportive of construct validity with only 3/9 hypotheses significant [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref]. The BASC was able to discriminate between male and female caregivers and between different relationships between caregivers and patients. The negative personal impact subscale, but not the total score, differentiated between caregivers with and without mental health conditions. There were weak correlations overall with depression, high blood pressure and gastrointestinal complaints. All short forms of the ZBI were shown to have good discriminative validity to correctly classify participants as those with and without burden (contrasting to the classification on the 22-item version as gold standard). Two studies [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] [bib_ref] Quality of life of patients with advanced cancer and acquired immune deficiency..., Sherman [/bib_ref] report comparisons between groups (e.g. male/female; spouse/non-spouse), but these were not established a priori as known-group differences for hypothesis testing. One did not examine construct validity with convergent, divergent or known-group analysis [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref]. ## Internal consistency Internal consistency was assessed in all papers. All subscales of the ACS had a [ 0.7 in both papers [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref] with the exception of the challenge subscale, which was subsequently dropped from the measure [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] ; the threat subscale slightly exceeded the upper limit of a at 0.91 in one paper. [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] Overall a for the BASC was just acceptable (0.7); the negative personal impact factor, which can be used as an independent subscale, was 0.8. For the CQOLC, a approaches and slightly exceeds the upper limit (a = 0.87 and 0.91, respectively) [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) Scale: Revalidation in a home..., Weitzner [/bib_ref]. The five subscales of the CRA range from a = 0.8 to 0.9 [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] and the four subscales of the FACQ-PC from a = 0.73 to 0.86. Item total correlations were all in excess of 0.2, the strongest 0.78 [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref]. Overall a for the QOLLTI-F was 0.86. The individual subscales were generally weaker ranging from a = 0.48 to 0.81 which may reflect the small number of items in some subscales. The measure also includes two single-item subscales [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref]. Internal consistency for the QOL-FV was a = 0.89. Finally, internal consistency for the ZBI 22-item version as gold standard was a = 0.88 and ranged from 0.69 for the 4-item short version to 0.85 for the 12-item version. ## Test-retest reliability Three of the five papers with at least two time points did not attempt to assess test-retest reliability [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref] [bib_ref] Quality of life of patients with advanced cancer and acquired immune deficiency..., Sherman [/bib_ref]. Test-retest reliability of the CQOLC was found to be excellent (0.95) [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref]. For the total QOLLTI-F, test-retest reliability was found to be acceptable between T1 and T2 (0.77) and T2 and T3 (0.80). Intraclass correlations for individual subscales were below an acceptable level in 10 out of 14 cases, which may reflect the small number of items in the subscales [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref]. ## Responsiveness Responsiveness of the QOLLTI-F [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] was assessed by contrasting subscale scores on days that participants considered to be bad, average and good. These differences were statistically significant in all comparisons with the exception of the financial concerns subscale between average and good days. All differences between good and bad days exceeded 0.5 s.d. for minimal important difference. Only 3/8 comparisons did so between good and average and 4/8 between average and bad days. The ''potential to be responsive to change'' of the CQOLC was assessed by using CQOLC to predict patient performance status at a single time point rather than measure responsiveness to change over time. The studies report contradictory findings: in one [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] , the predicted significant negative correlation between CQOLC scores and patient performance status is reported as significant (r = -0.46, p \ 0.0001), but in the other [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) Scale: Revalidation in a home..., Weitzner [/bib_ref] , this correlation approaches zero (r = 0.09). ## Precision None of the included studies conducted Rasch analysis or an assessment of measurement error. Floor and ceiling effects were not formally reported in any paper although two subscales of the QOLLTI-F [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] were described as having a lack of variance due to ceiling effects which made them less predictive of global quality of life. The subscales, quality of care and relationships, comprised two items each. ## Acceptability The acceptability of measures was not consistently reported and was difficult to assess using missing data and participation rates, as the measure is often given as part of a pack and information is not assessed separately. No information pertaining to acceptability was provided by four studies [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] [bib_ref] Short-form Zarit Caregiver Burden Interviews were valid in advanced conditions, Higginson [/bib_ref] [bib_ref] Measuring appraisal during advanced cancer: Psychometric testing of the appraisal of caregiving..., Lambert [/bib_ref] [bib_ref] Quality of life of patients with advanced cancer and acquired immune deficiency..., Sherman [/bib_ref]. In five studies, acceptability was appraised as only partially evidenced due to high dropout or incomplete data [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref] [bib_ref] Development of a brief assessment scale for caregivers of the medically ill, Glajchen [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] [bib_ref] The caregiver reaction assessment (CRA) for caregivers to persons with chronic physical..., Given [/bib_ref] [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) Scale: Revalidation in a home..., Weitzner [/bib_ref] , surprising for the QOLLTI-F which had thoroughly tested acceptability in the development phase [bib_ref] QOLLTI-F: Measuring family carer quality of life, Cohen [/bib_ref]. For the ACS [bib_ref] Caregiving demands and appraisal of stress among family caregivers, Oberst [/bib_ref] , overall response rate was 74 % (including postal responses) and only 3/50 participants were eliminated due to missing data, suggesting the questionnaire was acceptable. ## Examination of instrument content and categorisation into related domains The 8 included instruments yielded 194 individual items. These were categorised into 16 conceptual domains under 5 overarching themes of approximately equal size: lifestyle disruption (22 % of items); well-being (22 %); health of the caregiver (21 %); managing the situation (18 %) and relationships (18 %). Most dominant domains were ''confidence, self-esteem and self-efficacy'' (24 items across 7 measures) and ''psychological health of the caregiver'' (22 items across 6 measures). Least represented were ''impact on other family members'' (2 items across 2 measures) and ''impact on paid employment'' (2 items across 2 measures). The distribution and total number of items across the different domains along with example items are given in. # Discussion This systematic review was conducted to investigate instruments commonly used to measure caregiver impact in cancer. Specifically, we sought to identify (1) what caregivers were being asked about, and (2) whether the measures performed well in psychometric evaluation. Psychometric appraisal is critical to establish the quality and standards of a measure in a given context. With so many instruments available to researchers, this review is intended as a resource to enable researchers to judge for themselves whether the content and quality of the instruments described match their requirements. For 24 of the 32 identified measures, we found no evidence of psychometric performance using English language versions with cancer caregivers (see electronic supplementary material for a list of these measures). This is not to say that the measures have not been evaluated, but that we found no evidence in cancer. When assessing the performance of an instrument, the context is critical as it may perform differently in other populations. Without evaluation in cancer, researchers cannot be sure that Items categorised under the domain ''spirituality'' make direct reference to spirituality, faith, prayer, church/temple, etc instruments are reliably measuring the intended constructs. For 6 of the remaining 8 questionnaires, evidence of psychometric performance was identified in only a single study. The small number of studies identified meant that the evidence for psychometric performance was appraised for each individual paper, rather than allowing the appraisal to consider performance across a group of studies. In particular, the lack of studies beyond the initial validation of some measures is of concern. Most studies collected data at a single time point, and so, responsiveness to change, test-retest reliability and measurement error were not assessed. For the most part, content validity and internal consistency were reported and were adequate. Structural validity was assessed using factor analysis in five studies; four met these criteria, one only partially. Construct validity was assessed using different approaches to hypothesis testing in seven papers, fully meeting the criteria in only three. In the limited number of papers included, strongest support for psychometric performance was reported for the CRA and CQOLC. We set out to examine not only the psychometric performance of these measures in a cancer population, but also to understand what concepts and domains were being assessed. Only one of the 16 conceptual domains, time for self, social life and leisure, was represented in all eight instruments. There was considerable overlap in the domains measured, however, with 8/16 domains being assessed in at least 6/8 measures. We have identified several areas which are not well captured by the instruments included in this review. ## Paid employment Impact on paid employment was assessed with a single question on two measures, neither of which addressed impact on career aspiration and planning or career progression, simply whether paid employment had been affected (FACQ-PC [bib_ref] Development and initial validation of a family appraisal of caregiving questionnaire for..., Cooper [/bib_ref] , QOL-F [bib_ref] Family caregiving in cancer pain management, Ferrell [/bib_ref]. ## Sexual activity Only two questionnaires ask about relationships in terms of sexual activity (CQOLC [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] , QOL-F [bib_ref] Family caregiving in cancer pain management, Ferrell [/bib_ref]. ## Family members We also found that impact on the family as a unit was not well covered in the current measures. Impact on other family members was only addressed with a single question on two questionnaires (again CQOLC [bib_ref] The Caregiver Quality of Life Index-Cancer (CQOLC) scale: Development and validation of..., Weitzner [/bib_ref] and QOL-F [bib_ref] Family caregiving in cancer pain management, Ferrell [/bib_ref]. Current scales do not adequately capture role changes and responsibilities in the household and family routines, for example the impact on other caregiving responsibility such as to children or parents. Taking on new roles and responsibilities can raise issues around feelings of competence in the role, role strain and conflict and family cohesion. Such changes may be fluid as the cared-for member of the family moves through different phases of their cancer and treatment. Five of the eight measures included in this review were developed between 1980 and 1999. There has been considerable societal change in the intervening years; families, roles and responsibilities are structured differently. It is not clear how appropriate some of the older questionnaires are for the present day. The activities associated with informal caregiving incorporate a range of tasks affecting different aspects of the life of the caregiver and the whole family [bib_ref] Caregivers of cancer survivors: The state of the field, Girgis [/bib_ref] [bib_ref] Physical, psychosocial, relationship, and economic burden of caring for people with cancer:..., Girgis [/bib_ref]. The impact of caregiving will vary depending not only on the patient's situation but also on family make-up, in terms of other caregiving responsibilities, financial and occupation role responsibilities and time of life [bib_ref] Lifestyle interference and emotional distress in family caregivers of advanced cancer patients, Cameron [/bib_ref] [bib_ref] The cancer family caregiving experience: An updated and expanded conceptual model, Fletcher [/bib_ref] [bib_ref] The family caregiver of the older cancer patient, Weitzner [/bib_ref]. Impact is also likely affected by the number of other social roles, such as employment and other caregiving responsibilities that the caregiver has [bib_ref] Psychological adjustment of cancer caregivers with multiple roles, Kim [/bib_ref]. There is limited research about how the effect on variables such as employment and role strain might change over time, as caring responsibilities likely vary in line with different lines of treatment or transition to palliative care [bib_ref] The cancer family caregiving experience: An updated and expanded conceptual model, Fletcher [/bib_ref] [bib_ref] Review of the literature on the effects of caring for a patient..., Stenberg [/bib_ref] [bib_ref] Support for caregivers of cancer patients: Transition after active treatment, Given [/bib_ref]. Future measures should attempt to capture the changing nature of caregiver impact. We have identified a number of areas which are currently not well captured by measures that have been evaluated in cancer. These gaps may exist for several reasons. First, some measures were not initially developed for this population, and so, constructs important to cancer caregivers may not have the same salience. Second, some measures are old and may not reflect what is important in current society. Third, advances in cancer treatments mean that many more people are living a long life with cancer. For some, initial therapy is just the start of a journey that will involve repeated lines of treatment over time. The patient and the whole family have to continually adjust to a fluid situation and will be impacted variably at different times while trying to maintain a sense of normality in other aspects of their lives. Better treatments mean a longer life with cancer is a possibility for patients; however, we need ways to measure the longer-term impacts of cancer and cancer treatment for them and their informal caregivers. At the very least, we would suggest caregiver input into updating some of the older content if not the development of a new measure to capture the broader impacts we have described. # Limitations The search strategy may have limited the number of papers identified in two ways: (1) searching for measures by name and acronym. The precise wording of the measure name and even the acronym sometimes varied; (2) reporting standards have changed; some older papers have poor use of keywords and do not always include psychometric terms or the names of measures in title/abstract/keywords. The impact of both of these limitations is mitigated by thorough backwards and forwards citation chasing. We intentionally restricted the review to studies that reported on the psychometric properties of the English version of measures. This decision was taken as we felt we could not assume cultural equivalence for the caregiving role or the salience of different aspects of burden and impact in diverse populations. We took the decision to exclude all non-English versions of the measures rather than make subjective decisions as to whether one culture was sufficiently similar, while another was not. We are aware, however, that there are a number of studies reporting on the psychometric properties of other language versions of measures included in this review, e.g. [bib_ref] Preliminary assessment of the psychometric properties of the abridged Arabic version of..., Bachner [/bib_ref] [bib_ref] A psychometric validation study of the Quality of Life and FAMCARE scales..., Can [/bib_ref] [bib_ref] The caregiver reaction assessment: Psychometrics, and temporal stability in primary caregivers of..., Grov [/bib_ref] [bib_ref] Psychometric testing of the Caregiver Quality of Life Index-Cancer scale in an..., Khanjari [/bib_ref] [bib_ref] The Caregivers Quality of Life Cancer index scale (CQoLC): An exploratory factor..., Lafaye [/bib_ref] [bib_ref] Measuring both negative and positive reactions to giving care to cancer patients:..., Nijboer [/bib_ref] [bib_ref] Korean version of the caregiver quality of life index-cancer (CQOLC-K), Rhee [/bib_ref] [bib_ref] Validation of the ''Quality of Life in Life-Threatening Illness-Family Carer Version'' (QOLLTI-F)..., Schur [/bib_ref]. We acknowledge there may be cultural differences between and within different countries where English is commonly spoken and where measures developed in English have been used. While this is an extremely important area of research, it is beyond the remit of the current review and it is not an aim of this study to investigate these potential differences. In this review, 7/10 included studies were conducted in the USA and 1 study each in the UK, Australia and Canada. We also recognise that the pool of individual items identified is restricted by our stringent inclusion criteria for measures. Measures developed in other contexts, e.g. family function in a paediatric setting , for economic evaluation [bib_ref] The CarerQol instrument: A new instrument to measure care-related quality of life..., Brouwer [/bib_ref] , domain-specific measures [bib_ref] Construct validity of the Work Productivity and Activity Impairment questionnaire across informal..., Giovannetti [/bib_ref] and multidimensional measures which have not been subject to psychometric evaluation in cancer caregivers in the English language [bib_ref] Lifestyle interference and emotional distress in family caregivers of advanced cancer patients, Cameron [/bib_ref] [bib_ref] The CareGiver Oncology Quality of Life questionnaire (CarGOQoL): Development and validation of..., Minaya [/bib_ref] [bib_ref] The COPE index-a first stage assessment of negative impact, positive value and..., Mckee [/bib_ref] may include concepts and items that are pertinent but which would need to be evaluated in appropriate studies. # Conclusions A large number of measures purport to assess caregiver impact, but most have not been subject to psychometric evaluation in cancer populations. Few studies met our inclusion criteria so it was not possible to consider psychometric performance of the measures across a group of studies. Our content analysis identified several areas which are currently not well captured. These include changes to career aspiration and planning, changes in roles and responsibilities within the family and the way the family functions as a unit. We also note that some of the measures were developed up to 35 years ago, and their relevance to the current day may need to be reviewed. Strategies to overcome some of these limitations could include caregiver input into revising existing measures or using two or more measures to cover a broader range of outcome domains. However, our review suggests there is a need for a new measure capturing the impacts on broader areas of life for the caregiver and the family unit. [fig] Figure 2: Flow chart showing study selection in search stage 2. a All breaches of exclusion criteria were recorded; articles were excluded for multiple reasons were returned. The combined searches for each of the remaining 22 individual measures resulted in 365 records. [/fig] [table] Table 1: General characteristics of identified measures [/table] [table] Table 2: Studies evaluating psychometric performance of measures in the cancer caregiver population [/table] [table] Table 3: Appraisal of measure performance and characteristics in the cancer caregiver population [/table]

Long-term survival of gastric mixed neuroendocrine-non-neuroendocrine neoplasm: Two case reports BACKGROUNDGastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN), which consists of neuroendocrine and non-neuroendocrine components, is quite rare. Until now, most data on gastric MiNEN come from clinical cases, without largescale retrospective studies or controlled clinical trials. Consequently, no consensus regarding the origin, molecular characteristics, or appropriate treatment of MiNEN has been reached so far. We conducted chemotherapy of irinotecan plus cisplatin (IP regimen) and surgery in two patients with gastric MiNEN, which had never been used in treating this kind of tumor, leading to their long-term survival for more than 3 and 7 years, respectively.CASE SUMMARYWe present two patients (one male and one female) with gastric MiNEN, with the primary manifestation of recurrent upper abdominal pain. After they were referred to our hospital, a diagnosis of gastric MiNEN was defined with the help of CT scan, and histopathological and immunohistochemical examinations on the samples of gastrointestinal endoscopy or radical surgery. The male patient (case 1) were found to have metastases in the reginal lymph nodes and the left liver. He received four cycles of IP regimens first, then the gastrectomy and partial left liver resection, followed by additional two cycles of IP chemotherapy. The female patient (case 2) underwent a laparoscopic gastrectomy, and received six cycles of IP regimen. She was found to have metastatic lesions in the right lung 2 years after that, and underwent video-assisted thoracoscopic surgery (VATS) of the lower lobe of the right lung. The two patients have now survived for more than 3 years and 7 years, respectively, without any evidence of recurrence or metastases.CONCLUSIONIP regimen, combined with curative-intent surgery if feasible, could be considered as the priority in the choice of front-line chemotherapy for gastric MiNEN. Woo LT et al. Front-line chemotherapy for gastric MiNENs WJCC https://www.wjgnet.com 7937Core Tip: Mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) is a rare, highly aggressive tumor with a poor prognosis (median overall survival less than 12 mo), and no consensus regarding the appropriate treatment has been reached so far. We conducted chemotherapy of irinotecan plus cisplatin regimen and surgery in two patients with gastric MiNEN, which had not been used to treat this kind of tumor before, leading to their long-term survival for more than 3 and 7 years, respectively. Our reports may provide a reference for other clinicians. Citation: Woo LT, Ding YF, Mao CY, Qian J, Zhang XM, Xu N. Long-term survival of gastric mixed neuroendocrine-non-neuroendocrine neoplasm: Two case reports. World J Clin Cases 2022; 10(22): 7936-7943 CONCLUSION Gastric MiNEN is a rare malignant tumor without specific clinical symptoms. Histopathological and immunohistochemical examinations are requisite for pathologists and physicians to make diagnosis. Palliative surgery remains essential even when patients have undergone distant metastases. In the choice of front-line chemotherapy, we believe that IP regimen could be considered as the priority. More prospective studies are urgently needed to explore better treatment options for patients with gastric MiNEN.FOOTNOTESAuthor contributions: Woo LT performed the bibliographic retrieval and wrote the paper; Ding YF contributed to the paper revision; Mao CY and Qian J provided the data and detailed information of the patients; Zhang XM performed the pathological examination and immunohistochemistry of the specimens; Xu N conceived the whole idea and contributed to the manuscript revision. # Introduction Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN), which consists of neuroendocrine and non-neuroendocrine components, is quite rare, accounting for about 7% of all gastric neuroendocrine neoplasms and 25% of all gastric poor differentiated neuroendocrine carcinomas, but their prevalence has not been explored specifically so far . Gastrointestinal tumor with an exocrine and a neuroendocrine component was first described by Cordier in 1924 [bib_ref] Mixed Adenoneuroendocrine Carcinomas (MANECs) of the Gastrointestinal Tract: An Update, Rosa [/bib_ref]. Many different names had been used since then, causing confusion among clinicians, surgeons, and pathologists, such as composite carcinoid, mucin-producing carcinoid argentaffin cell adenocarcinoma, mixed exocrineendocrine tumors, mixed adenoneuroendocrine carcinomas, and so on [bib_ref] WHO Classification of Tumours Editorial Board. The 2019 WHO classification of tumours..., Nagtegaal [/bib_ref]. In the 2019 WHO Classification of Tumors of the Digestive System, the term MiNEN has been used when referring to this kind of tumor [bib_ref] A Case Report: Gastric Mixed Neuroendocrine-Nonneuroendocrine Neoplasm with Aggressive Neuroendocrine Component, Pham [/bib_ref]. Until now, most data on gastric MiNEN come from clinical cases [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref] [bib_ref] Oropharyngeal Mixed Neuroendocrine-Nonneuroendocrine Neoplasm (MiNEN): A Case Report and Literature Review, Robinson [/bib_ref] [bib_ref] A case of primary hepatic mixed neuroendocrine-non-neuroendocrine tumor (MiNEN) associated with gallbladder..., Kaneko [/bib_ref] [bib_ref] Mixed Neuroendocrine-Nonneuroendocrine Neoplasms (MiNENs): Unifying the Concept of a Heterogeneous Group of..., Rosa [/bib_ref] , without large-scale retrospective studies or controlled clinical trials. Consequently, no consensus regarding the origin, molecular characteristics, or appropriate treatment of MiNEN has been reached so far. Due to the lack of knowledge of gastric MiNEN, this tumor has a quite poor prognosis, presenting with a short median survival of less than 12 mo [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref]. The preferred treatment for high-grade MiNENs is currently suggested to be combining etoposide and a platinum salt (EP regimen) or the combinations of 5-fluorouracil and irinotecan or temozolomide or amrubicin[1]. However, we conducted chemotherapy of irinotecan plus cisplatin (IP regimen) and surgery in two patients with gastric MiNEN, leading to their long-term survival for more than 3 and 7 years, respectively. Here, we present the process of diagnosis and treatment and a brief review of the literature to improve our understanding of the tumor. ## Case presentation ## Chief complaints Case 1: A 63-year-old man was admitted to the hospital because of frequent upper abdominal pain for over 1 mo. ## Case 2: A 54-year-old female patient was admitted to the hospital with recurrent epigastric pain for more than 7 years. ## History of present illness Case 1: The patient felt frequent upper abdominal pain for over 1 mo, so he underwent upper gastrointestinal endoscopy and magnetic resonance imaging at a local hospital. Then he was diagnosed as having gastric MiNEN with metastases in the regional lymph nodes and the left liver. He came to our hospital soon after, and was admitted because of "gastric cancer". ## Case 2: The patient had recurrent epigastric pain for 7 years, and the pain got worse on an empty stomach. She took omeprazole herself without obvious relief. Then she underwent upper gastrointestinal endoscopy at a local hospital and was diagnosed as having gastric cancer. So the patient came to our hospital for surgery and was admitted because of "gastric cancer". Volume 10 Issue 22 ## History of past illness Case 1: This patient had a history of hypertension for more than 10 years and herniorrhaphy surgery 5 years ago. ## Case 2: The patient was diagnosed with chronic nasosinusitis, thyroiditis, cholecystolithiasis, hepatic cyst, and hepatic haemangioma. ## Personal and family history Case 1: The patient's father was dead, and his mother was healthy. ## Case 2: The patient's father was dead; her mother and little brother were alive. ## Physical examination Case 1: The physical examination revealed the following: Temperature: 36.5 °C; pulse: 86/min; respiration rate: 14/min; blood pressure: 122/82mmHg. In the upper gastrointestinal endoscopy, no enlarged superficial lymph nodes, no abdominal wall varicosis, and no gastrointestinal peristalsis [fig_ref] Case 1: Laboratory examinations revealed the following [/fig_ref]. ## Case 2: The physical examination revealed the following: Temperature: 37.1 °C; pulse: 80/min; respiration rate: 16/min; blood pressure: 118/76mmHg. Upper gastrointestinal endoscopy confirmed the gastric cancer [fig_ref] Case 1: Laboratory examinations revealed the following [/fig_ref]. ## Laboratory examinations ## Imaging examinations Case 1: CT revealed the tumor infiltration into the omentum majus, with metastases to regional lymph nodes and the left liver (stage IV). Subsequently, the patient received four cycles of IP regimen as firstline chemotherapy. CT after the second and third cycles of chemotherapy revealed that the lesion in the left liver and regional nodes decreased markedly [fig_ref] Figure 3: Computed tomography images of case 1 [/fig_ref]. Then, gastrectomy and partial left liver resection were performed and the histopathological examination confirmed that the neuroendocrine component of those lesions basically disappeared, only with adenocarcinoma component remaining in one regional lymph node [fig_ref] Case 1: Laboratory examinations revealed the following [/fig_ref] and B2). Metastases in the left liver totally disappeared (pT1aN1M0 stage). Two cycles of IP chemotherapy ensued after the operation. ## Case 2: CT showed that the tumor infiltrated into the stomach wall and metastasized to regional lymph nodes. ## Final diagnosis Case 1: Gastric MiNEN (metastases to the left liver). ## Case 2: This patient underwent a total of six cycles of IP regimens without serious adverse effects. ## Outcome and follow-up Case 1: The patient has survived for more than 3 years without any evidence of recurrence or metastases. Case 2: Two years after treatment, CT re-examination revealed metastatic lesions in the lower lobe of the right lung and video-assisted thoracoscopic surgery (VATS) was performed. Histopathological examination confirmed the neuroendocrine carcinoma (positive for CK7, CgA, and Syn; Ki67 index 30%) infiltration, with no metastases in regional lymph nodes. After the surgery, the patient did not undergo any further chemotherapy or radiotherapy and has survived for more than 7 years without any evidence of recurrence or metastases. # Discussion MiNEN is rare, especially in the stomach. To date, there is no consensus on the definition of MiNEN, especially the minimum proportion of each component. According to the WHO classification of digestive system tumors, MiNEN should contain both adenocarcinoma and neuroendocrine carcinoma components and each component is not less than 30%. However, this cutoff value has not been universally accepted, as it is defined arbitrarily rather than on proven clinical evidence and a minor (i.e., < 30%) poorly differentiated neuroendocrine carcinoma (PDNEC) component can impair prognosis[1,9, 10]. Pham et al [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref] once reported a case in which the adenocarcinoma component accounted for 10%-20% of the tumor, just as the case in our two patients. Park et al found that a minor proportion (10%-30%) of PDNEC component would negatively influence the prognosis of patients with gastric MiNENs in a study including 88 patients. Consequently, the current 30% threshold, without sufficient prognostic value, may be not mandatory for defining MiNEN. Most gastrointestinal MiNENs are highly aggressive, with a poor prognosis and median survival of less than 12 mo [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref]. At present, the diagnosis mainly relies on pathological examination and IHC of surgical specimen [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref]. CK, carcinoembryonic antigen, and caudal type homeobox 2 are used as markers for adenocarcinoma components, and Syn, CgA, and CD56 for neuroendocrine components . In our two cases, the adenocarcinoma components were positive for CK18 or CKpan, and neuroendocrine component positive for CgA and Syn. Until now, most studies suggest that surgical resection should be the main treatment for gastrointestinal MiNENs. Pham et al [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref] argued that palliative surgery remains essential even if the patients have developed distant metastases. Our two patients underwent resection of the primary lesion and metastatic lesion, respectively, and both of them achieved long-term survival, being in good condition, without any evidence of recurrence to date. Therefore, we believe that curative-intent surgery if feasible, is crucial for the treatment of MiNEN, as recommended by other authors [bib_ref] Surgical treatment and prognosis of gastric neuroendocrine neoplasms: a single-center experience, Shen [/bib_ref] [bib_ref] Current Treatment Options in Gastroenteropancreatic Neuroendocrine Carcinoma, Thomas [/bib_ref]. Volume 10 Issue 22 There is still no consensus regarding the standard front-line chemotherapy against MiNENs [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref]. Platinum combined with etoposide (EP) regimen is found to be the most recommended first-line therapy for gastroenteropancreatic neuroendocrine carcinomas (GEPNECs) [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref] [bib_ref] Diagnosis, Assessment, and Therapeutic Strategy for Colorectal Mixed Adenoneuroendocrine Carcinoma, Tanaka [/bib_ref]. The preferred treatment for high-grade MiNENs is also suggested to be EP regimen or the combinations of 5fluorouracil and irinotecan or temozolomide or amrubicin[1]. Yamaguchi et al compared IP regimen and EP regimen in treating GEPNECs, discovering that the IP group had a higher response rate (50% vs 28%, respectively; P = 0.001). When it comes to irinotecan and etoposide, there were some studies demonstrating a lower incidence of grade 4 adverse events and treatment-related deaths in the irinotecan group than in the etoposide group when treating digestive neuroendocrine carcinoma [bib_ref] Diagnosis, Assessment, and Therapeutic Strategy for Colorectal Mixed Adenoneuroendocrine Carcinoma, Tanaka [/bib_ref]. IP regimen is also better than irinotecan monotherapy when comparing progression-free survival and disease control rate [bib_ref] Variability of the Ki-67 proliferation index in gastroenteropancreatic neuroendocrine neoplasms -a single-center..., Shi [/bib_ref]. Therefore, we thought that IP regimen could be used for our two patients. Surprisingly, both of them achieved long-time survival for more than 3 years and 7 years, respectively, Volume 10 Issue 22 which are much longer than those in other studies [bib_ref] Gastric mixed neuroendocrine-non-neuroendocrine neoplasm (MiNEN) with pancreatic acinar differentiation: a case report, Fujita [/bib_ref]. It may suggest that IP regimen could be considered as the priority in the choice of front-line chemotherapy for gastric MiNEN. To the best of our knowledge, we were the first to use IP regimen along with surgical resection for patients with gastric MiNENs. To date, the effect of Ki67 proliferation index variation on prognosis remains unclear. Shi et al [bib_ref] Impact of Ki67 re-assessment at time of disease progression in patients with..., Panzuto [/bib_ref] discovered that the Ki67 index would rise in 40% (n = 30) patients and decline in 13.3% patients with gastroenteropancreatic NEC during the treatment. In addition, Panzuto, Botling, and their colleagues [bib_ref] High-Grade Progression Confers Poor Survival in Pancreatic Neuroendocrine Tumors, Botling [/bib_ref] found that the Ki67 index of patients tends to rise at time of disease progression, and median OS was significantly shorter in patients with rising Ki67 index (50.2 vs 115.1 m, hazard ratio = 3.89, 95% confidence interval [CI]: 1.91-7.94, P < 0.001). The Ki67 index of the patient in case 2 declined from 60% to 30% after IP regimen treatment, which was associated with a long-term survival. This, to some extent, may indicate that the decrease of Ki67 index is related to a better prognosis, which still needs further study. At present, the most common genetic changes found in MiNENs include TP53, KRAS, BRAF, APC, PIK3CA, MYC, etc [bib_ref] Characterization of genome-wide copy number aberrations in colonic mixed adenoneuroendocrine carcinoma and..., Sinha [/bib_ref] [bib_ref] Concordance of genetic alterations in poorly differentiated colorectal neuroendocrine carcinomas and associated..., Vortmeyer [/bib_ref]. We wonder if our two patients share some common genetic changes, which could be part of the reason for their long-term survival. Next-generation sequencing tests were performed on the surgical specimens of them, revealing that they were all proved to be microsatellite stable (MSS), and the tumor mutation burden (TMB) was 4.06 mut/Mb and 2.03 mut/Mb, respectively. TP53 mutation was found in patient 1, and BRCA2 mutation, along with copy number increase in nine genes (MET, FGFR1, FGFR4, CDK4, CDK6, CDKN2A, ERBB3, RIT1, and VEGFA) in patient 2. We may assume that MSS and TMB fewer than 10 mut/Mb could be associated with improved response to IP regimen from the tests result. It still needs further studies to explore which genetic changes may indicate a better prognosis in patients with MiNEN receiving IP regimen treatment. [fig] Case 1: Laboratory examinations revealed the following: Red blood cell count (RBC) 4.2 × 10 12 /L; hemoglobin (Hb) 110 g/L; white blood cell count (WBC) 6.8 × 10 9 /L; platelet count (PLT) 126 × 10 9 /L. The pathological examination and immunohistochemistry (IHC) confirmed the gastric MiNEN and the tumor was composed of two different components. The adenocarcinoma component was positive for cytokeratin 18 (CK18), and the neuroendocrine carcinoma component was positive for chromogranin A (CgA) and synaptophysin (Syn) (Ki67 index 80%) (Figure 2A1-A5). A high mitotic activity was seen (> 20 mitoses/10 high power fields [HPFs]).Case 2:Laboratory examinations revealed the following: RBC 3.8 × 10 12 /L; Hb 102g/L; WBC 8.4 × 10 9 /L; PLT 208 × 10 9 /L. The histopathological examination revealed tumor infiltration into the subserosal layer, with 11 regional lymph node metastases (pT4aN3aM0 stage). The tumor was composed of two different components, of which the adenocarcinoma component (positive for CKpan and CK18) accounted for 20% and neuroendocrine carcinoma component (positive for CKpan, CK18, CgA, and Syn; Ki67 index 60%) accounted for 80% (Figure 2C1-3). The mitotic activity was high (about 40 mitoses/10 HPFs). [/fig] [fig] Case 2, Figure 1: Gastric MiNEN.TREATMENTCase 1: The patient received four cycles of IP regimen as first-line chemotherapy, then gastrectomy and partial left liver resection were performed. Upper gastrointestinal endoscopy. A: There was a large mass in the greater curvature of the stomach with unclear borders, accompanied by ulcers (case 1); B: There was a pitted ulcer in the corner of the stomach, and the surrounding gastric mucosa was markedly congested and edematous (case 2). [/fig] [fig] Figure 2: Immunoprofile and hematoxylin-eosin staining. A: Immunoprofile of endoscopy specimen in case 1. A1: Hematoxylin-eosin (H&E) staining; A2-4: CK18 (A2), Syn (A3), and CgA (A4) were positive in the solid component; A5: A high Ki67 index (about 80%) can be seen; B: H&E staining of the surgery specimen in case 1. B1: A small amount of atypical glands in the muscle layer at the bottom of the ulcer area, and the surrounding fibrous tissue hyperplasia accompanied by infiltration of interstitial inflammatory cells; B2: Adenocarcinoma metastases can be seen in surrounding lymph nodes; C: Immunoprofile of surgical specimen in case 2. C1: The neuroendocrine carcinoma component was positive for CgA; C2: A high Ki67 proliferation index (about 60%) can be seen; C3: Neuroendocrine carcinoma component was dominant (about 80%). [/fig] [fig] Figure 3: Computed tomography images of case 1. A: Metastasis of the left liver. A1: At the baseline; A2: After the 2 nd cycle of chemotherapy; A3: After the 3 rd cycle of chemotherapy; B: Surrounding lymph nodes. B1: At the baseline, B2: After the 2 nd cycle of chemotherapy; B3: After the 3 rd cycle of chemotherapy. [/fig]

Complete chloroplast genome of seven Fritillaria species, variable DNA markers identification and phylogenetic relationships within the genus Fritillaria spp. constitute important traditional Chinese medicinal plants. Xinjiang is one of two diversity hotspots in China in which eight Fritillaria species occur, two of which are endemic to the region. Furthermore, the phylogenetic relationships of Xinjiang Fritillaria species (including F. yuminensis) within the genus are unclear. In the present study, we sequenced the chloroplast (cp) genomes of seven Fritillaria species in Xinjiang using the Illumina HiSeq platform, with the aim of assessing the global structural patterns of the seven cp genomes and identifying highly variable cp DNA sequences. These were compared to previously sequenced Fritillaria cp genomes. Phylogenetic analysis was then used to evaluate the relationships of the Xinjiang species and assess the evolution of an undivided stigma. The seven cp genomes ranged from 151,764 to 152,112 bp, presenting a traditional quadripartite structure. The gene order and gene content of the seven cp genomes were identical. A comparison of the 13 cp genomes indicated that the structure is highly conserved. Ten highly divergent regions were identified that could be valuable in phylogenetic and population genetic studies. The phylogenetic relationships of the 13 Fritillaria species inferred from the protein-coding genes, large single-copy, small single-copy, and inverted repeat regions were identical and highly resolved. The phylogenetic relationships of the species corresponded with their geographic distribution patterns, in that the north group (consisting of eight species from Xinjiang and Heilongjiang in North China) and the south group (including six species from South China) were basically divided at 40˚N. Species with an undivided stigma were not monophyletic, suggesting that this trait might have evolved several times in the genus. PLOS ONE | https://doi.org/10.1371/journal.pone.0194613 March 15, 2018 1 / 17 OPEN ACCESS Citation: Li Y, Zhang Z, Yang J, Lv G (2018) Complete chloroplast genome of seven Fritillaria species, variable DNA markers identification and phylogenetic relationships within the genus. PLoS ONE 13(3): e0194613. https://doi.org/10. [formula] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [/formula] # Introduction The genus Fritillaria L. (Liliaceae) consists of approximately 140 species and is widely distributed in Europe (mostly in the Mediterranean region), Central Asia, China, Japan, and North America [bib_ref] Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium Liliaceae; Liliales)..., Rønsted [/bib_ref]. Twenty-four species occur in China, of which 15 are endemic. They are distributed throughout most provinces in China, among which Sichuan and Xinjiang constitute two diversity hotspots. Seven species occur in Xinjiang, and F. tortifolia X.Z.Duan & X.J.Zheng and F. yuminensis X.Z.Duan are endemic to this region. Two further species, F. tachengensis X.Z. Duan & X.J.Zheng (endemic) and F. ferganensis Losinsk, recorded in Flora Xinjiangensis, were reduced to the synonyms of F. yuminensis and F. walujewii Regel, respectively, in the Flora of China (FOC, http://foc.eflora.cn/). The morphological traits of Fritillaria species, particularly the Fritillaria cirrhosa D.Don complex (referring to F. cirrhosa and closely-related species in morphology), which are widely distributed in southwest China [bib_ref] A revision of Fritillaria L. (Liliaceae) in the Hengduan Mountains and adjacent..., Luo [/bib_ref] , are complex due to the high variability of several characters, including leaf width; leaf curling; petals tessellated or not, and bract number. However, the mechanism of the variation is not clear and the current classification of some species is only temporary. More comprehensive studies into the morphological variation in the genus are required to facilitate a precise and reasonable species classification [bib_ref] A revision of Fritillaria L. (Liliaceae) in the Hengduan Mountains and adjacent..., Luo [/bib_ref]. Furthermore, the species occurring in Xinjiang also exhibit significant morphological variation due to the diversity of microclimates (mountains, swamps, saline conditions, and other habitats). Currently, 16 variants are recorded in Flora Xinjiangensis, though they are treated as synonyms of the corresponding accepted species names in the FOC and The Plant List (www.theplantlist.org). Certain character variations of some individuals are prominent and beyond the characteristic range of the genus, such as 8-12 petals, 4-8 stamens, and a 3-5-lobed stigma. Moreover, the stigma of most Fritillaria species is 3-lobed, but in a few species, i.e., F. yuminensis and F. karelinii (Fisch. ex D.Don) Baker, it is undivided. It has been proposed that an undivided stigma is a primitive characteristic, but physiological and molecular evidence is required to test this hypothesis and to assess the evolution of this trait within the genus. The bulbs of some Fritillaria species, including F. thunbergii Miq., F. cirrhosa, F. walujewii, and F. pallidiflora Schrenk, have long been used in traditional Chinese medicine. As a result, long-term excessive harvesting has led to substantial declines in the size of wild Fritillaria populations. At present, all of the eight species in Xinjiang have been classified as vulnerable according to the list of rare endangered endemic higher plants of Xinjiang, which has attracted scientific interest. The genetic diversity of some species in the genus was previously assessed, and corresponding conservation areas were proposed [bib_ref] Conservation genetics and geographic patterns of genetic variation of the endangered officinal..., Su [/bib_ref] [bib_ref] Conservation genetics and geographic patterns of genetic variation of the vulnerable officinal..., Su [/bib_ref] ; however, some species with very narrow distributions and greater extinction threat require evaluation. A scientific approach to conservation requires an accurate understanding of the population genetic diversity and structure. The diversity estimated by different markers, such as plastid DNA, genomic inter-simple sequence repeats (ISSRs), and single nucleotide polymorphisms (SNPs), can be used to comprehensively inform conservation strategies. The classification of the genus was previously revised where it was subdivided into eight subgenera, including Davidii, Liliorhiza, Japonica, Fritillaria, Rhinopetalum, Petilium, Theresia, and Korolkowia. A later phylogenetic analysis of 37 Fritillaria species using matK, trnK intron, rp116 intron, and nrDNA ITS [bib_ref] Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium Liliaceae; Liliales)..., Rønsted [/bib_ref] supported this subgeneric classification. Khourang et al. investigated the phylogenetic position of nine species in Iran using the ITS and trnL-F regions [bib_ref] Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and..., Khourang [/bib_ref] , and showed that members of the subgenera Fritillaria and Rhinopetalum formed one clade. However, a phylogenetic study of 92 species using matK, rbcL, and rpl16 [bib_ref] Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae), Day [/bib_ref] indicated that, in contrast to the results of [bib_ref] Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium Liliaceae; Liliales)..., Rønsted [/bib_ref] [bib_ref] Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and..., Khourang [/bib_ref] , Fritillaria appeared to be polyphyletic. Additionally, the monophyly of seven out of the eight newly classified subgenera by Rix(F. subgenus davidii, Liliorhiza, Japonica, Rhinopetalum, Petilium, Theresia, and Korolkowia) was well supported. The largest subgenus (F. subgenus Fritillaria) formed two strongly supported clades, with one clade comprising taxa that occur mainly in Europe, the Middle East, and North Africa, and the other clade comprising taxa occurring in China and Central Asia [bib_ref] Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae), Day [/bib_ref]. However, the relationships of some of these species were not well resolved, particularly F. thunbergii Miq. and F. cirrhosa. The phylogenetic position of the Xinjiang-endemic species F. yuminensis remains unclear. The chloroplast (cp) genome in angiosperms is highly conserved, with a quadripartite structure consisting of a large single copy (LSC) region, a small single copy (SSC) region, and two copies of a larger inverted repeat (IR). The gene orders in these regions are also similar; however, structural rearrangements and gene losses can be found in some lineages [bib_ref] The evolution of the plastid chromosome in land plants: gene content, gene..., Wicke [/bib_ref] [bib_ref] Plastome phylogeny and early diversification of Brassicaceae, Guo [/bib_ref]. Plastid sequences have been widely used for deciphering phylogenetic relationships and in DNA barcoding to identify plant species [bib_ref] Use of DNA barcodes to identify flowering plants, Kress [/bib_ref]. However, DNA barcoding for species identification and phylogenetic analysis is hampered by weak resolution in some plants [bib_ref] Failure of DNA barcoding in discriminating Calligonum species, Li [/bib_ref] [bib_ref] Understanding the spectacular failure of DNA barcoding in willows (Salix): does this..., Percy [/bib_ref] [bib_ref] Evolutionary relationships in Panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae), Burke [/bib_ref]. Complete cp genomes have therefore emerged as a means of improving the resolution of phylogenies that have varied among, or been unresolved in, earlier single-and multi-gene studies [bib_ref] Chloroplast phylogenomics resolves key relationships in ferns, Lu [/bib_ref] [bib_ref] Plastid phylogenomics of the cool-season grass subfamily: clarification of relationships among early-diverging..., Saarela [/bib_ref] [bib_ref] Complete chloroplast genome of Gracilaria firma (Gracilariaceae, Rhodophyta), with discussion on the..., Ng [/bib_ref] [bib_ref] Plastid phylogenomics resolve deep relationships among eupolypod II ferns with rapid radiation..., Wei [/bib_ref]. With the rapid development of next-generation sequencing techniques, it is now more convenient and relatively inexpensive to obtain cp genome sequences and extend gene-based phylogenetics to phylogenomics. To date, a total of six Fritillaria cp genomes have been sequenced and are available on Gen-Bank. Park et al. reported the cp genomes of F. ussuriensis and F. cirrhosa and performed a comparative analysis with four Fritillaria cp genomes available on GenBank, the outcome of which has provided a basic understanding of the cp genome characteristics of the genus [bib_ref] The complete chloroplast genome sequences of Fritillaria ussuriensis Maxim. and Fritillaria cirrhosa..., Park [/bib_ref]. In the present study, we sequenced the cp genomes of seven Fritillaria species from Xinjiang using the Illumina HiSeq platform. The aims of this study were to (1) analyze the global structural patterns of the seven cp genomes and compare them with the six cp genomes available on GenBank; (2) discover highly divergent DNA markers that can be used for population genetics; and (3) evaluate the phylogenetic relationships of the Xinjiang species, particularly the position of F. yuminensis, and assess the evolution of an undivided stigma in the genus. # Materials and methods # Plant materials Fresh leaves of seven Fritillaria species were collected from Tacheng and Yili Prefecture of Xinjiang Uygur Autonomous Region, China. The geographic origin and coordinates of sampling locations were listed in S1 [fig_ref] Table: Sampled species and their voucher specimens [/fig_ref] The sample collection was approved by the Forestry Bureau of Tacheng Prefecture and Yili Prefecture. For each species, two to five individuals were sampled. Voucher specimens were deposited at the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences (S1 . ## Genome sequencing Total DNA was extracted from approximately 100 mg of fresh leaves using the CTAB method following Yao et al. [bib_ref] Chloroplast genome structure in Ilex (Aquifoliaceae), Yao [/bib_ref]. Illumina paired-end libraries were constructed and sequenced by the Illumina HiSeq X-Ten platform (Illumina Inc., USA) at the Germplasm Bank of Wild Species in Southwest China, Kunming Institution of Botany, Chinese Academy of Sciences. Each individual of each species was sequenced independently. In total, 22 individuals of seven species were sequenced. Because the cp genome sequences of repeat individuals of each species were almost identical, therefore, we reported only one genome of each species. ## Genome assembly and annotation The raw reads were trimmed and assembled into contigs using SPAdes [bib_ref] SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing, Bankevich [/bib_ref]. Contigs representing the cp genome were obtained after a BLAST search using the cp genome sequence of F. cirrhosa (GenBank No. KY646167) as a reference sequence. The resulting contigs were assembled after being aligned to the reference genome using Geneious 4.8 [bib_ref] Geneious Basic: An integrated and extendable desktop software platform for the organization..., Kearse [/bib_ref] and annotated using the Dual Organellar GenoMe Annotator (DOGMA) database. The cp genome map was generated using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/) [bib_ref] OrganellarGenomeDRAW-a suite of tools for generating physical maps of plastid and mitochondrial..., Lohse [/bib_ref]. The raw sequencing data were deposited in GenBank SRA database (SAMN08348372-SAMN08348378, https://submit.ncbi.nlm.nih.gov/subs/sra). The annotated seven cp genomes were deposited in GenBank (accession number MG200070, MG211818-MG211823). ## Genome comparison A comparative plot consisting of full alignments of the cp genomes with annotations was produced by mVISTA using F. cirrhosa as the reference. The sequences were aligned using MEGA 6 [bib_ref] Molecular evolutionary genetics analysis version 6.0, Tamura [/bib_ref] and then manually adjusted using BioEdit software (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). Subsequently, a sliding window analysis was conducted to evaluate the nucleotide diversity (Pi) of the cp genome using DnaSP 5.1 [bib_ref] DnaSP v5: a software for comprehensive analysis of DNA polymorphism data, Librado [/bib_ref]. The step size and window length was set to 200 bp and 600 bp, respectively. The number of variable sites and the Pi across the complete cp genomes, LSC, SSC, and IR regions were calculated using DnaSP 5.1. The p-distance among species was calculated in MEGA 6 to evaluate the divergence of Fritillaria species. ## Phylogenetic analyses Sequences of the 13 Fritillaria species and three Lilium species were aligned using MEGA 6. Phylogenies were constructed by maximum likelihood (ML) and Bayesian Inference (BI) analyses using the protein-coding genes (PCGs), LSC, SSC, and IR regions. ML analyses were conducted in MEGA 6, while BI analyses were conducted using BEAST 1.7 [bib_ref] Bayesian phylogenetics with BEAUti and the BEAST 1.7, Drummond [/bib_ref]. GTR+G+I and GTR+G were selected as the best substitution models for the ML and BI analyses according to the Akaike information criterion (AIC) [bib_ref] Information theory and an extension of the maximum likelihood principle, Akaike [/bib_ref] and Bayesian information criterion (BIC) [bib_ref] Estimating the dimension of a model, Schwarz [/bib_ref] , respectively, and were estimated using MrModeltest 2.3 [bib_ref] MrModeltest v2. Program distributed by the author, Nylander [/bib_ref]. For ML, initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. All alignment positions containing more than 5% gaps were eliminated. For BI, two independent Markov Chain Monte Carlo chains were conducted simultaneously for 2 × 10 7 generations and sampled every 1,000 generations. Each run was assessed using Tracer 1.6to evaluate whether a sufficient effective sample size (ESS) had been reached. The two runs were considered as converged when the ESS of all relevant parameters was above 200. A consensus maximum clade credibility (MCC) tree was generated from the 75% post-burn-in trees using TreeAnnotator 1.7. # Results ## Genome sequencing, assembly, and genome features Illumina sequencing generated 3.5 to 7.1 Gb of raw reads and 164,351 to 697,002 paired-end reads for the seven Fritillaria species. After the de novo assembly, eight to 19 contigs covering the whole chloroplast genome were used to generate a complete cp genome (S1 . Using reference-guided assembly, seven Fritillaria cp genomes were obtained, with coverage of 162× to 688× for each species. The gene content and order were identical in the seven species. A total of 114 distinct genes were annotated, including 78 PCGs, 30 tRNA genes, four rRNA genes, infA (translation initiation factor gene), and hypothetical ORF ycf15 (S2 . Eighteen genes were duplicated in the cp genome, including eight tRNAs (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC, trnH-GUG), four rRNAs (rrn16, rrn23, rrn4.5, rrn5), and six PCGs (ndhB, rpl2, rpl23, rps12, ycf2, rps7). Gene rps12 was trans-spliced because the 5 0 end was located in the LSC region and the 3 0 end in the IR region. Gene ycf1 in the junction region between SSC and IRb was the only pseudogene found due to the incomplete duplication of the normal copy in the junction region [fig_ref] Fig 1: Gene map of the Fritillaria cp genome [/fig_ref]. There were 18 intron-containing genes, among which two genes (ycf3 and clpP) had two introns each, while the other 16 had one intron, including 10 PCGs (atpF, rpoC1, rpl2, ndhB, ndhA, petB, petD, rpl16, rps16, rps12) and six tRNA genes (trnA-UGC, trnG-GCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC) [fig_ref] Fig 1: Gene map of the Fritillaria cp genome [/fig_ref]. The Pi of the seven species was 0.00648. SSC had the highest Pi value, while IR had the lowest value [fig_ref] Table 2: Variable site analyses in Fritillaria chloroplast genomes [/fig_ref]. The mean p-distance among the seven species was 0.00558, ranging from 0.003 to 0.01. The distance between F. karelinii /F. meleagroides and the other five species was larger than that between the five species (S3 , indicating that F. karelinii and F. meleagroides were most divergent. ## Genome sequence divergence We compared the Pi of the LSC, SSC, and IR regions of the cp genome. In total, 3,199 variable sites were found (Pi = 0.00557), indicating moderate genetic divergence of the Fritillaria cp genomes. The IR regions exhibited the lowest Pi (0.00148), while SSC had the highest Pi (0.01044) [fig_ref] Table 2: Variable site analyses in Fritillaria chloroplast genomes [/fig_ref]. The p-distances among the Fritillaria species ranged from 0.0001 to 0.01, and F. karelinii, F. meleagroides, and F. ussuriensis exhibited the greatest sequence divergence. The Pi of the Xinjiang species (0.00648) was higher than that of the species from the other regions (0.00419), as the two highly divergent species F. karelinii and F. meleagroides are from Xinjiang. ## Junction characteristics The junction of the LSC, SSC, and IR regions of the seven species are shown in [fig_ref] Fig 2: Comparison of LSC, SSC, and IR border regions among the 13 Fritillaria... [/fig_ref] The rps19 gene located in the LSC was extended into the IRa by 11-43 bp. The border between IRa/SSC and SSC/IRb extended into the ycf1 genes. Overlaps of 17 bp were found between the ycf1 pseudogene and the ndhF gene. The trnH genes were all located in the IR region, 158-189 bp from the IRb/LSC boundary. ## Genome-wide comparative analyses We aligned the 13 Fritillaria cp genomes using mVISTA, and found that the gene order and clusters were very similar in all the species [fig_ref] Fig 3: Comparison of 13 Fritillaria cp genomes with F [/fig_ref]. Using sliding window analysis, we identified the 10 most divergent regions that could be utilized as potential molecular markers for population genetic and phylogenetic studies in Fritillaria. These regions included matK-rps16, trnS-trnG, atpH-atpI, trnC-petN, trnE-trnT-psbT, trnT-trnL-trnF, rps12-psbB, rpl32-trnL in IGS, and the petB intron and ycf1 in the coding region [fig_ref] Fig 4: Sliding window analysis of the entire chloroplast genome of 13 Fritillaria species [/fig_ref]. Additionally, psbB-psbH, petD-rpoA, ycf4-cemA, and ycf2 also constitute potential candidates. ## Phylogenetic analyses The phylogenetic analyses were conducted with the PCGs, LSC, SSC, and IR regions using ML and BI inference methods. All the analyses revealed congruent tree topologies, and all branches were highly supported (S1 # Discussion In this study, seven new cp genomes of Fritillaria were sequenced and ranged in size from 151,764 to 152,112 bp. The reported Fritillaria cp genome size in this study is consistent with previously sequenced Fritillaria cp genomes, and is also within the cp genome size range of angiosperms. The gene content and gene order were the same in the seven Xinjiang species, containing 78 PCGs, 30 tRNA genes, four rRNA genes, and the infA and ycf15 genes. Compared with the other six species, the tRNA and rRNA genes were identical, but the PCGs differed, ranging from 77 to 78 due to the absence of the clpP gene in the cp genome of F. hupehensis. The hypothetical gene ycf68 was present in the cp genome of F. unibracteata, while ycf15 was absent from F. taipaiensis, F. thunbergii, and F. ussuriensis. The absence or presence of some genes in a particular species of a genus has also been observed in Ipomoea [bib_ref] Analyses of the complete genome and gene expression of chloroplast of sweet..., Yan [/bib_ref]. The functions of ycf15 and ycf68 are ambiguous in various land plants; for instance, ycf15 in Ipomoea purpurea and Ageratina adenophora encode a complete RF15 protein, but the former has no ycf68, while the latter has one incomplete ycf68 open reading frame. In Musa acuminata, these two genes were determined as non-functional due to the presence of several stop codons in the gene sequence [bib_ref] Analyses of the complete genome and gene expression of chloroplast of sweet..., Yan [/bib_ref]. The boundaries between IR and LSC or SSC were identical, except in F. taipaiensis. The LSC/IRa boundary of Fritillaria is located in the rps19 gene, and a small section of the 5 0 end of rps19 is in the IRb region, which is similar to Ilex [bib_ref] Chloroplast genome structure in Ilex (Aquifoliaceae), Yao [/bib_ref] and Brassicaceae species [bib_ref] Analysis of chloroplast genome of two cytoplasmic male sterile lines derived from..., Ohta [/bib_ref] [bib_ref] Characterization of the complete chloroplast genome of two sister species of Pugionium..., Hu [/bib_ref] [bib_ref] Characterization of the complete chloroplast genome of two sister species of salt..., Shang [/bib_ref] [bib_ref] Characterization of the complete chloroplast genome sequence of Lepidium meyenii (Brassicaceae), Zhou [/bib_ref]. In contrast, rps19 does not extend into the IR in Lupinus luteus [bib_ref] The first complete chloroplast genome of the Genistoid legume Lupinus luteus: evidence..., Martin [/bib_ref] and Millettia pinnata [bib_ref] Capturing the biofuel wellhead and powerhouse: the chloroplast and mitochondrial genomes of..., Kazakoff [/bib_ref] , while in others, such as Phaseolus vulgaris [bib_ref] Rapid evolutionary change of common bean (Phaseolus vulgaris L) plastome, and the..., Guo [/bib_ref] and Oryza [bib_ref] Relationships of wild and domesticated rices (Oryza AA genome species) based upon..., Wambugu [/bib_ref] , the whole gene is contained inside the IR. ψycf1 spans the SSC/IRa boundary and overlaps with the ndhF gene in most of the Fritillaria species. However, these are separated and located at each side of the boundary in the F. taipaiensis cp genome, which has also been observed in Petroselinum crispum (HM596073), Tiedemania filiformis (HM596071), and Panax ginseng (AY582139). The SSC/IRb boundary is inside the ycf1 gene, which is consistent with many plants, including those from Asteraceae [bib_ref] Complete chloroplast genome of the multifunctional crop globe artichoke and comparison with..., Curci [/bib_ref] , Ilex [bib_ref] Chloroplast genome structure in Ilex (Aquifoliaceae), Yao [/bib_ref] , Lilium [bib_ref] Complete chloroplast genome sequences of Lilium: insights into evolutionary dynamics and phylogenetic..., Du [/bib_ref] , and Ananas [bib_ref] Complete chloroplast genome sequence of pineapple (Ananas comosus), Nashima [/bib_ref]. Conversely, in Cryptochloa strictiflora [bib_ref] Plastome sequences of two New World bamboos-Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)-extend..., Burke [/bib_ref] and Ipomoea batata [bib_ref] Analyses of the complete genome and gene expression of chloroplast of sweet..., Yan [/bib_ref] , the junction falls into the ndhF gene due to the loss of the ycf1 gene. The trnH gene is duplicated in the IRs in Fritillaria, as observed in Lilium [bib_ref] Complete chloroplast genome sequences of Lilium: insights into evolutionary dynamics and phylogenetic..., Du [/bib_ref] , whereas trnH is a single cope gene located in the LSC of other species, such as Ipomoea batata [bib_ref] Analyses of the complete genome and gene expression of chloroplast of sweet..., Yan [/bib_ref] , Datura stramonium [bib_ref] Complete chloroplast genome sequence of Poisonous and medicinal plant Datura stramonium: organizations..., Yang [/bib_ref] , and Citrus aurantiifolia [bib_ref] Complete chloroplast genome sequence of omani lime (Citrus aurantiifolia) and comparative analysis..., Su [/bib_ref]. The genomic structure and gene order of the Fritillaria cp genomes are highly conserved, and no rearrangement has occurred. The IRs of the Fritillaria species were about 26 kb, which is within the size range of most angiosperm cp genomes (20-28 kb). The IR usually varies between 200 and 300 nucleotides in seed plants. However, the extreme expansion of the IRs has been observed in Oenothera (54 kb) [bib_ref] Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I..., Hupfer [/bib_ref] , Fabaceae (50 kb) [bib_ref] The distribution and phylogenetic significance of a 50-kb chloroplast DNA inversion in..., Doyle [/bib_ref] , and Pelargonium×hortorum (75 kb) [bib_ref] The complete chloroplast genome sequence of Pelargonium x hortorum: organization and evolution..., Chumley [/bib_ref]. In contrast, the loss or near loss of the IR has also been also detected in Erodium and Sarcocaulon [bib_ref] Chloroplast DNA variation in the Geraniaceae: a preliminary report, Price [/bib_ref]. These significant contractions and expansions of the IR contribute towards genome size variation. Several variable cp DNA markers have been used in phylogenetic studies of Fritillaria, for instance rbcL, matK, and atpB. Some divergent intergenic spacers, i.e., trnH-psbA, rpl32-trnL, psbB-psbH, and trnS-trnG, are more informative and suitable in lower taxonomic ranks [bib_ref] A Comparative analysis of whole plastid genomes from the Apiales: expansion and..., Downie [/bib_ref]. Upon comparison of the 13 cp genomes, the 10 most divergent regions were identified, and included matK-rps16, trnS-trnG, atpH-atpI, trnC-petN, trnE-trnT-psbT, trnT-trnL-trnF, rps12-psbB, and rpl32-trnL in IGS, and the petB intron and ycf1 in the coding region. The Pi of these regions ranged from 0.015 to 0.022. Additionally, psbB-psbH, petD-rpoA, ycf4-cemA, and ycf2 also constitute potential candidates, which corroborates previous studies [bib_ref] Complete chloroplast genome sequences of Lilium: insights into evolutionary dynamics and phylogenetic..., Du [/bib_ref]. These highly divergent regions (also called hotspots) in the cp genome are useful for further phylogenetic and population genetics studies. However, in contrast to Park et al. [bib_ref] The complete chloroplast genome sequences of Fritillaria ussuriensis Maxim. and Fritillaria cirrhosa..., Park [/bib_ref] , we found the petB intron to be highly divergent. Furthermore, the clpP intron was also found to be highly variable, as reported in Acacia ligulata [bib_ref] The complete sequence of the Acacia ligulata chloroplast genome reveals a highly..., Williams [/bib_ref]. Gene ycf1 is considered as the most promising plastid DNA barcode of land plants [bib_ref] ycf1, the most promising plastid DNA barcode of land plants, Dong [/bib_ref]. Universal DNA barcoding is widely used in the identification of plant species, but has several limitations [bib_ref] Failure of DNA barcoding in discriminating Calligonum species, Li [/bib_ref] [bib_ref] Understanding the spectacular failure of DNA barcoding in willows (Salix): does this..., Percy [/bib_ref] [bib_ref] Evolutionary relationships in Panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae), Burke [/bib_ref]. The complete cp genome, as a super DNA barcode, has been successfully used in numerous phylogenetic studies of seed plants [bib_ref] Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms..., Jansen [/bib_ref] [bib_ref] Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic..., Williams [/bib_ref] and in resolving species relationships at lower taxonomic levels [bib_ref] Plant DNA barcoding: from gene to genome, Li [/bib_ref]. Park et al. conducted a phylogenetic study of six Fritillaria species based on the cp genome and concluded that plastome phylogenies are suitable for uncovering relationships among Fritillaria species, and obtain good support with high bootstrap values [bib_ref] The complete chloroplast genome sequences of Fritillaria ussuriensis Maxim. and Fritillaria cirrhosa..., Park [/bib_ref]. We constructed phylogenetic trees of 13 Fritillaria species using the PCG, LSC, SSC, and IR datasets. The phylogenetic relationships within the genus were identical and strongly supported in all of the phylogenies. In this study, Fritillaria appears to be a monophyletic group, which differs from the results of Day et al. [bib_ref] Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae), Day [/bib_ref] and may be attributed to our smaller sampling size. However, the positions of F. cirrhosa and F. thunbergii are far more highly resolved in our study (S1 . With some exceptions, our phylogenies are largely consistent with Day et al. [bib_ref] Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae), Day [/bib_ref] and support the polyphyletic classification of F. subgenus Fritillaria by Rix. Two species of F. subgenus Fritillaria (F. meleagroides and F. ussuriensis) clustered together and are sister to F. karelinii of F. subgenus Rhinopetalum in clade I (Figs 5 and 6), which is similar to the results of Khourang et al. [bib_ref] Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and..., Khourang [/bib_ref] , and may be attributed to the small sample size. The other 10 species of F. subgenus Fritillaria formed a strongly supported clade (clade II), and two subclades were resolved in clade II. The five species from outside Xinjiang formed a strongly supported subclade (subclade I), which was sister to subclade II containing the other five Xinjiang species. This indicated that the Xinjiang species had a close genetic affinity. Interestingly, we found that the eight species in clade I and subclade II of clade II originate from Xinjiang and Heilongjiang in North China (named the "north group"), and the five species in subclade I of clade II originate from South China (named the "south group"). An alternative explanation of the phylogenetic pattern is that the southern taxa diverged from the northern taxa and become distinct due to limited seed flow or genetic contact. The seven Xinjiang species did not form a monophyletic group, as F. karelinii and F. meleagroides were highly divergent from the other five species, and, interestingly, also differ in their morphology and habitat. Specifically, F. meleagroides occurs in a variety of habitats, including hilly slopes, shallow waters in mountainous areas, saline areas, and shallow swamps, while F. karelinii can usually be found in the plains of Artemisia desert habitats (desert habitat dominated by some drought tolerant Artemisia species) or low gravel hills. This species has a style that is longer than the stamens, and the stigma is scarcely lobed and slightly inflated at the top [fig_ref] Fig 5: Map indicating the distribution of Fritillaria in China [/fig_ref]. Endemic species are often limited to specific geographic areas, and in many instances have evolved vicariantly. Previous studies have demonstrated that specific limiting factors in an environment can significantly influence the geographic distribution patterns of species, including physical factors (i.e., temperature, light, moisture, aridity) and biotic factors (i.e., competition, predation, food availability). These factors usually influence the survival and propagation ability of plants. For instance, Corynephorus canescens is widely distributed in mid and south Europe, and its northern distribution limit in Europe coincides with the 15˚C isotherm in July, as its germination and flowering are affected by low temperature [bib_ref] Factors limiting the survival of Corynephorus canescens (L.) Beauv. in Great Britain..., Marshall [/bib_ref]. The winter distribution and abundance patterns of several avian species are directly linked to their physiological limits, with the northern range limit being associated with the −4˚C isotherm of the average minimum January temperature [bib_ref] Energy constraints on Avian distributions and abundances, Root [/bib_ref]. As high solar radiation and temperature are most favorable for the C4 photosynthetic pathway, C4 grass abundance patterns in North America are separated at 40˚N, where the C4 grass abundance is above 50% north of 40˚N and below 50% south of 40˚N [bib_ref] Climatic patterns and the distribution of C4 grasses in North America, Teeri [/bib_ref]. Interestingly, we also discovered that the northern and southern groups were largely separated at 40˚N [fig_ref] Fig 5: Map indicating the distribution of Fritillaria in China [/fig_ref]. However, the determining factor(s) influencing the distribution of Fritillaria species are not investigated in the present study. However, we hypothesize that soil moisture is an important environmental constraint influencing the growth of Fritillaria and other spring ephemeral plants. A semi-arid or desert climate prevails in Xinjiang and the precipitation is very low. Adequate water supply is only available from snow melting during March to June. From late June, the climate turns dry and hot, and is not suitable for growth. They have therefore adapted to a complete growth cycle ahead of the hot summer. Conversely, in south China, such as Sichuan, Hubei, and Zhejiang, precipitation is greater in summer, and thus some species have much longer growth cycles and can thrive from August to October (i.e., F. cirrhosa) (S4 . The stigma in the majority of Fritillaria species is 3-lobed; however, in a few species, i.e., F. yuminensis and F. karelinii, the stigma is undivided [fig_ref] Fig 6: The evolutionary progression of stigmatic traits within the genus [/fig_ref]. We surveyed 48 species in FOC and Flora of USSR, and found that only four species possess a scarcely lobed stigma. It was proposed that the trait of an undivided stigma might be a primitive characteristic. Our results do not support this hypothesis. The phylogenies demonstrate that F. karelinii diverges early, while F. yuminensis does not, and F. karelinii is closely related to F. tortifolia and F. verticillata. Moreover, in comparison to the phylogeny of Day et al. [bib_ref] Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae), Day [/bib_ref] , F. karelinii is not resolved as a basal species. Therefore, at this stage, we cannot infer a definite evolutionary trend for this trait. More cp genomes need to be sequenced to gain a comprehensive and accurate assessment of the evolutionary progression of the stigma. Furthermore, as F. yuminensis and F. karelinii do not form a monophyletic clade, this suggests that this trait might have evolved independently several times in the genus. Additionally, wild Fritillaria populations have been dramatically reduced due to excessive harvesting in recent decades. During our field investigation, we noted that F. meleagroides and F. karelinii were rare in the wild. The endemic species F. yuminensis is now endangered and can only be found in remote areas that are uninhabited by humans and livestock. Small populations of the other endemic species F. tortifolia can only be found in remote areas and natural reserves. Although all seven species in Xinjiang are listed in the class I protection plant list of Xinjiang, conservation action is urgently required. Population diversity is an important index in the formulation of a scientific conservation strategy. The newly sequenced cp genomes of these seven Fritillaria species would be useful for the development of SSR markers, and together with the identified divergent regions DNA regions, could be used to comprehensively assess the genetic diversity of wild populations in order to inform the protection of these valuable medicinal resources. As there are more than 140 species in the genus, the currently sequenced species only represent a very limited sample. However, we provide evidence that the cp genome can increase the resolution of phylogenetic relationships within the genus. More cp genomes are required to clarify the taxonomic and phylogenetic relationships of Fritillaria species at lower taxonomic levels, and can be used to estimate the population genetic diversity in order to formulate effective protection strategies. Supporting information S1 [fig] Fig 1: Gene map of the Fritillaria cp genome. Genes belonging to different functional groups are color-coded. The dashed area in the inner circle indicates the GC content. Cp genome size ranges are provided for the seven Xinjiang Fritillaria species. https://doi.org/10.1371/journal.pone.0194613.g001 [/fig] [fig] Fig 2: Comparison of LSC, SSC, and IR border regions among the 13 Fritillaria cp genomes. Colored boxes for genes [/fig] [fig] Fig 3: Comparison of 13 Fritillaria cp genomes with F. cirrhosa as the reference. LSC: long single copy region; SSC: short single copy region; IRa and IRb: inverted regions. Gray arrow: gene and translation direction; blue block: exon of the gene; red block: conserved non-coding sequences (CNS). Sequence identities are labeled at the right side and range between 50%-100%. https://doi.org/10.1371/journal.pone.0194613.g003 [/fig] [fig] Fig 4: Sliding window analysis of the entire chloroplast genome of 13 Fritillaria species. https://doi.org/10.1371/journal.pone.0194613.g004 [/fig] [fig] Fig 5: Map indicating the distribution of Fritillaria in China. The distribution area of each species is drawn according to the records in the FOC and Flora Xinjiangensis. Photographs of the species are also provided. https://doi.org/10.1371/journal.pone.0194613.g005 [/fig] [fig] Fig 6: The evolutionary progression of stigmatic traits within the genus. [/fig] [fig] S4: Table. The distribution areas and habitats of the 13 Fritillaria species in China.(DOCX) S1 Fig. Phylogenetic relationships of the 13 Fritillaria species inferred from ML and BI analyses using different data partitions. (A) PCGs, (B) LSC region, (C) SSC region, and (D) IR region. Values above the branches represent ML bootstrap values/BI posterior probability values. Outgroup: Lilium brownii KY748296; L. bakerianum KY748301; L. henryi KY748302. (TIF) [/fig] [table] Table 1: The chloroplast genomic characteristics of 13 Fritillaria species. [/table] [table] Table 2: Variable site analyses in Fritillaria chloroplast genomes. [/table] [table] Table: Sampled species and their voucher specimens. (DOCX) S2Table. Genes present in the seven Fritillaria cp genomes. (DOCX) S3 Table. Paired genetic distance between seven Fritillaria species in Xinjiang. (DOCX) [/table]

Adaptation of Escherichia coli Biofilm Growth, Morphology, and Mechanical Properties to Substrate Water Content ## S2 : Averaged reduced Young modulus Er values, describing the measured rigidity of the substrate surface by nanoindentation (from 0.5% to 2.5% agar: 4.8 ± 0.5 kPa, 15.0 ± 2.1 kPa, 62.3 ± 2.4 kPa, 102.1 ± 3.4 kPa; n = 7-8 individual measurements) S3 [fig] S2 S4, Figure S3 S5, Figure S4 S6, Figure S5 S7, Figure S6 S8, Figure S7: Relative area spreading rates of E. coli AR3110 biofilms grown on agar of different agar concentrations, calculated from the derivative 1/Ai * dA/dt Wet and corresponding dry biofilm mass from gravimetric measurements Brightfield images of cross sections corresponding to the fluorescence intensity images shown in Fig. 3C Average biofilm thicknesses (n = 10) measured on brightfield images on central and peripheral biofilm cross sections Representative load-displacement curve during loading and unloading AR3110 biofilm surface (1.8% agar). Indicated are the areas used for calculating the plasticity indices (A1, A2). Cross-sectioning protocol of biofilms (A) Isolating of individual biofilms (B) Embedding of biofilms in liquid agar (1.8%) and cutting of region of interest (ROI) (C) Embedding of agarbiofilm-agar sandwich in wax and gluing to the sample holder (D) Performing slices with the VT1000 S vibratome vibrating blade with a lateral distance of 250 µm [/fig]

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAIcarrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown. # Introduction Helicobacter pylori persistently infects more than one half of all humans, and can cause ulcer disease, gastric cancer, and MALT lymphoma [bib_ref] Helicobacter pylori infection, Suerbaum [/bib_ref]. The H. pylori cag pathogenicity island (cagPAI) is an intriguing virulence module of this obligate host-associated bacterium [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] [bib_ref] Helicobacter pylori cag-Pathogenicity island-dependent early immunological response triggers later precancerous gastric changes..., Wiedemann [/bib_ref]. H. pylori strains that possess a functional cagPAI are particularly frequently associated with severe sequelae, notably gastric atrophy and cancer [bib_ref] Helicobacter pylori cag-Pathogenicity island-dependent early immunological response triggers later precancerous gastric changes..., Wiedemann [/bib_ref] [bib_ref] Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify highrisk individuals..., Figueiredo [/bib_ref] [bib_ref] Host-bacterial interactions in Helicobacter pylori infection, Amieva [/bib_ref] [bib_ref] SagA of CagA in Helicobacter pylori pathogenesis, Hatakeyama [/bib_ref]. The cagPAI is ,37 kb long, and contains ,28 genes [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref]. These genes encode multiple structural components of a bacterial type IV secretion system (t4ss) as well as the 128 kDa effector protein, CagA [bib_ref] SagA of CagA in Helicobacter pylori pathogenesis, Hatakeyama [/bib_ref]. After H. pylori has adhered to a host cell, the Cag t4ss translocates CagA into that cell. CagA is subsequently phosphorylated by host cell kinases and interacts with multiple targets (e.g. SHP-2, Grb2, FAK), profoundly altering host cellular functions [bib_ref] Helicobacter pylori and gastric carcinogenesis, Hatakeyama [/bib_ref] [bib_ref] Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagA, Lu [/bib_ref]. The alterations induced by the cagPAI are thought to ultimately contribute to malignant transformation [bib_ref] Helicobacter pylori cag-Pathogenicity island-dependent early immunological response triggers later precancerous gastric changes..., Wiedemann [/bib_ref] [bib_ref] Helicobacter infection and gastric neoplasia, Peek [/bib_ref] , and CagA has been designated a bacterial oncoprotein [bib_ref] Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in..., Ohnishi [/bib_ref]. H. pylori has a high mutation rate, which has resulted in extensive genetic diversity [bib_ref] Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori, Bjorkholm [/bib_ref] , and also recombines frequently with other H. pylori [bib_ref] Free recombination within Helicobacter pylori, Suerbaum [/bib_ref]. H. pylori isolates have been subdivided into distinct biogeographic populations and subpopulations with specific geographical distributions that reflect ancient human migrations [bib_ref] Traces of human migrations in Helicobacter pylori populations, Falush [/bib_ref] [bib_ref] The peopling of the Pacific from a bacterial perspective, Moodley [/bib_ref] [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref]. The global population structure of H. pylori is now well understood based on multilocus haplotypes from seven housekeeping genes. However, very little is known about the biogeographic variation of virulence factors, such as the cagPAI, nor has the impact of genetic variation on disease outcome and host adaptation been adequately addressed. Previous analyses on the basis of comparative genome hybridization have demonstrated marked differences between biogeographic populations with respect to the cagPAI [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. Microarray analysis of 56 globally representative strains of H. pylori revealed that the cagPAI was present in almost all strains from some biogeographic populations and subpopulations in Africa and Asia, while it was variably present in other populations [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. The cagPAI was lacking in all isolates of hpAfrica2, which is distantly related to the other populations [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. Currently, nine complete cagPAI sequences are publicly available [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] [bib_ref] The complete genome sequence of the gastric pathogen Helicobacter pylori, Tomb [/bib_ref] [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref] [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] [bib_ref] The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain:..., Oh [/bib_ref] [bib_ref] Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter..., Alm [/bib_ref] , whose isolates belong to hpEurope (7 sequences), hspWAfrica (1) and hspEAsia (1) (see , and no sequence data is available for the cagPAI in the other six populations and subpopulations where the cagPAI is present. Here we analyze complete cagPAI sequences from 38 isolates representing all known H. pylori populations and subpopulations and compare their genetic polymorphisms with measures of functional expression. Our data show that the cagPAI has shared a long evolutionary history with the H. pylori core genome, and displays a remarkable global conservation of gene content, structure and function, with minor exceptions. We provide evidence that the cagPAI was acquired by ancestral H. pylori in a single event that occurred before modern humans migrated out of Africa. Sequence comparisons identified domains in multiple components of the t4ss that are likely to be under diversifying selection, and these findings can guide future research into the function of t4ss components. # Results ## Distribution of the cagpai in a global collection of h. pylori In order to define the occurrence of the cagPAI in H. pylori, we screened a globally representative collection of H. pylori isolates from 53 different geographical or ethnic sources [bib_ref] The peopling of the Pacific from a bacterial perspective, Moodley [/bib_ref] [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. 877 isolates were tested for the presence of the cagPAI by a PCR approach. Strains were classified as cagPAI-positive if we succeeded in separate PCR amplifications for the 59 and 39 ends of the cagPAI, or as cagPAI-negative if we succeeded in amplifying an empty site with primers from the flanking regions. The cagPAI was present in at least 95% of strains assigned to the hpAfrica1 (hspWAfrica plus hspSAfrica), hpEastAsia (hspEAsia, hspMaori) and hpAsia2 populations. In contrast, none of the hpAfrica2 strains possessed the cagPAI, and it was only variably present in strains from the populations hpEurope (225/330 strains; 58%), hpNEAfrica (58/72: 81%), and hpSahul or the hspAmerind subpopulation of hpEastAsia . Based on their multilocus sequence typing (MLST) haplotypes, seven strains with published cagPAI sequences belong to the hpEurope population (NCTC11638 from Australia [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] ; 26695 from England [bib_ref] The complete genome sequence of the gastric pathogen Helicobacter pylori, Tomb [/bib_ref] ; and DU23, DU52, Ca52, Ca73 [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] and HPAG1 [bib_ref] The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain:..., Oh [/bib_ref] from Sweden). J99 from the U.S.A. [bib_ref] Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter..., Alm [/bib_ref] belongs to hpAfrica1, and F32 [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref] from Japan belongs to the hspEAsia population of hpEastAsia. None of these published cagPAI sequences were from strains of the hpNEAfrica, hpSahul, or hpAsia2 populations, from the hpEastAsia subpopulations hspAmerind or hspMaori, or from the hpAfrica1 subpopulation hspSAfrica, although those populations are also potentially important for our understanding of the evolutionary history of H. pylori. We therefore selected 29 strains from our global strain collection to supplement these nine published cagPAI sequences and provide a globally representative sample of cagPAI diversity [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. These strains included all known biogeographic populations, except for the cag-negative hpAfrica2. The entire cagPAI, approximately 37 kilobasepairs in length, was sequenced and annotated from each of the 29 strains, either after shot-gun cloning of overlapping long-range PCR products or via direct amplification of multiple, smaller PCR products. ## Conserved synteny and low macrodiversity in the cagpai The 38 complete cagPAI sequences were compared by pairwise sequence alignments and by a multiple alignment in KODON relative to the cagPAI from J99 used as a scaffold sequence . The general pattern of gene content and gene order (signifying macrodiversity) was similar in most sequences, with only limited variation due to changed synteny or deletions. Synteny changes resulted from genomic rearrangements, horizontal genetic exchange (e.g. replacement of HP0521 by HP0521b), possibly in conjunction with IS (insertion sequence) element insertion, or gene inversions, such as for HP0535. Insertions, deletions, point mutations, frameshift mutations or disruption through insertion elements [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref] were also observed in some of the cagPAI sequences, some of which should have resulted in pseudogenes. We therefore tested all strains for their ability to induce interleukin-8 (IL-8) in gastric epithelial cells , [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref] , as an indicator of PAI function [bib_ref] Mucosal chemokines in Helicobacter pylori infection, Shimoyama [/bib_ref]. Most of the strains containing a cagPAI were able to induce IL-8, indicating that many of the mutations did not drastically reduce the general function of the cagPAI . ## Fixed and transient variants in cagpai sequence organization Most new mutations are deleterious, whether associated with single nucleotide polymorphisms, mobile elements or genomic rearrangements, and will be removed by purifying selection. However, mutations without a drastic effect on fitness, so-called neutral or nearly neutral mutations, can remain as rare variants within a population for long time periods. The vast majority of such mutations remain at low frequency until they are (usually) lost due to genetic drift. Rare neutral mutations can become more frequent over time, or even become fixed, also due to genetic drift [bib_ref] The neutral theory of molecular evolution: a review of recent evidence, Kimura [/bib_ref]. Still other mutations are under positive selection. These rapidly become frequent or fixed due to Darwinian selection. In isolated clonal populations, Muller's ratchet can even result in some deleterious mutations rising to high frequencyand the same is true of extreme bottlenecks, which can fix deleterious ## Author summary Most humans are infected with Helicobacter pylori. The H. pylori cag pathogenicity island (cagPAI) encodes a secretion apparatus that can translocate the CagA protein into host cells. Humans infected with cagPAI-carrying H. pylori are at increased risk of severe disease, including gastric cancer. We analyzed the nucleotide sequences and functional diversity of the cagPAI in a globally representative collection of isolates. Complete cagPAI sequences were obtained for 29 strains from all known H. pylori biogeographic populations. The gene content and arrangement of the cagPAI and its function were highly conserved. Diversity in most cag genes consisted in large part of synonymous polymorphisms. However some genes-in particular those that encode proteins predicted to be secreted or located on the outside of the bacterial cell-had particularly high frequencies of non-synonymous polymorphisms, suggesting that they were under diversifying selection. Our study provides evidence that the cagPAI was only acquired once and provides an important resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown. mutations immediately. These basic evolutionary principles indicate that the demographies of rare versus frequent mutations differ and should be examined separately. ## Frequent variants A number of frequent cagPAI macrodiversity variants were found, some of which were present in all isolates of at least one sub-population, or almost all isolates . These included insertion events due to one of three variants of IS606 [bib_ref] Sequence organization and insertion specificity of the novel chimeric ISHp609 transposable element..., Kersulyte [/bib_ref] or of a mini-IS605 insertion [bib_ref] Analyses of the cag pathogenicity island of Helicobacter pylori, Akopyants [/bib_ref] , an inversion of gene HP0535 plus its flanking non-coding DNA, a deletion of either the complete HP0521 ORF (D2; or part of that ORF, or the replacement of HP0521 by the unrelated ORF HP0521B , . Additionally, most of the 39 (right) half of the cagPAI is lacking in all three hspAmerind strains due to one of two similar 11.2 kb deletions with distinct 39 ends (D4, D5; . These large deletions terminate within HP0546, and are associated with a second (intergenic) deletion of 410 bp or a 620 bp deletion that terminates within the N-terminal part of HP0547 (cagA). In strains V225 and HUI1769, a copy of the deleted segment plus the HP0546 and HP0547 ORFs have translocated to a separate, currently unidentified, location of the chromosome, leaving a shortened version of HP0546 at the original location . It is interesting to note that IL-8 . Conservation of the cagPAI genetic organization across H. pylori biogeographic populations. The sequences were aligned in KODON using the cagPAI of strain J99 as a scaffold sequence. Individual isolates are grouped according to biogeographic (sub-)populations. The continuity of the cagPAI was disrupted in isolates PAL3414, V225 and HUI1769, and fragments found in secondary locations are displayed in greyshaded boxes on separate lines. The two cagPAI sequences from reference strains J99 and 26695 were extracted from whole genomes. Genes essential for a basic function of the cagPAI type IV secretion system (IL-8 induction; [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] are labeled with an asterisk*. Activity of the Cag t4ss (IL-8 secretion; + or 2) was monitored during experimental infection of AGS cells with H. pylori. Obs., observed IL-8 secretion; exp., IL-8 secretion expected from the cagPAI sequence; red, genes in forward orientation; blue, genes in reverse orientation; light blue, shorter gene version; white, different gene HP521B [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] in this locus; yellow, pseudogenes; black, IS elements; green, cagPAI insertion sites. Diamonds: frameshift mutations leading to pseudogenes. D followed by numbers 1 through 10 indicate different deletions (manifestation of macrodiversity) and are consecutively numbered as mentioned in the text and . a,b,c,d: strains not functionally tested in this study possess functional cagPAIs according to the following references: a [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] ; b [bib_ref] The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain:..., Oh [/bib_ref] ; c [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] ; d [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref]. doi:10.1371/journal.pgen.1001069.g002 Neighbor joining (NJ) tree of neutral genetic relatedness of H. pylori strains, including information about the presence or absence of the cagPAI. The NJ tree was calculated from concatenated sequences of seven housekeeping genes (length 3406 bp) from 877 isolates of H. pylori [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] plus 9 additional isolates from which either cagPAI sequences [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] or whole genome sequences had been published (indicated by arrows; [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] [bib_ref] The complete genome sequence of the gastric pathogen Helicobacter pylori, Tomb [/bib_ref] [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref] [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref] [bib_ref] The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain:..., Oh [/bib_ref] [bib_ref] Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter..., Alm [/bib_ref]. Each strain was scored for presence (filled triangles) or absence (empty circles) of the cagPAI based on the results of PCR reactions that span the ends of the cagPAI. Population assignments based on Bayesian analyses [bib_ref] The peopling of the Pacific from a bacterial perspective, Moodley [/bib_ref] [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] are indicated by the color coding of symbols that correspond to the labels next to the tree; red symbols indicate all strains whose cagPAI sequences are now available, including the 29 strains that have been newly selected for cagPAI sequence analysis. (B) Geographic sources of strains whose cagPAI sequences are now available. Each dot indicates the source of isolation of one of the 38 cagPAI sequences that were analyzed. The dots are color-coded by population or subpopulation as in (A). doi:10.1371/journal.pgen.1001069.g001 pylori in gastric epithelial cells (AGS, shown here, and MKN28, data not shown) was determined as a read-out for Cag t4ss activity. The two strains J99 and 26695A, for which entire genome sequences are available, were included as positive controls. CagA EPIYA motifs for each strain are indicated on top of the graph. Exceptions in the genetic integrity of some of the islands and other explanations for an observed loss of functionality are indicated above the single bars. Colored bars designate the population assignments of strains. Coincubation experiments were performed independently at least three times for each strain, with similar results, and one representative experiment, performed in triplicates for each strain, is shown. IL-8 secretion is depicted in relative values, as a multiple of the negative control (mock), which was set to 1. (B,C) Assessing underlying causes of loss of induction was not eliminated by any of these frequent mutations , [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref] , , suggesting that they are not deleterious to cagPAI function, and might be neutral or even under positive selection. ## Rare variants Rare variants were present in only one or two strains, are probably transient, and will tend to disappear during genetic drift [bib_ref] Near-neutrality in evolution of genes and gene regulation, Ohta [/bib_ref]. The rare variants included frameshift mutations in multiple ORFs within three single isolates (CC42C, HPAG1 and L72) and IS elements (mini-IS605, IS605, IS606, IS607 or IS608 [bib_ref] Sequence organization and insertion specificity of the novel chimeric ISHp609 transposable element..., Kersulyte [/bib_ref] that have integrated at distinct locations in 7 other isolates (Table 1; [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. Our dataset consisted of only 38 isolates, and it was possible that these rare mutations might be more widely distributed. We therefore screened 95 other globally representative strains for the presence of IS605, IS606, IS607 or IS608 at those locations, but only identified two additional strains with IS element insertions, one each for IS605 (MOR3055 -hspWAfrica) and IS607 (BASQ9523 -hpEurope) (data not shown). Thus, strains carrying these particular insertion mutations really are rare. We also found two rare, distinct genomic rearrangements . One of these was in strain NCTC11638 from Australia and has been reported previously [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref]. It splits the cagPAI between ORFs HP0534 and HP0535 into two segments, one of which is translocated elsewhere in the genome, and is distinct from the split of the cagPAI in the hspAmerind strains. Previous analyses identified the same rearrangement in 4/40 strains from Italy [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] , but it was not found in any of the other 38 cagPAI sequences analyzed here nor in any of the 95 other, globally representative strains that we investigated by PCR. The other rearrangement separated HP0547 (cagA) through HP0549 plus flanking DNA from the rest of the cagPAI. It has been previously described for two hpEurope strains from Sweden and one from Australia [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref]. We found the same pattern in a fourth hpEurope strain isolated in Palestine (PAL3414). Both of these rearrangements were present in less than 5% of isolates. The 17 rare mutations were identified in a total of 12 isolates. Only three of those, CC42C, HUI1692 and L72, did not induce IL-8, indicating that the majority of the rare sequence changes also did not cause a severe loss of cagPAI function. This observation is compatible with most of the rare mutations being selective neutral or near-neutral. ## Genomic decay Three overlapping small deletions (D1, D2, D3) that removed the HP521 ORF were found in all but one hpEastAsia isolate, one hpEurope isolate and the hpSahul strain ; , but those did not abolish cagPAI function (see above). Eight other deletions were found in four individual strains . Two of these isolates were unable to induce IL-8: CC42C (hspSAfrica) contains multiple frameshift mutations and an insertion of IS606 as well as deletion D11, which removes part of cagA (HP547). D4 and D6 deleted half of the cagPAI in hspAmerind strain HUI1692. The cagPAI is clearly decaying in both CC42C and HUI1692. In contrast, although deletions D5 and D7-D10 also removed large parts of the cagPAI in hspAmerind strains V225 and HUI1769, these deletions occurred in a segment that has been duplicated to a separate location (see above) and these two isolates remain able to induce IL-8. Thus, with one exception (D1), these deletions are rare and seem to be associated with accelerated decay of nonfunctional cagPAI genes. In addition, the cagPAI in non IL-8inducing strain L72 also contained one frameshift and one premature stop codon in a coding region, and seems to be undergoing decay. ## Signatures of selection within individual cagpai genes Darwinian selection for variation in coding regions can also be exerted at the nucleotide or protein level. We therefore analyzed sequence polymorphisms (microdiversity) in individual cagPAI genes for traces of such selection (Materials and Methods). Similar to housekeeping genes [bib_ref] Horizontal versus familial transmission of Helicobacter pylori, Schwarz [/bib_ref] , almost all alleles of each cagPAI ORF were unique to one isolate among the 38 strains. Exceptionally, we identified duplicates of a single allelic sequence in six genes; in each case, the strains possessing the duplicate alleles were from a common population . Occasional duplicate alleles within populations have also been described for housekeeping genes [bib_ref] Horizontal versus familial transmission of Helicobacter pylori, Schwarz [/bib_ref] and are considered to represent homologous recombination. Again, similar to housekeeping genes, most cagPAI genes seemed to be under purifying selection because their Ka/Ks ratios were #0.2 [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. However, five genes (HP0534-0535, HP0538, HP0546-0547) showed signs of positive or diversifying selection because their overall Ka/Ks ratios were greater than 0.2; of these, cagA (HP0547) had the highest proportion of nonsynonymous polymorphisms (Ka/Ks = 0.45). However, Ka/Ks ratios are relatively insensitive indicators of Darwinian selection, which can act at the level of single protein epitopes or conformational domains. We therefore used a Bayesian method (PAML/CODEML [bib_ref] PAML: a program package for phylogenetic analysis by maximum likelihood, Yang [/bib_ref] to search MLST and cagPAI genes for codons that might be under diversifying selection (indicated by v .1). Only two of the seven MLST housekeeping genes (trpC, yphC) contained an appreciable frequency (3.9%; 5.3%) of codons with posterior probabilities of v .1 being above 0.95 [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. In contrast, .5.3% of the codons matched this criterion in 10 of the 28 cagPAI ORFs [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref] , including four of the five ORFs with high overall Ka/Ks ratios (HP0535, HP0538, HP0546, HP0547). We also tested eleven cagPAI ORFs, including nine with high frequencies of codons under selection according to PAML, and two with lower frequencies (HP0524, HP0525) with a second Bayesian program, OmegaMap [bib_ref] Estimating diversifying selection and functional constraint in the presence of recombination, Wilson [/bib_ref] [bib_ref] Pilus operon evolution in Streptococcus pneumoniae is driven by positive selection and..., Muzzi [/bib_ref] , which unlike PAML also takes into account the occurrence of recombination (r) between different alleles . OmegaMap detected fewer codons with high probabilities of positive selection, but the codons that it identified often overlapped with codons that had been identified as being under positive selection by PAML function of cagPAIs in some H. pylori strains. (B) CagA translocation assays performed after infection of AGS cells with the two selected H. pylori strains D3A and M49. These displayed loss of cagPAI-related activity in IL-8 release assays. Both strains were unable to translocate CagA into human gastric epithelial cells. Strains SU2, N6, and 26695A wild type (wt) were used as positive controls for CagA translocation. Strains SU2Dcag and 26695ADcag (isogenic cagPAI deletion mutants to SU2 and 26695A) were included as negative controls. (C) transcript amounts of single cagPAI genes. 30 strains (4 strains shown here -for complete results see were studied using semiquantitative RT PCR for each gene with known function in the Cag t4ss (refer to [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref] for gene names). Two strains with loss of t4ss function, CC72C, and M49, are shown. TAI196 and 26695A are depicted as positive controls. TAI196, a strain with a high propensity to induce IL-8, shows relatively high transcript amounts for the majority of genes. Strains CC42C and L72 (not shown) which have pseudogenes and lost the ability to induce IL-8, showed low or undetectable transcript amounts for some genes including the pseudogenes. M49 displayed low transcript amounts for a number of essential genes of the t4ss located predominantly in the right half of the cagPAI (genes HP0528, and HP0537 to HP0544). doi:10.1371/journal.pgen.1001069.g003 . Genetic macro-and minidiversity variants (gene order and orientation, gene identity, insertion elements) within the H. pylori cagPAI with regard to population assignments. [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] 357 bp versus 659 bp for HP0521 in J99. [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] Also in 34/63 strains from Sweden [bib_ref] Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter..., Blomstergren [/bib_ref]. [bib_ref] Helicobacter pylori cag-Pathogenicity island-dependent early immunological response triggers later precancerous gastric changes..., Wiedemann [/bib_ref] IL-8 induction is according to data published by Oh et al. [bib_ref] The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain:..., Oh [/bib_ref]. However, HP0527 and HP0544 possess frameshift mutations that would normally prevent induction of IL-8. [bib_ref] Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify highrisk individuals..., Figueiredo [/bib_ref] Found in 1/95 additional strains from a global survey (this study) and 11/40 strains from Italy [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref]. [bib_ref] Host-bacterial interactions in Helicobacter pylori infection, Amieva [/bib_ref] in 1/95 additional strains from a global survey (this study). [bib_ref] SagA of CagA in Helicobacter pylori pathogenesis, Hatakeyama [/bib_ref] also found in 4/40 strains from Italy [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref]. [bib_ref] Helicobacter pylori and gastric carcinogenesis, Hatakeyama [/bib_ref] Deletion would prevent IL-8 induction. IL-8 induction is observed because of the presence of HP0536 -HP0547 in another genomic location. doi:10.1371/journal.pgen.1001069.t001 . Finally, we employed a sliding window along codons of PAML posterior probabilities of v to identify clusters of sites with signs of diversifying selection [fig_ref] Figure 4: Sliding window map of maximum likelihood analysis of codons to be under... [/fig_ref]. In addition to a high frequency of putative codons under diversifying selection, HP0527 (cagY) and HP0547 (cagA) also exhibited variable gene lengths. This was due to variable numbers of repetitive modules within the genes, as previously reported [bib_ref] Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system..., Aras [/bib_ref] [bib_ref] Oncogenic mechanisms of the Helicobacter pylori CagA protein, Hatakeyama [/bib_ref]. In the CagA protein, the number of phosphorylation sites (C-terminal EPIYA repeat motifs) differed, as did the types of these repeats [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref]. As previously described [bib_ref] Oncogenic mechanisms of the Helicobacter pylori CagA protein, Hatakeyama [/bib_ref] , the third EPIYA motif of CagA was type D in most (13/17) Asian strains whereas type D was not found in isolates from any other population. This reflected the preponderance of type D EPIYA in isolates assigned to the hpEastAsia and hpAsia2 populations. If the EPIYA type D motif were ancestral in Asian populations, this finding might reflect horizontal acquisition of cagA by the four exceptional Asian strains from Western strains. Homologous recombination involving the cagPAI has also been reported in isolates from Mestizos in Peru [bib_ref] Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention, Ahmed [/bib_ref] and might reflect selection due to functional differences that are related to ethnic specificity. ## Comparison of cagpai and housekeeping gene phylogeny We next asked whether the phylogeny of cagPAI genes was similar to that of housekeeping genes. Concatenated sequences of the cagPAI genes yielded a tree [fig_ref] Figure 5: Pairwise correlation of genetic distances and phylogeographic diversity between H [/fig_ref] that is very similar to the tree based on a concatenate of the seven MLST housekeeping genes [fig_ref] Figure 5: Pairwise correlation of genetic distances and phylogeographic diversity between H [/fig_ref]. Similarly, matrices of pairwise genetic distances of the concatenated cagPAI genes were highly correlated with corresponding matrices of pairwise distances of concatenated housekeeping genes (R = 0.65, p,0.001) [fig_ref] Figure 5: Pairwise correlation of genetic distances and phylogeographic diversity between H [/fig_ref]. These data show that 42% of the variance among cagPAI genes can be attributed to a linear relationship with housekeeping genes. The correlations for individual cagPAI genes ranged from R = 0.17 to R = 0.74 [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. While most cagPAI genes thus fell into the range observed for the individual housekeeping genes (0.46 to 0.69), the correlations were lower for particular cagPAI genes (e.g. cagL, R = 0.17), which might reflect selection and/or recombination between cagPAIs from different bacterial populations. These observations indicate a generally similar genealogy of cagPAI and housekeeping genes, which would imply that the cagPAI has accompanied H. pylori since before human migrations out of Africa some 60,000 years ago [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. In agreement, the genetic diversity of the cagPAI genes per population decreased significantly with distance from Northeast Africa (data not shown). ## Cagpai sequence variation and type iv secretion system function Only five of the strains tested here were not able to induce IL-8 [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref]. The same five strains did not translocate CagA into AGS cells, a second marker of t4ss function [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref]. For three of the five strains (CC42, L72 and HUI1692), a lack of function can be explained by sequence features of coding sequence (CDS) decay. The cagPAI of CC42C contains multiple pseudogenes, some of which are crucial for t4ss function [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref]. Half of the cagPAI including numerous essential t4ss genes is lacking in strain HUI1692. For strain L72, a point mutation results in a premature stop codon in gene HP0530, which is essential for t4ss function. In contrast, the cagPAI sequences did not offer obvious explanations for the lack of induction of IL-8 by strains M49 and D3a. We therefore investigated the transcript abundance of all 14 genes involved in IL-8 induction and of cagA for 28 sequenced strains as well as for the reference strains 26695A and J99 [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref] ; . The inability of strain M49 to induce IL-8 can be accounted for by very low transcript levels for 7/15 cagPAI genes [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref] ; ; the cause of this low transcription is unknown. However, we are unable to explain the inability of strain D3a to induce IL-8, because it was not impaired in cagPAI transcription . We are also not readily able to explain the considerable variation of transcript levels among the other strains that did induce IL-8 , except that it did not correlate with the macrodiversity patterns described above (data not shown). Similar to the variable transcript levels, the levels of IL-8 induction also varied dramatically [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref]. This variation did not correlate with strain assignments to biogeographic populations or with the type and number of EPIYA motifs within CagA [fig_ref] Figure 3: Variability of Cag t4ss function in H [/fig_ref] ; [bib_ref] Oncogenic mechanisms of the Helicobacter pylori CagA protein, Hatakeyama [/bib_ref]. Nor did they correlate with quantitative values for adhesion of the strains to AGS or MKN28 gastric epithelial cells (data not shown). # Discussion Since its discovery in 1996 [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] , the cagPAI has probably been the most intensively studied segment of the H. pylori genome. The virulence functions of the Cag t4ss and its translocated effector, CagA, have been investigated in great detail, and numerous studies have correlated cagPAI-associated polymorphic markers with disease risk. However, all these studies focused on one or only few genes within the cagPAI (such as cagA), and were performed with strains from one or few geographic regions. We therefore anticipated that a comparative analysis of complete cagPAI sequences from a globally representative and well characterized collection of strains would provide valuable information about the evolutionary history of the cagPAI and its variability within a phylogeographic context. The complete cagPAI sequences of 29 strains were determined and combined with 9 published complete sequences to yield a large and comprehensive dataset of cagPAI diversity, which was analysed at the levels of both macrodiversity (differences in gene content, synteny and function), and microdiversity (sequence polymorphisms). ## Phylogeographic implications of h. pylori cagpai diversity It has previously been noted from limited samples that different populations of H. pylori differ in the frequency of possession of the cagPAI [bib_ref] Traces of human migrations in Helicobacter pylori populations, Falush [/bib_ref] [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. Our data on 877 isolates from all known H. pylori populations and subpopulations provide unambiguous evidence for this variability. Carriage of the cagPAI varies from almost universal presence in hpEastAsia and hpAfrica1 through intermediate presence (hpEurope) to complete absence (hpAfrica2) [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. The cagPAI is also absent in the related species H. acinonychis [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref] , which resulted from a host jump from humans to large felines [bib_ref] Who ate whom? Adaptive Helicobacter genomic changes that accompanied a host jump..., Eppinger [/bib_ref]. The absence of the cagPAI from hpAfrica2 and H. acinonychis has been interpreted as the ancestral state, i.e. H. pylori acquired this genomic island by horizontal gene transfer from an unknown source after H. pylori had established itself in humans [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref]. But when was it acquired, and on how many occasions? The data presented here indicate that the cagPAI was only acquired once because its microdiversity correlated with microdiversity within housekeeping genes [fig_ref] Figure 5: Pairwise correlation of genetic distances and phylogeographic diversity between H [/fig_ref]. That acquisition was prior to 60,000 years ago, the time when H. pylori accompanied modern humans during their migrations ''out of Africa'' [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] , because cagPAI sequence microdiversity diminished with distance from North East Africa. An important implication of this conclusion is that, with the exception of hpAfrica2, the variable presence of the cagPAI in H. pylori populations usually reflects secondary loss, rather than inheritance of the ancestral virgin state. ## Macrodiversity versus fitness and function Previous analyses have shown that strains that circulate within the same communities, and even within the same stomach, can be mixed in respect to possession of the cagPAI [bib_ref] Horizontal versus familial transmission of Helicobacter pylori, Schwarz [/bib_ref]. This observation indicates that cag positive bacteria do not outcompete cag negative bacteria in all environments. Nevertheless, our data support the inference [bib_ref] Gain and loss of multiple genes during the evolution of Helicobacter pylori, Gressmann [/bib_ref] that a functional cagPAI provides a fitness advantage to H. pylori in most human populations: macrodiversity variants that inactivated t4ss function through deletions or insertion of IS elements were rare, whereas macrodiversity variants that were frequent did not affect t4ss function. For instance, shortening, complete loss or replacement (by HP0521b) of gene HP0521 was observed in almost all populations but this did not reduce cagPAI functionality, suggesting that this gene is not important for t4ss functions. Similarly, the genetic organization of the cagPAI was in general strongly conserved, and insertion elements did not play a decisive evolutionary role for the cagPAIs, unlike previous conclusions [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref]. Even separation of the cagPAI in two parts did not lead to loss of function, except when a deletion was involved. ## Cagpai t4ss microdiversity and signatures of positive selection High variation at the level of sequence microdiversity was found along the cagPAI, but this is also true of housekeeping genes, and might possibly result from the high frequencies of mutation and recombination in H. pylori [bib_ref] Traces of human migrations in Helicobacter pylori populations, Falush [/bib_ref] [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref]. However, unlike most housekeeping genes, multiple cagPAI ORFs showed signs of Darwinian diversifying selection, as indicated by higher Ka/Ks values and codon-based analyses, which identified specific amino acids or regions of particularly high non-synonymous diversity in 13 cagPAI genes [fig_ref] Figure 4: Sliding window map of maximum likelihood analysis of codons to be under... [/fig_ref] , [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. In the following we attempt to interpret these measures of selection by mapping them onto known components including structural features of the t4ss encoded by the cagPAI. Seventeen of the cagPAI genes are essential for the known t4ss functions (IL-8 induction, CagA translocation [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] , of which 12 have been characterized in structural or functional terms (virB1, [bib_ref] ) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and..., Censini [/bib_ref] [bib_ref] Helicobacter pylori cag-Pathogenicity island-dependent early immunological response triggers later precancerous gastric changes..., Wiedemann [/bib_ref] [bib_ref] Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify highrisk individuals..., Figueiredo [/bib_ref] [bib_ref] Host-bacterial interactions in Helicobacter pylori infection, Amieva [/bib_ref] [bib_ref] SagA of CagA in Helicobacter pylori pathogenesis, Hatakeyama [/bib_ref] [bib_ref] Helicobacter pylori and gastric carcinogenesis, Hatakeyama [/bib_ref] [bib_ref] Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagA, Lu [/bib_ref] [bib_ref] Helicobacter infection and gastric neoplasia, Peek [/bib_ref] [bib_ref] Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in..., Ohnishi [/bib_ref] and virD4 orthologs, cagA). In [fig_ref] Figure 6: Model of the Cag t4ss of H [/fig_ref] , we present a schematic structural model of the cagPAI t4ss apparatus including all known structural Cag proteins plus the effector CagA. Different shades of grey indicate the proportion of amino acids which are likely to have undergone diversifying selection according to PAML. ## Caga The translocated effector protein CagA (HP0547), which interacts with various host proteins [bib_ref] Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel..., Jimenez-Soto [/bib_ref] , had the highest proportion of such amino acids of the entire cagPAI. These were distributed along its entire length, suggesting functional adaptation or modulation. CagA binds to host cell integrins [bib_ref] Helicobacter pylori type IV secretion apparatus exploits beta1 integrin in a novel..., Jimenez-Soto [/bib_ref] and is translocated into host cells by the cagPAI t4ss. Within the host cell, individual domains of CagA interact with intracellular proteins such as SH-2 proteins and protein kinases (e.g. Src, Abl [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref] , MARK2/PAR1b kinase family [bib_ref] SagA of CagA in Helicobacter pylori pathogenesis, Hatakeyama [/bib_ref] [bib_ref] Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagA, Lu [/bib_ref]. These interactions render it potentially subject to diversifying or positive selection due to host polymorphisms which could even result in modified host protein interactions. A prominent example of amino acid diversity noted previously are the EPIYA motifs in the C-terminal half of CagA, which differ between Asian (hpAsia2; hpEastAsia) (type D) and all other populations [bib_ref] Helicobacter pylori evolution and phenotypic diversification in a changing host, Suerbaum [/bib_ref]. The D type EPIYA repeat binds SHP-2 phosphatase more avidly than other types [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref]. A clear bipartite ''Eastern''/''Western'' separation in the present global dataset was not only observed in phylogenetic trees based on the C-terminal half of CagA containing the divergent EPIYA repeat motifs, but also in its less well-characterized N-terminal moiety. Interestingly, CagA from the ancient and isolated hpSahul population [bib_ref] The peopling of the Pacific from a bacterial perspective, Moodley [/bib_ref] localised in between the Eastern and Western type CagA clusters (not shown). The global strain selection provided further evidence of functional adaptation in a different CagA motif. Recently, structural analyses of a second CagA subdomain (CM domain, aa 885 to 1005) in complex with its interaction partner from the human host, the cellular kinase MARK2, were performed [bib_ref] Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates, Nesic [/bib_ref]. This analysis revealed the crucial contribution of specific residues in CagA (MKI motif; [bib_ref] Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates, Nesic [/bib_ref] to the physical interaction with the kinase. The short CagA peptide that could be mapped in the cocrystal (Phe 948 -Lys 961 ) is characterized in our strain collection by high amino acid variability [fig_ref] Figure 7: Diversifying selection in the MARK2 kinase binding domain of CagA [/fig_ref]. Superposition of the amino acids under selection (according to PAML) onto the structure of the peptide [bib_ref] Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates, Nesic [/bib_ref] revealed that all but five of the 14 amino acids in this MARK2 binding domain of CagA have a high posterior probability of being under diversifying selection [fig_ref] Figure 7: Diversifying selection in the MARK2 kinase binding domain of CagA [/fig_ref]. Interestingly, Arg 952 and Val 956 , which both strongly influence MARK2 binding [bib_ref] Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates, Nesic [/bib_ref] , have a likelihood of 1.0 and 0.81, respectively, of being under positive selection whereas two other MARK2 binding residues, Leu 950 and Leu 959 , were not under diversifying selection. This result suggests that, although some specific MARK2 binding sites in CagA do have a lower propensity of being under positive selection, the binding strength of CagA to MARK2 can still be influenced by H. pylori protein variation, indicative of functional fine-tuning. These predicted functional implications of global variation in the MKI motif are in agreement with an earlier study by Lu et al. [bib_ref] Structural and functional diversity in the PAR1b/MARK2-binding region of Helicobacter pylori CagA, Lu [/bib_ref] who observed differences in CagA PAR1b binding and function when they exchanged two Western and Eastern phylogeographic variants of the CagA MARK2/PAR1b binding region within CagA chimeras. We therefore expect that other regions of CagA that are under selection [fig_ref] Figure 4: Sliding window map of maximum likelihood analysis of codons to be under... [/fig_ref] also warrant detailed structural and functional analyses. The observed CagA diversity, which is proposed to allow functional fine-tuning, may not only be associated with different host ethnicities but also with niche-dependent intrahost diversification during long-term colonization (e.g. stomach antrum versus corpus) [bib_ref] Heterogeneity in the activity of Mexican Helicobacter pylori strains in gastric epithelial..., Reyes-Leon [/bib_ref] [bib_ref] Helicobacter pylori cag pathogenicity island genotype diversity within the gastric niche of..., Matteo [/bib_ref]. ## Other cag genes A prior general comparison of component diversity in type III and IV secretion systems from different bacterial species [bib_ref] Protein homology network families reveal step-wise diversification of Type III and Type..., Medini [/bib_ref] found that core structural proteins located in the bacterial cytoplasm or the inner membrane exhibit significantly lower diversity than do structural proteins exposed on the surface of the bacteria or secreted effector proteins [bib_ref] Protein homology network families reveal step-wise diversification of Type III and Type..., Medini [/bib_ref]. Two well-characterized cag genes whose gene products are exposed on the cell surface have experienced strong selection: cagY (HP0527), which encodes a VirB10 ortholog that is a structural component of the cagPAI t4ss [bib_ref] A novel sheathed surface organelle of the Helicobacter pylori cag type IV..., Rohde [/bib_ref] , and cagC (HP0546), which encodes a VirB2 pilin subunit ortholog [bib_ref] Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system..., Aras [/bib_ref] [bib_ref] Characterization of the pilin ortholog of the Helicobacter pylori type IV cag..., Andrzejewska [/bib_ref]. CagY is under selection due to host antibodies and/or direct host interactions [bib_ref] Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system..., Aras [/bib_ref] [bib_ref] A novel sheathed surface organelle of the Helicobacter pylori cag type IV..., Rohde [/bib_ref]. In cagC, those codons with the highest likelihood of diversifying selection (amino acids 21 to 42; overlap with codons forming surface-exposed and highly strain-specific epitopes in the N-terminus of mature CagC [bib_ref] Characterization of the pilin ortholog of the Helicobacter pylori type IV cag..., Andrzejewska [/bib_ref]. The virB2 (HP0546) and virB5 (HP0539) orthologs of the cagPAI show signatures of diversifying selection in the present study; they encode surface-exposed pilin and pilus tip structural components of the Cag apparatus [bib_ref] VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems?, Backert [/bib_ref] and their sequence homology with functionally related VirB2 and VirB5 proteins from other bacteria is so low that they had to be identified by nonsequence-based approaches [bib_ref] Helicobacter exploits integrin for type IV secretion and kinase activation, Kwok [/bib_ref] [bib_ref] Characterization of the pilin ortholog of the Helicobacter pylori type IV cag..., Andrzejewska [/bib_ref]. We also find that 9 other cagPAI genes are under diversifying selection but their function is largely unclear. These include HP0520, HP0522 (part of the Cag outer membrane subcomplex [bib_ref] Cag3 is a novel essential component of the Helicobacter pylori Cag type..., Pinto-Santini [/bib_ref] , HP0523 (cagc; proposed to code for a virB1 orthologous peptidyglycan hydrolase [bib_ref] Characterization of peptidoglycan hydrolase in Cag pathogenicity island of Helicobacter pylori, Zhong [/bib_ref] [bib_ref] Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed..., Kutter [/bib_ref] , HP0528 (virB9), HP0534, HP0535, HP0536, HP0538 (encodes a membrane protein [bib_ref] Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed..., Kutter [/bib_ref] [bib_ref] The Helicobacter pylori cag pathogenicity island protein CagN is a bacterial membrane-associated..., Bourzac [/bib_ref] , and HP0540 [bib_ref] The Helicobacter pylori CagF protein is a type IV secretion chaperone-like molecule..., Pattis [/bib_ref]. Of these, HP0535 exhibits extensive non-synonymous variation and a clear bipartite Eastern-Western subdivision, similar to cagA. This gene is not involved in IL-8 induction or CagA translocation and is not predicted to possess a signal peptide. It may be a non-canonical secreted protein (score of 0.48 by SecretomeP). Based on the signs of selection and high diversity, we hypothesize that the HP0535encoded protein interacts closely with CagA or is a novel effector protein that is translocated into host cells by the Cag t4ss. Of the other genes under diversifying selection whose function is unknown, HP0520 might be a non-canonical secreted protein because its SecretomeP score was also high (0.92). In contrast to the genes just described, genes encoding cagPAI proteins that are not thought to be exposed on the bacterial surface [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] should be subject to purifying selection. In agreement with this expectation, other cagPAI genes including virD4 (HP0524) and virB11 (HP0525) orthologs [bib_ref] A novel sheathed surface organelle of the Helicobacter pylori cag type IV..., Rohde [/bib_ref] [bib_ref] Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed..., Kutter [/bib_ref] , displayed lower non-synonymous diversity and fewer codons under positive selection [fig_ref] Figure 6: Model of the Cag t4ss of H [/fig_ref] ; . [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. 10 cagPAI genes which do not participate in the structure or are of unknown function are not included in the model. The model of the Cag t4ss is based on [bib_ref] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for..., Fischer [/bib_ref] [bib_ref] VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems?, Backert [/bib_ref] [bib_ref] Cag3 is a novel essential component of the Helicobacter pylori Cag type..., Pinto-Santini [/bib_ref] [bib_ref] Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed..., Kutter [/bib_ref] [bib_ref] Interaction with CagF is required for translocation of CagA into the host..., Couturier [/bib_ref] [bib_ref] Protein-protein interactions among Helicobacter pylori cag proteins, Busler [/bib_ref]. doi: In conclusion, the present work reports a genetic and functional approach within a global population genetic perspective to study diversity in a complex secretion system. This comprehensive library of data allowed the identification of genes with a high probability of having undergone diversifying selection. cagPAI genetic diversity is accompanied by modulations in functionality, but rarely by complete loss of function. Functional modulation of the t4ss appears to be an important feature in vivo and is predicted to rely not only on protein diversification but also on straindependent transcript level diversity in the cagPAI. These data will be a resource for future research on the biological roles and variable host interactions of individual cagPAI proteins. It will also foster research on the phylogeographic variability and evolution of determinants of host interaction in other microbes. The diversity in this dataset will also be useful to evaluating predictions by recent evolutionary models based on the structure of proteins, such as neutral networks of protein folds [bib_ref] Connectivity of neutral networks, overdispersion, and structural conservation in protein evolution, Bastolla [/bib_ref] [bib_ref] Neutral evolution of proteins: The superfunnel in sequence space and its relation..., Noirel [/bib_ref] , which might be able to distinguish selection processes that favor structural versus functional conservation. # Materials and methods Bacterial isolates, sequencing, and RT-PCR Bacterial isolates and sequences of seven housekeeping gene fragments (atpA, efp, mutY, ppa, trpC, ureI, yphC) have been described previously [bib_ref] Free recombination within Helicobacter pylori, Suerbaum [/bib_ref] [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] [bib_ref] Recombination and clonal groupings within Helicobacter pylori from different geographic regions, Achtman [/bib_ref]. Strains were checked for the presence of the cagPAI by PCR, amplifying the 59 (Primers O2872 + O2902) and 39 (O2899 + O3326) flanking regions, or for absence (empty site) (primers O2872 + O3326). Primer sequences are provided in [fig_ref] Table S1: List of primers [/fig_ref]. Strains were chosen to represent all currently defined H. pylori populations possessing the cagPAI [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref]. The complete cagPAI was amplified for sequencing as two overlapping long range PCR products of ,20 kb each with primers O2903 + O3048 and O3047 + O2904 [fig_ref] Table S1: List of primers [/fig_ref] , respectively in 50 ml reactions with the EXL long range polymerase kit (Stratagene) using the following conditions: bacterial DNA 20 ng, Primers 20 mM each, 6 ml of 2mM dNTPs, 5 ml Buffer 1, 1 ml stabilizing solution, 1 ml EXL Polymerase, H 2 O to 50 ml. An initial denaturation for 1 min at 94uC was followed by 30 cycles of 45 sec at 94uC, 1 min at 65uC and 17 min 30 sec at 68uC. Long range PCR fragments were subjected to shotgun cloning. DNA fragments ranging from 0.8 to 1.2 kb were end repaired and cloned into the pGEM T-Easy vector (Promega), inserts were sequenced to 10-fold coverage by MWG Biotech. Alternatively, the cagPAIs were amplified as overlapping PCR products of ,5 kb each with additional primers listed in [fig_ref] Table S1: List of primers [/fig_ref] (primer combinations available on request) and sequenced with an extended set of primers [fig_ref] Table S1: List of primers [/fig_ref] by gene walking. The cagPAI sequence of strain PNGhigh85 was obtained by shotgun 454 sequencing of the whole genome (unpublished). Sequences were assembled with GAP4 (Staden Package, GCG Wisconsin). The individual cagPAI sequences have been submitted to the EMBL Nucleotide Sequence Database (accession numbers FR666825 -FR666857). Details for RNA preparation and RT-PCR are given in Text S1. RT-PCR primers and cycling conditions for transcript analyses of the cagPAIs are listed in [fig_ref] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection... [/fig_ref]. Multiple sequence alignment, sequence analysis, annotation, and phylogenetic analyses CDSs were annotated in ACT and in KODON (Applied Maths BVBA, Sint-Martens-Latem, Belgium), automatic multiple sequence alignment of individual cagPAI genes was performed in BIONUMERICS (Applied Maths BVBA, Sint-Martens-Latem, Belgium) and corrected manually after visual inspection, where necessary. Sequence comparison and graphical output of multiple complete cagPAI sequences was performed in KODON. We only included one of eleven cagPAI sequences (F32) available from Japanese strains [bib_ref] Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in..., Azuma [/bib_ref] because information is lacking on the phylogeographic population assignment of the remaining 10 strains. Pairwise genetic distances, phylogenetic trees and F ST were calculated in MEGA3 [bib_ref] MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences, Kumar [/bib_ref] and in Arlequin [bib_ref] Arlequin (version 3.0): An integrated software package for population genetics data analysis, Excoffier [/bib_ref] , respectively. Pairwise geographic distances and distance from North East Africa (Addis Ababa, Ethiopia), as well as confidence intervals were calculated as previously described [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref]. For analyses of increasing diversity with geographic distance from East Africa, the dataset was stripped of recent migrants [bib_ref] An African origin for the intimate association between humans and Helicobacter pylori, Linz [/bib_ref] which resulted in the use of 33 out of the 37 cagPAI sequences. Pseudogenes were excluded from the dataset in all phylogenetic analyses. ## Evolutionary analyses Ks/Ka ratios were determined in DNASP4.0 [bib_ref] DnaSP version 3: an integrated program for molecular population genetics and molecular..., Rozas [/bib_ref] and SWAAP, including a sliding window analysis. The number and location of potential codons under selection (v) in each cagPAI gene were determined using the program CODEML in PAML 3.15 [bib_ref] PAML 4: phylogenetic analysis by maximum likelihood, Yang [/bib_ref] , implementing a sliding windows graphic representation. This software calculates the ratio of maximum likelihood of different evolutionary algorithms (models) for each codon (site) of a coding sequence to be under positive selection (v.1), followed by Naive Empirical Bayes (NEB) and Bayes Empirical Bayes (BEB) analyses of posterior probabilities. Sites with a posterior probability P.0.95 by the CODEML codon substitution models M3 (discrete) or M8 (beta and v) of v.1 were considered as being under positive or diversifying selection. The likelihood of codons under diversifying selection in the presence of recombination was further analyzed using OmegaMap (V 0.5; [bib_ref] Estimating diversifying selection and functional constraint in the presence of recombination, Wilson [/bib_ref]. This software uses a Bayesian modeling algorithm to calculate the probability of codons to evolve under diversifying selection (v.1) in the presence of recombination (r). By explicitly modeling recombination, this method has a low rate to detect false positives. The settings used in the program were: norders = 100, thinning = 100, rhoprior = inverse, omegaprior = inverse, block length = 3 and 100,000 or 250,000 iterations. 5,000 iterations were deduced after each calculation as the burn-in phase. The model type used for both v and r was ''variable''. Three repetitions of the calculations with different settings were initially performed for control genes of defined structural properties and where some information is available about their function (e.g. HP0546), to exclude high variations in the calculations due to inadequate settings. Pseudogenes were excluded from the dataset. ## Housekeeping genes and population structure Fragments of the housekeeping genes atpA, efp, mutY, ppa, trpC, ureI, and yphC were amplified and both strands were sequenced from independent PCR products as described [bib_ref] Recombination and clonal groupings within Helicobacter pylori from different geographic regions, Achtman [/bib_ref]. Alternatively, comparable sequences were extracted from the published genomes (26695, HPAG1, J99). These sequences were assigned to populations and subpopulations by STRUCTURE [bib_ref] Traces of human migrations in Helicobacter pylori populations, Falush [/bib_ref]. Functional assays of the cagPAI t4ss IL-8 induction assay using the human gastric epithelial carcinoma cell line AGS (isolated from adenocarcinoma from a Caucasian patient) was performed for all strains of the sequencing project. Strain 26695A [bib_ref] Switching of flagellar motility in Helicobacter pylori by reversible length variation of..., Josenhans [/bib_ref] was used as a reference. Cells were cultured in RPMI 1640 medium (buffered with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum (medium and serum: Biochrom, Berlin, Germany). Details for bacterial culture conditions are given in Text S1. Cell infection experiments for IL-8 secretion measurement were performed on subconfluent cell layers (70%-90% confluence) in 24-well tissue culture plates. Cells were washed three times and preincubated in fresh medium with serum for 30 min prior to infection. By the addition of exponentially growing bacteria that were resuspended in cell culture medium (RPMI 1640, 25 mM HEPES, 10% heatinactivated serum), the infection was started (MOI of 50). To synchronize the infection, the incubation plates were centrifuged at 500 x g, 20uC, for 3 min. The coincubation was carried out for 20 h. Non-infected cells (mock coincubated) were used as negative control. Supernatants were harvested, cleared of cell debris by centifugation, immediately frozen and stored at 220uC until use. Release of IL-8 into the cell supernatants was quantified by using BD OptEIA IL-8 enzyme-linked immunosorbent assay kit (BD Pharmingen; San Diego, USA) according to the company's instructions, using appropriate dilutions. The assays were performed in triplicate and the means and standard deviations of at least six independent coincubations were calculated. Adherence of the strains was tested in a high throughput assay, but no correlation was found between adherence and the IL-8 induction (data not shown). To study CagA translocation, AGS cells were cultured in sixwell plates and infected with H. pylori at a multiplicity of infection (MOI) of 100. After 4 h of coincubaction, non-adherent bacteria were removed by washing twice with PBS-Dulbecco (pH 7.4; Biochrom, Berlin, Germany). Cells were harvested with a cell scraper and resuspended in 1 ml PBS (pH = 7.4; Biochrom, Berlin, Germany). After centrifugation (250 x g, 4uC, 5 min), cells were resuspended in 300 ml of modified RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate, 1 mM sodium orthovanadate, 1 protease inhibitor tablet per 10 ml buffer (Complete, Roche, Mannheim, Germany), 1 mM PMSF). During lysis, cells were incubated on ice for 30 min. Lysates were cleared by centrifugation (10 min, 21,900 x g, 4u) and the pellets were carefully separated from the supernatants. The pellet fraction was resuspended in 100 ml RIPA buffer and the fractions were immediately frozen at 280uC. To determine the amount of protein, a BCA protein assay was performed using the BCA Protein Assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. ## Western blot analysis of caga translocation Equal amounts of cleared cell lysates (see above; corresponding to 10 mg of protein) of infected cells were resuspended in 5 x SDS loading buffer (0.31M Tris-HCl, pH6.8, 37.5% glycerol, 10% SDS, 0.05% bromophenol blue, 20% b-mercaptoethanol) and boiled for 10 min. For determination of molecular mass, BenchMark pre-stained Protein Ladder (Invitrogen, Karlsruhe, Germany) was used. Samples were separated on 10.4% denaturing SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Protran BA 85, Whatman, Dassel, Germany) by semi-dry blotting. Membranes were blocked with 5% non-fat dried milk in TBS-T (20 mM Tris-HCl, 13.7 mM NaCl, 0.1% Tween 20, pH 7.4) for 1 h and subsequently incubated with specific primary antibody. Anti-CagA-antibody (Rabbit anti-H. pylori Cag antigen IgG fraction [polyclonal], Austral Biologicals, San Ramon, USA) was used at a dilution of 1/1,000 for the detection of CagA protein. To detect phosphorylated CagA, PY99-antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was used (dilution 1/ 250). Goat-anti-Rabbit-HRP antibody (dilution 1/10,000, Jackson Immunoresearch Laboratories, Suffolk, Great Britain) or Goatanti-mouse-HRP-antibody (dilution 1/5,000, Dianova, Hamburg, Germany) were used as secondary antibodies. Signal detection was performed with Enhanced SuperSignal West chemiluminescence substrate (Pierce, Rockford, IL, USA), and detection was on X-ray film (Hyperfilm, Amersham Biosciences, Buckinghamshire, UK). [fig_ref] Figure 1: Distribution of the cag pathogenicity island in a global collection of H [/fig_ref] Distribution of IS and mini IS elements and repetitive sequences in diverse cagPAIs. Repetitive sequences and sites where insertion (IS) elements and mini IS elements have integrated are indicated by symbols. Green: cagPAI insertion site containing repetitive sequence; red rectangles: mini IS606 insertions; blue triangles: mini IS605 insertion sites. Mini-IS607 and mini IS608 elements were not identified. a,b,c,d,e: different genetic variants of IS606 insertion elements. Found at: doi:10.1371/journal.pgen.1001069.s001 (0.14 MB PDF) ## Supporting information [fig] Figure 1: Distribution of the cag pathogenicity island in a global collection of H. pylori strains from different populations. (A) [/fig] [fig] Figure 3: Variability of Cag t4ss function in H. pylori strains from different biogeographic populations. (A) IL-8 induction in human gastric epithelial cells by diverse H. pylori strains from different biogeographic populations. IL-8 secretion induced at 20 h post infection by live H. [/fig] [fig] Figure 4: Sliding window map of maximum likelihood analysis of codons to be under diversifying selection for complete cagPAIs and housekeeping genes. Codons calculated by CODEML (model M3) to have a high likelihood p.95% of being under diversifying selection in each gene of the cagPAI or housekeeping genes of all analyzed strains are highlighted by black symbols. doi:10.1371/journal.pgen.1001069.g004 [/fig] [fig] Figure 5: Pairwise correlation of genetic distances and phylogeographic diversity between H. pylori housekeeping genes and concatenated cagPAI genes. (A) neighbor-joining (NJ) tree analysis of concatenated housekeeping genes for all strains, whose complete cagPAIs were analyzed. (B) NJ tree analysis of concatenated cagPAI genes for all strains. (C) Mantel comparison of pairwise genetic distances in housekeeping genes and cagPAI genes. doi:10.1371/journal.pgen.1001069.g005 [/fig] [fig] Figure 7: Diversifying selection in the MARK2 kinase binding domain of CagA. (A) amino acids (aa) in CagA predicted to be under diversifying selection (PAML, Model 3) were mapped onto the crystal structure of a short peptide within the CagA C-terminal domain (aa 948 to aa 961, MK1 peptide; aa under positive selection colored in pink, aa not predicted to be under positive selection colored in green), in complex with its interaction partner, the human kinase MARK2 [44]. Four residues critically involved in this interaction (Leu950, Arg952, Val954, Leu959) are labelled. Several amino acids involved in this interaction (e.g. Arg952 and Lys955, Ref. 44) are predicted to be under diversifying selection and are highly variable in our global strain collection. (B) amino acid alignment of the MARK2 binding region in the analyzed global strain collection. Black asterisks: amino acids involved in MARK2 binding. Pink dots: amino acids predicted to be under positive selection. Small dots in alignment: residue identical with reference strain 26695 (blue line on top). Hyphen: aa missing in respective strain. doi:10.1371/journal.pgen.1001069.g007 [/fig] [fig] Figure 6: Model of the Cag t4ss of H. pylori, highlighting diversifying selection on outer and secreted components of the t4ss apparatus. Each defined component of the cagPAI-encoded secretion system was shaded in grey according to averaged probability values, indicating the proportion of amino acids likely to be under diversifying selection for each individual protein; the probability values were calculated for each gene by the software CODEML [/fig] [table] Table 2: Sequence diversity, K s /K a ratios, and codons under diversifying selection in cagPAI and housekeeping genes (37 strains). Pearson correlation coefficient of p-distance matrices from individual genes versus concatenated housekeeping genes. R values for the housekeeping genes were calculated from matrices of concatenated sequences jackknifing from the respective gene. 1 cag genes predicted to be under diversifying.selection (p.95% in . = 5.3% of codons in PAML). u = genes of partly or completely undefined function. Total number of codons per gene refers to alignment length used for PAML. doi:10.1371/journal.pgen.1001069.t002 [/table] [table] Table S1: List of primers. Found at: doi:10.1371/journal.pgen.1001069.s002 (0.04 MB XLS) [/table] [table] Table S2: Primer list for transcript analyses of cagPAI genes. Found at: doi:10.1371/journal.pgen.1001069.s003 (0.02 MB XLS) Table S3 Transcript table for selected cag genes with a role in cag t4ss function (IL-8 induction) and for cagA. Found at: doi:10.1371/journal.pgen.1001069.s004 (0.02 MB XLS) Table S4 List of all identical alleles in single cag genes of the 38 analyzed cagPAIs. Found at: doi:10.1371/journal.pgen.1001069.s005 (0.03 MB DOC) Table S5 Congruence between PAML (CODEML model M8) and OmegaMap analyses for probabilities of diversifying selection of sites in H. pylori cagPAI genes. Found at: doi:10.1371/journal.pgen.1001069.s006 (0.04 MB XLS) Text S1 Supplementary Materials and Methods. Found at: doi:10.1371/journal.pgen.1001069.s007 (0.02 MB DOC) [/table]

The Impact of Meteorological Factors on Communicable Disease Incidence and Its Projection: A Systematic Review # Introduction In the last decades, the global burden of disease has shifted from communicable to non-communicable causes. The coronavirus disease 2019 (COVID-19) pandemic on the other hand has demonstrated how communicable disease remains a significant threat to global health, particularly as the climate crisis continues to influence disease spread in a variety of ways. The evidence shows that the global surface temperature during the most recent decade (2011-2020) was 1.09 [0.95 to 1.20] - C higher relative to the preindustrial period (1850-1900), which was driven by human activities . Based on the Sixth Assessment Report of the Intergovernmental Panel on Climate Change, the global surface temperature will continue to rise, ranging from 1.5 to 4.4 - C in the twenty-first century. The global warming of 2 - C is likely to occur in the mid-term period under the high greenhouse gases emissions scenario (Shared Socioeconomic Pathways, SSP3-7.0) . A warming of 2 - C and more poses greater risk to human health, particularly on vulnerable subpopulations such as the elderly, low-income populations, and people with comorbidities. Climate change poses a real challenge to public health and has contributed to causing the emergence and re-emergence of many communicable diseases of public health impor- frame,statistical analysis and climate model,findings related to meteorological factors and climate change prediction, and (7) adjustment for confounding and crossvalidation.shows the PRISMA flow diagram. All of the studies that were chosen followed an ecological design. Due to nature of the data, which is too heterogenous in term of statistically methods, study outcomes, and settings, the quantitative synthesis and analysis was not carried out. on 11 items, with a maximum overall score of 15 points. Supplementary Materials S2 presents the assessment scale adapted from Dufault and Klar. The article q was graded as low-(≤5 points), medium-(6-10 points), and high-relevance (≥11 po 2. ## . data extraction and synthesis MB and NA extracted the data independently using a standardized data extr form and organized it in a standard Microsoft Excel 2019 spreadsheet. The da information collected included: (1) authors, (2) year of publication (3) country, (4 frame,statistical analysis and climate model,findings related to meteoro factors and climate change prediction, and (7) adjustment for confounding and validation.shows the PRISMA flow diagram. All of the studies that were c followed an ecological design. Due to nature of the data, which is too heterogen term of statistically methods, study outcomes, and settings, the quantitative synthes analysis was not carried out. # Results ## Background of the eligible studies A total of 38 studies were included in this systematic review.Records excluded (n = 585) Reports sought for retrieval (n = 81) Reports not retrieved (n = 0) Reports assessed for eligibility (n = 81) Reports excluded: (n = 43) Spatial distributionDifferent study outcomeVector distributionStudies included in review (n = 38) Identification Screening Included. PRISMA flow diagram. # Results ## Background of the eligible studies A total of 38 studies were included in this systematic review.shows a descriptive summary of the included studies. The 38 studies were conducted in Bangladesh, Brazil, China, Indonesia, India, Iran, Korea, Malaysia, Mexico, Nepal, Nigeria, Philippines, Puerto Rico, Sri Lanka, Singapore, Sudan, Taiwan, Tanzania, Thailand, and Vietnam. When categorized into World Health Organization (WHO) regions, the majority of the studies had been conducted in the Western Pacific Region (WEPRO) and South-East Asia Region (SEARO). The analyzed articles were published between 2007 and 2020. More than half of the studies (60.5%) were conducted between 2015 and 2020. The study time frame varied from ≤5 years (10.5%) to 6-10 years (52.6%). Most of the studies explored the association of meteorological factors with vector-borne diseases: 23 studies (60.5%) focused on dengue and 11 studies (29%) focused on malaria as the health outcome. Three studies and one study examined the impact of meteorological factors on cholera (7.9%) and leptospirosis (2.6%), respectively.confounding factors, validation, and quality appraisal scoring. ## Meteorological factor variables The meteorological factor variables used in the included studies mainly consisted of rainfall/precipitation (34 studies), average/minimum/maximum temperature (33 studies), relative humidity (20 studies), and wind properties (three studies). Of these four meteorological factors, precipitation was most frequently associated with the selected climate-sensitive communicable diseases, followed by temperature, relative humidity, and wind properties. Of 23 studies reporting on dengue incidence, more than half reported a positive association between temperature (n = 17, 74%) and precipitation (n = 16, 70%) with dengue incidence. Furthermore, ten studies (43.5%) and three studies (13%) reported a positive association between relative humidity and wind properties, respectively, with dengue incidence. For malaria incidence, the majority of the studies reported a positive association with temperature (ten studies, 91%) and precipitation (nine studies, 81.8%). For the association between relative humidity and malaria incidence, five studies (45.5%) reported a positive association and three studies (27.3%) reported a negative association. No study reported an association between wind properties with malaria incidence. Two of three studies showed a positive association between temperature and precipitation with cholera cases. Additionally, Magny et al.reported a positive association between chlorophyll A anomaly with cholera cases, while Reyburn et al.reported a negative association. Lastly, Dhewantara et al. reported a positive association between temperature and precipitation with leptospirosis.summarizes the meteorological factors associated with the selected climate-sensitive communicable diseases. The other covariates included in the studies were duration of sunshine, El Niño-Southern Oscillation (ENSO), Indian Ocean Dipole (IOD), sea surface height (SSH), sea surface temperature (SST), sea level pressure, ocean chlorophyll concentration (OCC), and average river level. Some of the studies also included covariates related to landscape, such as normalized difference vegetation index (NDVI) and modified normalized difference water index (MNDWI), as well as covariates related to socio-economics, such as piped-water access, human migration, and population growth. ## Projection of climate-sensitive communicable diseases For the projection of dengue incidence in the event of climate change, Colon-Gonzales et al. reported that, under three climate scenarios (A1B, A2, B1), the mean annual dengue incidence across Mexico would increase around 12-18% by 2030, 22-31% by 2050, and 33-42% by 2080. A similar study conducted in Dhaka, Bangladesh, reported that dengue incidence would increase by 1.5 times if ambient temperatures increased by 1 - C in 2100 relative to 2010. If the temperature increases by 2 - C, the incidence of dengue would increase by seven times, and the worst-case scenario of a 3.3 - C rise would increase dengue incidence in Dhaka by 43 times in 2100 relative to 2010. In Guangzhou, China, Li et al. reported that under climate scenario Representative Concentration Pathway (RCP) 2.6, the overall incidence of dengue fever would be low, as would the occurrence of high numbers of cases. However, the overall incidence and the occurrence of high numbers of cases would increase under climate scenario RCP8.5. For malaria projection, Kwak et al. reported a gradually increased trend of malaria in Korea during a simulation using the RCP4.5 climate change scenario and the CNCM3 climate model. The maximum occurrence shifted from August (during 2010-2011) to July (using simulation data of 2011-2100). In the future, malaria occurrence would continually increase between April and July (before the rainy season in the summer) compared to between June and August in 2010-2011. Asadgol et al. reported that, under RCP8.5, the trend of cholera cases in Iran would increase by 2050. For the next 30 years, the seasonal pattern of cholera will change and the highest cases will be observed during spring and summer. The average monthly cholera cases will be highest in August if compared to the baseline data. ## Critical appraisal of the studies The studies were appraised using a modified scale of a checklist for assessing ecological research quality. None of the studies were scored as low relevance: 20 studies (52.6%) and 18 studies (47.4%) were scored as high and medium relevance on the assess-ment scale. For level of data aggregation, 8 (21%) studies were conducted at the national level, 11 (29%) conducted at regional or state level, and the remaining 19 (50%) involved province, county, district, or city as population unit. Only two (5.3%) studies used basic Spearman's rank and Pearson correlation as analytical methods. The rest of the studies applied advance statistical analysis such as Linear regression or Poisson regression, Autoregressive integrated moving average (ARIMA), and Multilevel Distributed Lag Non-linear Model (MDLNM). A total of 27 (71%) studies were conducted with a proper adjustment for covariates as suggested for ecological studies. For "quality of reporting", only eight (38%) studies include a statement of ecological study design. However, 13 (34.2%) studies explicitly justified study design, and most (97.4%) of the studies discussed the risk of ecological bias. The justification of study design allows readers to understand the rationale of choosing the design and apply the ecologic analysis. The risk of ecological bias needs to be explained clearly so that the authors sufficiently caution the readers in interpreting the results, representing the aggregated level.presents the scores of modified scale for each quality assessment item adapted from Dufault and Klar. # Discussion The aim of the present systematic review was to summarize key findings related to the relationship between meteorological factors and the occurrence of dengue, malaria, cholera, and leptospirosis, and to review recent communicable disease projection in the event of global warming. The present systematic review of 38 publications demonstrates that dengue, malaria, cholera, and leptospirosis transmission can be influenced by meteorological variables such as temperature, precipitation, relative humidity, and wind properties. The actions of these climate variables in influencing the transmission of communicable diseases are rarely independent. The combinations of a few climatic variables appear to be related to climatological niches for optimal disease transmission. Although the effects of climate change have been observed worldwide, the extent and patterns of the effect differ based on the country's location and socio-economic conditions. The present review shows that both precipitation and temperature are the most important meteorological factors for climate-sensitive communicable diseases, especially dengue and malaria. Several studies of different ecological units varying from city, province, regional, to nationallevels have demonstrated a positive association between temperature and precipitation with dengue incidence. This result is not limited to studies conducted in countries with tropical climates, but also includes studies conducted in Guangzhou, which has a subtropical monsoon climate. However, meteorological factors do not directly influence the incidence of dengue. Instead, meteorological variables such as temperatures, rainfall, and relative humidity have a direct impact on the larval development period, larval and adult mosquito survival, and the duration of the gonotrophic cycle of the primary dengue vector, and affect the general activity of the dengue vector, including host-seeking and blood meal intake. ## Relationship between metreological factors and dengue The ambient temperature alters the vector population dynamic by affecting the development of immature stages and reproductive behavior. The ideal temperature for Aedes aegypti development is between 22 and 32°C, while that for the A. aegypti adult lifespan and fecundity is between 22 and 28°C. Increasing the temperature will shorten the egg-laying time of A. aegypti, thereby increasing egg quantity. Moreover, higher temperatures are a favorable survival range for the vector and reduce the extrinsic incubation periods of the dengue virus. This will result in higher rates of viral transmission that can lead to increased dengue incidence. On the other hand, three studies have demonstrated a negative association between temperature and dengueand dengue haemorrhagic fever (DHF). According to Duarte et al., the monthly incidence of dengue will decrease by 32% with every 1 - C increment in the monthly average maximum temperature. This effect is particularly for increasing the maximum temperature to >32 - C, which is higher than the temperatures considered optimum for the vector. These temperatures help hasten the evaporation and drying of wastewater distributed around the city that would otherwise create mosquito breeding grounds. Rainfall has also been highly associated with dengue fever incidence. Rainfall provides abundant outdoor breeding sites for A. aegypti, for example, containers such as drums, discarded tires, and leaf axils that are naturally filled with rainwater. However, rainfall has also been negatively associated with dengue incidence in French Guiana, Singapore, Sulawesi, Indonesia, and Rio Branco, Brazil. According to the literature, heavy rainfall may reduce mosquito density because most mosquito eggs and larvae are carried away from breeding sites. This theory might explain why dengue in the abovementioned areas is reduced as rainfall increases. Relative humidity has also been associated with dengue incidence. Relative humidity affects all stages of the mosquito life cycle, the survival rate of the mosquito, the number of blood meals, and eventually its capacity to become infected and transmit dengue. Wind speed and direction are also important climatic factors. According to Chumpu et al. the best-fit model of Phayao province, Thailand, which incorporated wind direction and wind power, showed the highest dengue occurrences at wind speeds of 5-6 knots. This indicates that wind power is crucial for the spreading of dengue by mosquitoes. A higher wind power may affect dengue fever cases. More wind power on the sea surface results in a greater evaporation zone. Adult mosquitos may be able to survive longer and spread dengue as a result of the increased humidity. For mountainous areas, the most significant meteorological factors are wind direction variables. On the other hand, two studies conducted in the central region in Malaysiaand in Guangzhoufound that strong wind may suppress mosquito host-seeking activity and consequently reduce dengue transmission risk. However, only three studies included utilizing the wind properties as the independent variables. Future studies are recommended to explore wind properties as a possible meteorological factor related to dengue to overcome this limitation. ## Relationship between metreological factors and malaria Additionally, the majority of the 11 studies on malaria included in the present review reported a positive association between temperature and precipitation with malaria incidence. According to the data, increases in temperature, humidity, and rainfall facilitate the proliferation of mosquito populations at high altitudes. This expands the geographical distribution of malaria, allowing it to spread to new areas where mosquito populations previously did not exist. Furthermore, rising temperatures at lower altitudes, where mosquitoes and malaria are already endemic, alter the development cycle of the parasite that causes the Anopheles mosquito to transmit the disease, allowing it to develop malaria faster and therefore raising transmission rates. Other than climatic factors, factors such as human migration, population growth, and deforestation are associated with malaria transmission. The relative contribution of these factors may vary between countries and regions. Furthermore, malaria transmission can be exacerbated by human behavior, such as actively storing water in open containers, routine outdoor socializing during peak hours of Anopheles biting time (dawn and dusk), and other activities such as farming and fishing, which may increase the risk of exposure to mosquitoes and malaria infection. ## Relationship between metreological factors with cholera and leptospirosis There is substantial evidencethat cholera infection is linked to meteorological factors, such as low precipitation and high temperatures during the summer months, which might facilitate bacterial reproduction and increase cholera incidence. However, in Zanzibar, East Africa, Reyburn et al.reported that a 1 - C increase (at a four-month lag) would lead to two-fold increased cholera cases. Meanwhile, an increase of 200 mm rainfall (at a two-month lag) might increase cholera cases by 1.6-fold. Interestingly, a study in Matlab, Bangladesh, demonstrated a statistically significant one-month lag between OCC anomaly and cholera cases. Therefore, ocean and climatic trends are good predictors of cholera epidemics. For leptospirosis, a study in China has shown that land surface temperature and rainfall are significantly associated with leptospirosis notification. Warm temperature aids leptospire survival in the environment. Hot weather encourages some activities, such as people and animals swimming in the same pool of water, e.g., rivers. Besides, high humidity is a favorable condition for leptospire survival; however, Dhewantara et al.did not include relative humidity as one of the covariates. ## Projection of selected climate-senstive communicable disease The association between human activities and climate change has drawn increasing attention in recent years. It has been confirmed that human activity has a significant impact on present global warming. The rising emission of greenhouse gases has led to global warming and climate change, which have had various impacts, including on health, particularly toward communicable diseases. Projection of the geographic distribution of A. aegypti and A. albopictus has revealed that the abundance of mosquitoes will increase by the 2030s and beyond compared to the present, suggesting that more individuals will be at risk of dengue fever. A few studies have projected that exposure to the Aedes mosquito and the Aedes-transmitted virus would increase with 1.5/2.0 - C global warming. For example, most of the tropics are now ideal for virus transmission year-round for both Aedes aegypti and Aedes albopictus, with suitability decreasing along latitudinal gradients. However, projected warming temperatures in 2050 will substantially increase the potential for year-round transmission in the tropics, even into previously protected high-elevation locations. In addition, many temperate regions are presently devoid of significant Aedes vectors. However, in 2050, the risk of Ae. albopictus transmission is projected to increase significantly in temperate countries, particularly in high-latitude portions of Eurasia and North America. Elementary modelling predicts that the rising of global temperatures would increase the rate of malaria transmission and expand its geographical distribution. Several studies have reported that the increased malaria transmissionor its re-emergenceis associated with global warming. Khormi and Kumar projected that the southern regions of China might become susceptible to malaria mosquito infection in the future, in which suitability is expected to increase. Anopheles would be able to survive in large regions of southern China that are now unsuitable or marginal. Due to the high population density in these highly suitable areas, the number of individuals exposed to the Anopheles mosquito and hence to malaria is considerably increased. The predictive results indicate that modelling aids understanding of the disease transmission mechanism and assists in communicable disease intervention and control programs. However, the fundamental challenge for predicting climate-sensitive communicable disease transmission is how future climates can be best modelled at regional and/or local level. In other words, how can the results of global climate models (GCM) be suitably downscaled to a regional and/or local level? The Intergovernmental Panel on Climate Change (IPCC) Fifth Assessment Report (AR5) describes different climate futures, all of which are considered possible depending on the volume of greenhouse gases emitted in the years to come. These scenarios are categorized into four classes: RCP2.6, RCP4.5, RCP6, and RCP8.5, labelled after a possible range of radiative forcing values in 2100. These scenarios can be used to project future climates based on GCM. # Strenghts and limitations This review highlights the current public health issues on climate-sensitive communicable disease. All papers included in this review had undergone systematic critical appraisal using an adapted appraisal tool suitable for ecological study design, as described by Dufault and Klar. Thus, we used and adapted the structured assessment scale reported in other ecological review studies. None of the papers included in this review were low relevance, as based on the quality appraisal score. However, we recommend caution when estimating the relationships between climate variables and dengue in the following aspects: use of time lags, analysis of extreme climatic events, differences between seasonal and long-term trends, nonlinear effects and threshold effects in the associations. In addition, there should be more emphasis on data quality and the use of information for decision making. One limitation of this review is the included articles related to leptospirosis and cholera are limited. Therefore, caution is advised for interpretation when utilizing findings related to leptospirosis and cholera. Another limitation is that very few studies use the IPCC standardized climate change scenarios to predict future dengue, malaria, and cholera incidence. Besides, the exclusion criteria of non-English language articles could be one of the limitations of this review. Nearly half of the included studies were from WE-PRO, which majority comprises of non-English speaking countries. Therefore, this review might miss the wealth of related literature in particular published in Chinese. Due to the "English-language bias", this review could have bias estimates of effect, therefore reduce its generalizability. However, including studies published in non-English language may pose additional resources with respect to cost, time, and non-English language proficiency. One of the strengths of using the IPCC AR5 climate model is its ability to predict climates over a longer time or glacial year. The disadvantage is that it only considers the natural Earth systems and not the interaction between humans and nature. Furthermore, most of the individual studies assumed that human hosts are immobile. However, mass migration may contribute to dengue and malaria infection dynamics, especially at scales that exceed the limits of mosquito dispersal. We recommend that for future research to better understand dengue, malaria, cholera, and leptospirosis ecology to be directed at predicting the climate-biological relationships on disease transmission. Uncertainties due to confounding effects of urbanization, population growth, and human migration are required to develop scenarios based on future projections of population growth and socio-economic development, including human behavior. Future projection of climate-sensitive communicable diseases is greatly essential to aid planning and mitigation strategies by stakeholders, hence the need for scientific consensus on data potentially used in modelling. # Conclusions This review provides robust evidence of an association between meteorological factors and the incidence of climate-sensitive communicable diseases, i.e., dengue, malaria, cholera, and leptospirosis. Precipitation and temperature are important meteorological factors that influence the incidence of climate-sensitive communicable diseases. Future studies need to consider more determinants affecting precipitation and temperature fluctuations for better simulation and prediction of the incidence of climate-sensitive communicable diseases. In addition to future forecasts, accounting for alternative climate factor variables, considering climate change scenarios and other non-climatic drivers such as the presence/absence of dengue and malaria vectors, human migration, population growth, and socio-economics as crucial factors triggering communicable disease transmission would be beneficial. This would strengthen projection realism and act as a platform for academic and policymaker consensus on provisions to mitigate future climate-sensitive communicable diseases incidence.

A Rare Case of Erythema Gyratum Repens Associated With Esophageal Carcinoma # Introduction Erythema gyratum repens (EGR) is a distinctive rash often indicative of an underlying internal malignancy. However, recent reports showed 30% of the time it is idiopathic. EGR is associated with a variety of genitourinary, gastrointestinal, and hematological malignancies notably bronchogenic, esophageal, and breast cancer. It is also seen in patients with nonneoplastic conditions such as tuberculosis, hypereosinophilic syndrome, pregnancy, calcinosis, CREST syndrome, bullous pemphigoid, pemphigus vulgaris, systemic lupus erythematosus, ulcerative colitis, and rheumatoid arthritis. EGR was first described by in 1952 in a patient nine months before the appearance of breast cancer. The average age of onset is 63 years with male predominance. Most of the reported cases are Caucasians. ## Case presentation A 61-year-old African-American gentleman with no significant medical history presented to our clinic with dysphagia and progressive weight loss. He had a history of tobacco abuse. He was hemodynamically stable and physical examination was unremarkable. Esophagogastroduodenoscopy revealed esophageal carcinoma and CT abdomen revealed metastasis to the pancreas. He was started on chemotherapy with carboplatin and paclitaxel. Two months later he developed a rapidly progressive pruritic rash on his left thigh that progressively worsened to involve most of the trunk. On examination, distinctive serpiginous scaling patches with wood-grained appearance were noted on the thighs and trunk . A clinical diagnosis of EGR was made. A five-millimeter punch biopsy was obtained from the lateral thigh. Histology showed mild chronic perivascular lymphocytic infiltration with pigmented macrophages, epidermal parakeratosis, and focal spongiosis . The patient was educated about EGR and reassured. Chemotherapy was continued and the rash resolved in two months. # Discussion Erythema gyratum repens is a rash that is predominantly associated with malignancy. A systematic review reported that EGR was associated with underlying malignancy in 70% of the cases. The exact pathogenesis is unknown but postulated to be secondary to an immune response triggered by the underlying malignancy. Clinically the lesions are serpiginous, macular (occasionally papular), and erythematous with desquamating edges. Numerous serpiginous figures give rise to a characteristic "wood-grained" appearance. These lesions can extend rapidly, estimated at 1 cm/d in some patients. The hands, feet, and face are commonly spared. Occasional features include volar hyperkeratosis, ichthyosis, bullae, and onychodystrophy. Pruritus is almost always seen. Differential diagnosis includes erythema annulare centrifugum (EAC), necrolytic migratory erythema (NME), and erythema migrans. Diagnosis is usually made clinically. Histopathology is nonspecific, often showing mild hyperkeratosis, parakeratosis, acanthosis, and spongiosis with a perivascular mononuclear inflammatory infiltrate in the dermis. The observed immunofluorescence patterns of immunoglobulin G, C3, and C4 at the basement membrane corroborate a possible immunologic mechanism. Eosinophilia is observed in approximately 60% of cases. There is no specific therapy for the EGR. Topical and systemic steroids, vitamin A, and azathioprine have proven to be not effective. If a patient presents with the characteristic rash, an underlying malignancy should be suspected and investigated. Recognition and treatment of the underlying condition usually but not always lead to resolution of the rash. # Conclusions Erythema gyratum repens is a rare distinctive rash associated predominantly with underlying malignancy. The diagnosis is made clinically. Identification of the rash should prompt investigation into underlying conditions that can trigger the rash. Addressing the underlying condition will lead to improvement of the rash. # Additional information disclosures Human subjects: Consent was obtained by all participants in this study. ## Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

Analyzing the Spatial Equity of Walking-Based Chronic Disease Pharmacies: A Case Study in Wuhan, China Chronic diseases place a substantial financial burden on both the patient and the state. As chronic diseases become increasingly prevalent with urbanization and aging, primary chronic disease pharmacies should be planned to ensure that patients receive an equitable distribution of resources. Here, the spatial equity of chronic disease pharmacies is investigated. In this study, planning radiuses and Web mapping are used to assess the walkability and accessibility of planned chronic disease pharmacies; Lorenz curves are used to evaluate the match between the service area of the pharmacies and population; location quotients are used to identify the spatial differences of the allocation of chronic disease pharmacies based on residents. Results show that chronic disease pharmacies have a planned service coverage of 38.09%, an overlap rate of 58.34%, and actual service coverage of 28.05% in Wuhan. Specifically, chronic disease pharmacies are spatially dispersed inconsistently with the population, especially the elderly. The allocation of chronic disease pharmacies is directly related to the standard of patients' livelihood. Despite this, urban development does not adequately address this group's equity in access to medication. Based on a case study in Wuhan, China, this study aims to fill this gap by investigating the spatial equity of chronic disease medication purchases. # Introduction The term chronic disease refers to chronic non-communicable diseases. They have an adverse impact on the economic development of a nation. With global urbanization, the prevalence of chronic diseases increases [bib_ref] Urbanisation and human health in China: Spatial features and a systemic perspective, Li [/bib_ref]. According to the academics of WHO, chronic diseases will cause around 69% of deaths by 2030 [bib_ref] Projections of Global Mortality and Burden of Disease from 2002 to 2030, Mathers [/bib_ref]. For China, chronic diseases have become a major public health problem at the national level. China's State Council issued the Medium and Long-term Plan for the Prevention and Treatment of Chronic Diseases (2017-2025) and the Health China Action (2019-2030), which emphasize the prevention and treatment of chronic diseases. One of the effective ways to promote public health is to promote equity in health and social services in urban governance. Echoing this, China is in the process of reforming its primary healthcare system combined with walking-based 15 min life units to ensure equitable access to public services and promote full coverage of health services. On this, Wuhan is building 15 min walking community circles with health service. This kind of implementation is derived from the concept of '15 min city' which emphasizes the proximity and accessibility of facilities and services around residences [bib_ref] Improving the Spatial Accessibility of Community-Level Healthcare Service toward the '15-Minute City'..., Song [/bib_ref]. Current urban research studies in China are concerned with public healthcare accessibility for the construction of the 15 min city while failing to consider the joining of non-public facilities, such as retail pharmacies. Furthermore, previous relevant studies have not adequately considered the daily medicine shopping needs of patients with chronic diseases. This paper aims to fill in these gaps. The specific purposes are to: (1) propose a ## Study on spatial equity Spatial equity emphasizes the differences in the facilities enjoyed by different areas or social groups [bib_ref] Visualizing Fairness: Equity Maps for Planners, Talen [/bib_ref]. As regards urban facilities planning, spatial equity refers to the relationships between residents and facilities, i.e., spatial equality or spatial proximity [bib_ref] An accessibility-based integrated measure of relative spatial equity in urban public facilities, Tsou [/bib_ref]. This spatial equity is, to some extent, an extension of the concept of accessibility [bib_ref] Developing a composite index of spatial accessibility across different health care sectors:..., Siegel [/bib_ref]. Therefore, spatial accessibility evaluation is widely used to evaluate the spatial equity of public service facilities [bib_ref] Geographical Accessibility of Community Health Assist Scheme General Practitioners for the Elderly..., Deborah [/bib_ref] [bib_ref] Assessing Spatial Accessibility to Primary Health Care Services in Beijing, China, Zhang [/bib_ref] [bib_ref] Using Geographic Information Systems to investigate variations in accessibility to 'extended hours'..., Higgs [/bib_ref] [bib_ref] Spatial Accessibility to Primary Healthcare Services by Multimodal Means of Travel: Synthesis..., Kaur Khakh [/bib_ref] [bib_ref] Spatio-Temporal Analysis of Spatial Accessibility to Primary Health Care in Bhutan, Jamtsho [/bib_ref] [bib_ref] Evaluating the Spatial Accessibility and Distribution Balance of Multi-Level Medical Service Facilities, Jin [/bib_ref]. Spatial accessibility refers to the ease with which different spatial units can overcome the costs (time, distance, etc.) involved in reaching the desired facility. One of the critical factors affecting spatial equity is spatial distribution characteristics [bib_ref] Analyzing collective accessibility using average space-time prisms, Lee [/bib_ref]. Assessing spatial accessibility can effectively identify areas where public services are scarce, thus providing a quantitative basis for spatial equity research [bib_ref] Future accessibility impacts of transport policy scenarios: Equity and sensitivity to travel..., Pereira [/bib_ref] [bib_ref] Public transport accessibility tools matter: A case study of Gothenburg, Curtis [/bib_ref]. In terms of methods, the development of GIS technology in the 1990s led to the ArcGIS being favored by different academics, with common ones being buffer analysis, potential models, and gravity models [bib_ref] Assessing Spatial Accessibility to Primary Health Care Services in Beijing, China, Zhang [/bib_ref] [bib_ref] Spatio-Temporal Analysis of Spatial Accessibility to Primary Health Care in Bhutan, Jamtsho [/bib_ref]. After the 2000, along with the continuous maturation of Internet technology, traditional ArcGIS analysis has also been gradually linked to 'big data' and Web mapping services. Online platforms, such as Google Maps and Yahoo Maps, provide publicly available data. The application programming interface (API) allows access to real-time optimal route choices under different travel modes, corresponding time cost information, social data, media data, user comment data, etc. This feature of Web mapping ensures the validity, convenience and accuracy of the accessibility metric results [bib_ref] Measuring Spatial Accessibility to Hospitals of Acute Myocardial Infarction in Multi Period..., Su [/bib_ref]. In fact, there are still limitations to assessing spatial equity if only considering accessibility alone. Thus, actual spatial equity evaluations are not only based on accessibility, but also complemented by consideration of the needs of different social groups. Commonly used measures of spatial equity include correlation analysis, the Gini coefficient, the Lorenz curve, and locational quotients [bib_ref] Assessing Spatial Accessibility to Primary Health Care Services in Beijing, China, Zhang [/bib_ref] [bib_ref] The Advantages of Using Group Means in Estimating the Lorenz Curve and..., Lyon [/bib_ref] [bib_ref] Evaluation of the spatial equity of medical facilities based on improved potential..., Rong [/bib_ref]. Following the review of primary healthcare facilities and relevant methods, this research set a workflow to evaluate the equity of medicine purchase for patients with chronic diseases: the main urban area of Wuhan, China, was selected as the study area; Web mapping was used to measure the walkability to pharmacies; Lorenz curves were used to evaluate the match between pharmacies' service coverage and population; and locational quotients were used to identify differences in the spatial distribution of pharmacies' coverage on populations. # Methodology ## Study area According to China's Sixth Health Services Statistics Report 2018, the central region has the highest prevalence of chronic diseases in urban areas, at 34.7%. Wuhan is the center of development in central China, a mega-city in the Yangtze River Economic Belt, and a major national strategic development area in China. As of 2021, Wuhan has a population of 13,648,900 and 1,456,200 residents over the age of 65 . About 32.47% of Wuhan's population suffers from chronic diseases [bib_ref] Periodic optimization of drug purchase needs on site for chronic diseases under..., Wang [/bib_ref]. The setting of this study is the main urban area of Wuhan [fig_ref] Figure 1: Location of the study area in Wuhan [/fig_ref] , including Jiangan District, Jianghan District, Qiaokou District, Hanyang District, Wuchang District, Hongshan District and Qingshan District (including the East Lake Scenic Area, Wuhan Economic and Technological Development Zone and East Lake High-Tech Development Zone), with a total study area of about 678 km 2 . characteristics [bib_ref] Analyzing collective accessibility using average space-time prisms, Lee [/bib_ref]. Assessing spatial accessibility can effectively identify areas where public services are scarce, thus providing a quantitative basis for spatial equity research [bib_ref] Future accessibility impacts of transport policy scenarios: Equity and sensitivity to travel..., Pereira [/bib_ref] [bib_ref] Public transport accessibility tools matter: A case study of Gothenburg, Curtis [/bib_ref]. In terms of methods, the development of GIS technology in the 1990s led to the ArcGIS being favored by different academics, with common ones being buffer analysis, potential models, and gravity models [bib_ref] Assessing Spatial Accessibility to Primary Health Care Services in Beijing, China, Zhang [/bib_ref] [bib_ref] Spatio-Temporal Analysis of Spatial Accessibility to Primary Health Care in Bhutan, Jamtsho [/bib_ref]. After the 2000, along with the continuous maturation of Internet technology, traditional ArcGIS analysis has also been gradually linked to 'big data' and Web mapping services. Online platforms, such as Google Maps and Yahoo Maps, provide publicly available data. The application programming interface (API) allows access to real-time optimal route choices under different travel modes, corresponding time cost information, social data, media data, user comment data, etc. This feature of Web mapping ensures the validity, convenience and accuracy of the accessibility metric results [bib_ref] Measuring Spatial Accessibility to Hospitals of Acute Myocardial Infarction in Multi Period..., Su [/bib_ref]. In fact, there are still limitations to assessing spatial equity if only considering accessibility alone. Thus, actual spatial equity evaluations are not only based on accessibility, but also complemented by consideration of the needs of different social groups. Commonly used measures of spatial equity include correlation analysis, the Gini coefficient, the Lorenz curve, and locational quotients [bib_ref] Assessing Spatial Accessibility to Primary Health Care Services in Beijing, China, Zhang [/bib_ref] [bib_ref] The Advantages of Using Group Means in Estimating the Lorenz Curve and..., Lyon [/bib_ref] [bib_ref] Evaluation of the spatial equity of medical facilities based on improved potential..., Rong [/bib_ref]. Following the review of primary healthcare facilities and relevant methods, this research set a workflow to evaluate the equity of medicine purchase for patients with chronic diseases: the main urban area of Wuhan, China, was selected as the study area; Web mapping was used to measure the walkability to pharmacies; Lorenz curves were used to evaluate the match between pharmacies' service coverage and population; and locational quotients were used to identify differences in the spatial distribution of pharmacies' coverage on populations. # Methodology ## Study area According to China's Sixth Health Services Statistics Report 2018, the central region has the highest prevalence of chronic diseases in urban areas, at 34.7%. Wuhan is the center of development in central China, a mega-city in the Yangtze River Economic Belt, and a major national strategic development area in China. As of 2021, Wuhan has a population of 13,648,900 and 1,456,200 residents over the age of 65 . About 32.47% of Wuhan's population suffers from chronic diseases [bib_ref] Periodic optimization of drug purchase needs on site for chronic diseases under..., Wang [/bib_ref]. The setting of this study is the main urban area of Wuhan [fig_ref] Figure 1: Location of the study area in Wuhan [/fig_ref] , including Jiangan District, Jianghan District, Qiaokou District, Hanyang District, Wuchang District, Hongshan District and Qingshan District (including the East Lake Scenic Area, Wuhan Economic and Technological Development Zone and East Lake High-Tech Development Zone), with a total study area of about 678 km 2 . ## Data preparation ## Pharmacy data According to the Spatial Planning Guidance to Community Life Unit in China, the service radius of a 15 min life unit is 1 km, so this study extracted chronic disease pharmacies from the main urban area and its 1 km buffer zone. A total of 254 chronic disease pharmacies (as of the early 2019) were identified based on the public announcement list of health and wellness bureaus in each district, along with official media websites such as Changjiang Daily, Jingchu.com [fig_ref] Figure 2: Distribution of chronic disease pharmacies and traffic road network in the main... [/fig_ref]. The physical addresses of pharmacies are converted from to geographic coordinates through GeoSharp (Version 2.0, https://www. udparty.com (accessed on 22 December 2020)). of pharmacies are converted from to geographic coordinates through GeoSharp (Version 2.0, https://www.udparty.com (accessed on 22 December 2020)). ## Population data The population data is the 2017 Wuhan city street-level population data provided by the Wuhan Land Resources and Planning Information Centre. ## Road data The road data was obtained from Gaode Map (https://ditu.amap.com (accessed on 23 January 2019)). The road data also requires to be fixed line errors by geodatabase topology in ArcGIS (Version 10.3, http://www.esri.com (accessed on 1 January 2019)). ## Population data The population data is the 2017 Wuhan city street-level population data provided by the Wuhan Land Resources and Planning Information Centre. ## Road data The road data was obtained from Gaode Map (https://ditu.amap.com (accessed on 23 January 2019)). The road data also requires to be fixed line errors by geodatabase topology in ArcGIS (Version 10.3, http://www.esri.com (accessed on 1 January 2019)). # Research method ## The framework for chronic disease pharmacies equity measurement The method framework is shown in [fig_ref] Figure 3: Methods workflow. [/fig_ref]. First, in terms of geospatial service coverage, ArcGIS 10.3 combined with Web mapping is used to measure the planned service area and walking accessibility of chronic disease pharmacies. A comparison of the two coverages interrogates the gap between planned and actual service coverage. Second, regarding the allocation of service coverage, the Lorentz curve is used to understand the match between the pharmacies' service coverage and the population. Finally, the location quotients are calculated to identify differences in the allocation of service coverage among regions. ## The framework for chronic disease pharmacies equity measurement The method framework is shown in [fig_ref] Figure 3: Methods workflow. [/fig_ref]. First, in terms of geospatial service coverage, ArcGIS 10.3 combined with Web mapping is used to measure the planned service area and walking accessibility of chronic disease pharmacies. A comparison of the two coverages interrogates the gap between planned and actual service coverage. Second, regarding the allocation of service coverage, the Lorentz curve is used to understand the match between the pharmacies' service coverage and the population. Finally, the location quotients are calculated to identify differences in the allocation of service coverage among regions. ## Measuring planned service coverage by buffer model The service radius of outpatient pharmacies is defined as 1000 m and 500 m, respectively, based on the settings defined in the Spatial Planning Guidance to Community Life Unit. In the case of retail chronic disease pharmacies, there is no relevant planning guidance. As they are oriented to serve the surrounding communities, the walking range radius corresponding to a 15 min life unit is set at 1000 m. The buffers were constructed in ArcGIS 10.3. Service coverage indicates the service area covered by the pharmacies within each district as a proportion of their district's area. If a pharmacy's service area covers external jurisdictions, the external service area will be calculated in the external jurisdictions, but the overlapped coverage will not be calculated. The service overlap rate is the ratio of the overlapped service area within a jurisdiction to the sum of the service areas of all pharmacies in that jurisdiction (effective service area). The lower the overlap rate, the less duplication and waste of resources. [formula] C = ∑ (1) O = ∑CO − ∑PA ∑CO (2) [/formula] where Ci denotes the service coverage of chronic disease pharmacies in jurisdiction i; Oi denotes the service overlap rate of chronic disease pharmacies in jurisdiction i; PAi denotes the service area of the vertical projection of chronic disease pharmacies in the region; COi denotes the service area of pharmacies in jurisdiction i, i.e., the effective service area, and Ai denotes the size of jurisdiction i. ## Measuring walking time by web mapping The process is shown in [fig_ref] Figure 4: The workflow of measuring walking time by Web mapping [/fig_ref]. Firstly, the fishnets are constructed using ArcGIS 10.3. To ensure accuracy, this study applies 100 m × 100 m uniform point arrays within the main urban area of Wuhan based on the walking distance of a 15 min life unit. Next, with Python, the walking navigation API of Gaode Map is used to calculate the walking time between each fishnet and the three nearest chronic disease pharmacies. Additionally, ## Measuring planned service coverage by buffer model The service radius of outpatient pharmacies is defined as 1000 m and 500 m, respectively, based on the settings defined in the Spatial Planning Guidance to Community Life Unit. In the case of retail chronic disease pharmacies, there is no relevant planning guidance. As they are oriented to serve the surrounding communities, the walking range radius corresponding to a 15 min life unit is set at 1000 m. The buffers were constructed in ArcGIS 10.3. Service coverage indicates the service area covered by the pharmacies within each district as a proportion of their district's area. If a pharmacy's service area covers external jurisdictions, the external service area will be calculated in the external jurisdictions, but the overlapped coverage will not be calculated. The service overlap rate is the ratio of the overlapped service area within a jurisdiction to the sum of the service areas of all pharmacies in that jurisdiction (effective service area). The lower the overlap rate, the less duplication and waste of resources. [formula] C i = ∑ PA i A i (1) O i = ∑ CO i − ∑ PA i ∑ CO i(2) [/formula] where C i denotes the service coverage of chronic disease pharmacies in jurisdiction i; O i denotes the service overlap rate of chronic disease pharmacies in jurisdiction i; PA i denotes the service area of the vertical projection of chronic disease pharmacies in the region; CO i denotes the service area of pharmacies in jurisdiction i, i.e., the effective service area, and A i denotes the size of jurisdiction i. ## Measuring walking time by web mapping The process is shown in [fig_ref] Figure 4: The workflow of measuring walking time by Web mapping [/fig_ref]. Firstly, the fishnets are constructed using ArcGIS 10.3. To ensure accuracy, this study applies 100 m × 100 m uniform point arrays within the main urban area of Wuhan based on the walking distance of a 15 min life unit. Next, with Python, the walking navigation API of Gaode Map is used to calculate the walking time between each fishnet and the three nearest chronic disease pharmacies. Additionally, then, this data is sorted and aggregated by Excel to filter out the shortest time from each fishnet to the nearest pharmacy (The essence of calculating accessibility using Web mapping is to solve for the time it takes for each point of fishnet to reach each facility. Solving directly for the optimal route solution means N1 × N2 calculations (N1 is the number of fishnets and N2 is the number of pharmacies), which is beyond the availability of map suppliers and the computing power of computers. In addition, most fishnets are not linked to pharmacies as they span multiple areas between them. Hence, to reduce the amount of data calculated online, the data needs to be pre-processed offline to reduce the number of calculations to N1 × 3 times). Finally, the fishnets are assigned attributes by jurisdiction. The number of fishnets in each jurisdiction is counted by filtering by attribute field and time interval. The actual coverage based on walking time is the fishnets within the 15 min life units as a proportion of the total number of fishnets in the area. Multi-temporal data extraction is not conducted because this research only examines walking medicine purchase behavior and real-time traffic conditions have little influence on the findings. Solving directly for the optimal route solution means N1 × N2 calculations (N1 is the number of fishnets and N2 is the number of pharmacies), which is beyond the availability of map suppliers and the computing power of computers. In addition, most fishnets are not linked to pharmacies as they span multiple areas between them. Hence, to reduce the amount of data calculated online, the data needs to be pre-processed offline to reduce the number of calculations to N1 × 3 times). Finally, the fishnets are assigned attributes by jurisdiction. The number of fishnets in each jurisdiction is counted by filtering by attribute field and time interval. The actual coverage based on walking time is the fishnets within the 15 min life units as a proportion of the total number of fishnets in the area. Multitemporal data extraction is not conducted because this research only examines walking medicine purchase behavior and real-time traffic conditions have little influence on the findings. ## Measuring equality of service on chronic diseases pharmacies Equity is assessed on two levels, namely, per-capita and inter-regional. In this study, the service buffer of chronic disease pharmacies is used to represent the service resources. By using ArcGIS extraction analysis, a proportion of the study area is calculated for each street's effective service area. The proportions are ranked from lowest to highest. Then, the cumulative percentage of the area served, as well as the resident population and the elderly population, are plotted to form the Lorenz curve and their fitted curves. The Lorenz curve does not reflect the spatial match between the distribution of pharmacies and the urban population. Therefore, this research applies location quotients to measure the match between the service coverage of chronic disease pharmacies and the population distribution. The calculation formula is as follows. [formula] LQi = (Mi/Pi)/(M/P)(3) [/formula] where, LQi is the location quotient of chronic disease pharmacies' service in the street i; Mi is the effective service area of chronic disease pharmacies in the street i; Pi is the number of resident populations in the street i; M is the sum of effective service area of chronic disease pharmacies in the study area; and P is the resident population in the study area. When LQi > 1, it means the per capita access to the resources in the street i is higher than the overall level. Additionally, there is a high probability that the facilities' resources match or are more adequate than the street population; When LQi < 1, it indicates the per capita access to resources is lower than the overall level and that there may be a lack of resources [bib_ref] Evaluation of the spatial equity of medical facilities based on improved potential..., Rong [/bib_ref]. # Results ## Accessibility by the planned service coverage ## Measuring equality of service on chronic diseases pharmacies Equity is assessed on two levels, namely, per-capita and inter-regional. In this study, the service buffer of chronic disease pharmacies is used to represent the service resources. By using ArcGIS extraction analysis, a proportion of the study area is calculated for each street's effective service area. The proportions are ranked from lowest to highest. Then, the cumulative percentage of the area served, as well as the resident population and the elderly population, are plotted to form the Lorenz curve and their fitted curves. The Lorenz curve does not reflect the spatial match between the distribution of pharmacies and the urban population. Therefore, this research applies location quotients to measure the match between the service coverage of chronic disease pharmacies and the population distribution. The calculation formula is as follows. [formula] LQ i = (M i /P i )/(M/P)(3) [/formula] where, LQ i is the location quotient of chronic disease pharmacies' service in the street i; M i is the effective service area of chronic disease pharmacies in the street i; P i is the number of resident populations in the street i; M is the sum of effective service area of chronic disease pharmacies in the study area; and P is the resident population in the study area. When LQ i > 1, it means the per capita access to the resources in the street i is higher than the overall level. Additionally, there is a high probability that the facilities' resources match or are more adequate than the street population; When LQ i < 1, it indicates the per capita access to resources is lower than the overall level and that there may be a lack of resources [bib_ref] Evaluation of the spatial equity of medical facilities based on improved potential..., Rong [/bib_ref]. [fig_ref] Figure 5: Chronic disease pharmacy service coverage and overlap based on planning radius [/fig_ref] shows that the service coverage of chronic disease pharmacies in the study area is 38.09%, while the service overlap rate is 58.34%, with the latter being 20.25% higher than the former. In the south of the Yangtze River, the service coverage of chronic disease pharmacies is lower than that north of the Yangtze River. However, the south's overlap rate is lower, with an average overlap rate of 43.04% for the south and 57.21% for the north. # Results ## Accessibility by the planned service coverage ## Accessibility by walking The coverage of services within a 30 min walk is 62.52% [fig_ref] Figure 6: Actual service areas by walking [/fig_ref]. The service area within 15 min walking time accounts for 28.05% of the district area, which is about half the size of the 15 min life units in the main urban area of Wuhan (53.94% of the district area). than the former. In the south of the Yangtze River, the service coverage of chronic disease pharmacies is lower than that north of the Yangtze River. However, the south's overlap rate is lower, with an average overlap rate of 43.04% for the south and 57.21% for the north. ## Accessibility by walking The coverage of services within a 30 min walk is 62.52% [fig_ref] Figure 6: Actual service areas by walking [/fig_ref]. The service area within 15 min walking time accounts for 28.05% of the district area, which is about half the size of the 15 min life units in the main urban area of Wuhan (53.94% of the district area). The actual service coverage based on 15 min walking time decreased by 10.04% compared to the planned service coverage . The change in coverage in Qingshan District and Jianghan District is below 6% (the former 5.12% and the latter 5.67%). The changes in Wuchang, Jiangan and Hanyang districts are at an intermediate level, at 9.74%, 7.64% and 7.44%, respectively. Hongshan District and Qiaokou District show more significant changes in coverage, with the former shrinking by 11.13% and the latter by 11.66%. The actual service coverage based on 15 min walking time decreased by 10.04% compared to the planned service coverage . The change in coverage in Qingshan District and Jianghan District is below 6% (the former 5.12% and the latter 5.67%). The changes in Wuchang, Jiangan and Hanyang districts are at an intermediate level, at 9.74%, 7.64% and 7.44%, respectively. Hongshan District and Qiaokou District show more significant changes in coverage, with the former shrinking by 11.13% and the latter by 11.66%. ## Equality of service on chronic diseases pharmacies ## Per capita equity by the lorenz curves The Lorenz curves for chronic disease pharmacy services by population are depicted in [fig_ref] Figure 8: Lorenz curve by resident/elderly population [/fig_ref]. The fitted curves constructed correspond to R-squared values of 0.9986 and 0.9984, respectively, indicating that they are close to the original curves. According to the fitted curves, 60% of residents shared 47% of the pharmacies' services, while the remaining 40% had 6% more resources than the former. The unevenness is much more evident in the calculations for the elderly. 70% of the old only shared 52% of the pharmacies' services, while the remaining 30% had almost as many resources as the former. ## Equality of service on chronic diseases pharmacies ## Per capita equity by the lorenz curves The Lorenz curves for chronic disease pharmacy services by population are depicted in [fig_ref] Figure 8: Lorenz curve by resident/elderly population [/fig_ref]. The fitted curves constructed correspond to R-squared values of 0.9986 and 0.9984, respectively, indicating that they are close to the original curves. According to the fitted curves, 60% of residents shared 47% of the pharmacies' services, while the remaining 40% had 6% more resources than the former. The unevenness is much more evident in the calculations for the elderly. 70% of the old only shared 52% of the pharmacies' services, while the remaining 30% had almost as many resources as the former. According to the fitted curves, 60% of residents shared 47% of the pharmacies' services, while the remaining 40% had 6% more resources than the former. The unevenness is much more evident in the calculations for the elderly. 70% of the old only shared 52% of the pharmacies' services, while the remaining 30% had almost as many resources as the former. ## Inter-regional equity by location quotients The location quotients are classified into five classes using natural discontinuity analysis [fig_ref] Figure 1: Location of the study area in Wuhan [/fig_ref]. In terms of location quotients based on resident population,.86% of streets in the study area are below the medium level of chronic disease pharmacies' services per capita; 11.83% of streets are in the extremely high or higher bracket; residents within streets in the exceptionally high bracket enjoy more than three times the overall average level of service area per capita. The results of the location quotients based on the elderly population show that 52.69% of the streets are below the overall level # Discussion This study simulated the currently planned service area of chronic disease pharmacies using buffers and measured the actual service area using Web mapping. It found a gap between the planned and actual use of chronic disease pharmacies. The overall coverage of the pharmacy service area is low, while the overlap is high, indicating the effective utilization of pharmacy resources is low. The accessibility of pharmacies decreases from the old city center to the periphery, and there is a spatial divergence between the north and south. This result supports the argument that old central districts provide higher pedestrian accessibility to public services [bib_ref] Walkability in urban landscapes: A comparative study of four large cities in..., Fan [/bib_ref] [bib_ref] Monitoring housing rental prices based on social media: An integrated approach of..., Hu [/bib_ref]. The results of the Lorenz curve and location quotients reveal a gap in the current per capita service level of chronic disease pharmacies in Wuhan's main urban areas. The distributional inequality is observed between groups with fewer service resources and those with more, and this inequality is particularly evident among the elderly. Recent research similarly indicates that there are pharmaceutical deprivation areas for the elderly, despite the illusion of good coverage implied at the metropolitan level [bib_ref] Geographical accessibility to community pharmacies by the elderly in metropolitan, Padeiro [/bib_ref]. ## Influencing factors of chronic disease pharmacies equality Three main factors contribute to differences in equity: the time of build-up of the area; the targets of the regional development; and the topography. (1) There are differences in the layout and type of residential areas and the demographic structure between the old and new urban areas. The main built environment of Wuhan is along the Yangtze River and gradually expanded outwards. The layout of the # Discussion This study simulated the currently planned service area of chronic disease pharmacies using buffers and measured the actual service area using Web mapping. It found a gap between the planned and actual use of chronic disease pharmacies. The overall coverage of the pharmacy service area is low, while the overlap is high, indicating the effective utilization of pharmacy resources is low. The accessibility of pharmacies decreases from the old city center to the periphery, and there is a spatial divergence between the north and south. This result supports the argument that old central districts provide higher pedestrian accessibility to public services [bib_ref] Walkability in urban landscapes: A comparative study of four large cities in..., Fan [/bib_ref] [bib_ref] Monitoring housing rental prices based on social media: An integrated approach of..., Hu [/bib_ref]. The results of the Lorenz curve and location quotients reveal a gap in the current per capita service level of chronic disease pharmacies in Wuhan's main urban areas. The distributional inequality is observed between groups with fewer service resources and those with more, and this inequality is particularly evident among the elderly. Recent research similarly indicates that there are pharmaceutical deprivation areas for the elderly, despite the illusion of good coverage implied at the metropolitan level [bib_ref] Geographical accessibility to community pharmacies by the elderly in metropolitan, Padeiro [/bib_ref]. ## Influencing factors of chronic disease pharmacies equality Three main factors contribute to differences in equity: the time of build-up of the area; the targets of the regional development; and the topography. (1) There are differences in the layout and type of residential areas and the demographic structure between the old and new urban areas. The main built environment of Wuhan is along the Yangtze River and gradually expanded outwards. The layout of the city is divided by the traffic ring roads and the Yangtze River. The first and second rings are dominated by old communities with dense housing and roads. Between the second and third rings, there are mainly old residential areas with high floor area ratios, low building density, and massive commercial areas due to urban renewal. Hence, walking accessibility is generally better within the second ring. The third ring and beyond are mainly new or under-construction high-end residential areas with low occupancy rates and poorly configured facilities. The clusters of residential areas are mostly separated by wide roads. firstly, the early construction of the city was limited by the lakes. Many of the new residential areas came about after the transport road network had been improved. Thus, most of those close to the third ring are upscale areas with an enormous footprint, and the construction of primary medical institutions has not yet been kept up. Secondly, the existence of an extensive lake system leads to road bypasses. Therefore, the layout of facilities is more dispersed, and the continuity of services is affected. ## Optimizing strategies for chronic disease pharmacies equity This study proposes optimization options that can be linked to three perspectives: demographic structure; adjustment of the pharmacies' layout; the form of pharmacy services. (1) With limited facility resources, priority should be given to elderly patients with lower location quotients. The leading group of chronic disease patients is the elderly, with limited mobility. Each district government should grasp the changes in the spatial distribution of chronic disease patients, especially the elderly. Governments should adjust the pharmacy locations according to the space of the life unit, traffic structure, patient population changes, and trends to plan. (2) The current public chronic disease pharmacy system in Wuhan has already been established. So, it is more feasible to adjust the layout of existing facilities than to re-establish a dispensing facility system. In terms of investment, pharmaceutical companies invest in retail pharmacies and do not require financial investment from the government. There is no lack of outpatient pharmacies on each street within the second ring. Hence, retail pharmacies are encouraged to be introduced in blind areas, effectively reducing the government's financial burden. Furthermore, some areas have a dense distribution of pharmacies and significant resource redundancy, requiring a moderate replacement of chronic disease pharmacies. Public outpatient pharmacies should be considered first for areas with insufficient chronic disease pharmacies. (3) Finally, optimizing facilities and services should also be linked to the Internet. Although most chronic disease medicines purchased online are not currently reimbursed in China, the future health insurance system will certainly interact more with the Internet, which will further enhance the resilience of cities in the future. It will play a pivotal role in the urban system, especially in response to public health emergencies such as the COVID-19 epidemic. The online purchase and reimbursement of medicines will lead to a new layout of primary healthcare facilities. # Conclusions This research analyses the spatial pattern of chronic disease pharmacy resources based on space and population, using the central city of Wuhan as a case site. The results show that Web mapping services can estimate the accessibility of facilities on a finer and more accurate scale. The approaches in this study can further examine the equity of service provision of public service facilities under planning in conjunction with population data. The timing of urban build-up, development targets, and topography determine the distribution and structure of the population and road networks. The city's distribution and structure of the population, as well as the road network, influence the accessibility and equity of pharmacy services. In conclusion, the main contribution of this study is to propose a workflow for evaluating the equity of the layout of chronic disease pharmacies based on the Web mapping API, Lorenz curves, and location quotients. It focuses on the needs of chronic disease patients and complements the study of Web mapping for evaluating community life unit facilities. This workflow is based on the available dataset and has the potential to be applied in other regions or facilities, considering a lot of countries and regions have accessible demographic, household, and point of interest (POI) data. For example, it could be used to explain the potential inequality of other facilities' locations in the 15 min city or life units, such as large grocery stores, parks, and recreational facilities [bib_ref] The 15-minute walkable neighborhoods: Measurement, social inequalities and implications for building healthy..., Weng [/bib_ref]. However, the workflow cannot be simply applied to other regions without considering the various travel modes, such as in the U.S., where people made 89% of their trips by automobile and only 8% on foot. For a region that heavily depends on vehicles, this framework is limited by ignoring real-time traffic data. A future study might focus on generalizing the model by including more types of travel modes. Meanwhile, considering that real-time traffic data are instantaneous, future studies might involve more informatics to achieve automatic or semi-automated results. On this, the automatic workflow could be combined with IoT collected-clinical data and be an effective tool in emergency medicine [bib_ref] Measuring Spatial Accessibility to Hospitals of Acute Myocardial Infarction in Multi Period..., Su [/bib_ref] [bib_ref] Secure IoT Analytics for Fast Deterioration Detection in Emergency Rooms, Caruccio [/bib_ref]. # Limitation There are still some limitations in this study, which are areas for further improvement. In terms of data acquisition, the data on the distribution of the population with chronic diseases is not publicly available and can only be estimated by the number of resident populations in each street. In terms of walking accessibility calculations, Web mapping fails to measure the walking speed of the elderly. Future research into the accessibility of facilities for patients of different ages will be more relevant in guiding the planning and layout of pharmacies for chronic diseases. [fig] Figure 1: Location of the study area in Wuhan. [/fig] [fig] Figure 2: Distribution of chronic disease pharmacies and traffic road network in the main urban area of Wuhan. [/fig] [fig] Figure 3: Methods workflow. [/fig] [fig] Figure 4: The workflow of measuring walking time by Web mapping. [/fig] [fig] Figure 5: Chronic disease pharmacy service coverage and overlap based on planning radius. [/fig] [fig] Figure 6: Actual service areas by walking. [/fig] [fig] of 16, Figure 7: Int. J. Environ. Res. Public Health 2023, 20, x FOR PEER REVIEW 9 Planned service areas for chronic disease pharmacies vs. actual service areas based on walking. [/fig] [fig] Figure 8: Lorenz curve by resident/elderly population. [/fig] [fig] Figure 7, Figure 7: Planned service areas for chronic disease pharmacies vs. actual service areas based on walking.4.3. Equality of Service on Chronic Diseases Pharmacies4.3.1. Per Capita Equity by the Lorenz CurvesThe Lorenz curves for chronic disease pharmacy services by population are depicted inFigure 8. The fitted curves constructed correspond to R-squared values of 0.9986 and 0.9984, respectively, indicating that they are close to the original curves.Int. J. Environ. Res. Public Health 2023, 20, Planned service areas for chronic disease pharmacies vs. actual service areas based on walking. [/fig] [fig] Figure 9: (a) The location quotient based on resident population; (b) The location quotient based on the elderly population. [/fig] [fig] 2: The different development targets of the seven jurisdictions have resulted in different plans for the built environment and public facilities. For example, Jianghan District has the highest economic power and ranks first in service coverage, while Hanyang District and Hongshan District are at the end of the list due to their late urban and economic development. In the Qiaokou District, Yijia Street and Gutian Street are mainly used for commercial services and public facilities, with many industrial parks and scattered residential land. Those areas are divided by railway lines and many urban highways with a width of 15 m or more, resulting in poor accessibility to the pharmacies.(3) Wuhan's many lakes and hilly terrain have two impacts on the layout of pharmacies: [/fig] [fig] Author: Contributions: Conceptualization, Y.L.; methodology, Y.L., Y.S. and X.L.; software, Y.L. and Y.S.; resources, Y.L.; data curation, Y.L.; writing-original draft preparation, Y.L. and Y.S.; writingreview and editing, Y.L. and X.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This research was funded by the China Scholarship Council, grant number 202210080009. The APC was funded by Queen's University Belfast. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig] [table] Table 1: Number and proportion of streets graded based on location quotient of the resident population.Table 2. Number and proportion of streets graded based on location quotient of the elderly population. [/table]

Deleterious Association of Inhalant Use on Sleep Quality during the COVID-19 Pandemic Citation: Gunge, D.; Marganski, J.; Advani, I.; Boddu, S.; Chen, Y.J.E.; Mehta, S.; Merz, W.; Fuentes, A.L.; Malhotra, A.; Banks, S.J.; et al. Deleterious Association of Inhalant Use on Sleep Quality during the COVID-19 Pandemic. Int. J. Environ. # Introduction While there is strong evidence linking conventional tobacco use with sleep disturbance, the effects of electronic (e)-cigarettes on sleep quality remain largely unknown. Nicotine is considered one of the causal factors in the adverse effects of tobacco on sleep quality due to its effects as a stimulant resulting in decreased sleep time and increased nighttime awakenings. Over ninety percent of e-cigarettes contain nicotine, and thus it is likely that vaping will also impact sleep quality due to the nicotine content. In our previous study, we found that female users of both conventional tobacco and e-cigarettes, known as dual users, had worsened sleep quality and higher sleep latency (amount of time to fall asleep) than never smokers/never vapers. This pilot study was replicated in a larger cohort, validating our findings that dual use is associated with increased sleep latency (manuscript in revision). Although there have been several studies published over the last few years attempting to address whether e-cigarettes or dual use impact sleep health, many of the studies focused on particular age groups or did not use validated questionnaires for evaluating sleep quality. Others only looked at one type of inhalant rather than all four groups. Of the eleven articles reviewed, one only alluded to a possible relationship between e-cigarette use, oxidative stress, and adverse effects on sleep. Only one study did not find a difference in sleep duration or quality in e-cigarette users; however, this study design had concerning limitations using unvalidated methods for assessing sleep. The remaining nine articles had findings supportive of a relationship between e-cigarette use and decreased sleep quality including sleep duration, onset, latency or regularity. In summary, although the articles all had unique limitations from study design to generalizability of findings, the vast majority of the articles concluded that e-cigarette or dual use leads to alterations in sleep that need further investigation. Increased life stressors are commonly associated with disruptions to the sleep cycle. During the COVID-19 pandemic, many lifestyle changes were required for the global population. These included changes to work environment, childcare, social interactions, physical activity, etc. These alterations to normal routine inevitably bring about new feelings that are sometimes positive but often negative, stressful, and anxiety-provoking. Further, the pandemic itself led to fear of dying, fear of spreading the virus, and fear of losing loved ones as another layer of stress. According to epidemiologic data provided by the Centers for Disease Control (CDC) and the World Health Organization (WHO), between the two timepoints of the surveys the globe was experiencing a trend of increasing daily COVID cases. In the same timeframe, the US was experiencing a spike in daily deaths and a plateau in daily cases of COVID-19. Because sleep is negatively impacted during times of increased stress, we hypothesized that the negative correlations between historical or active use of nicotine-based inhalants may be amplified as the COVID-19 pandemic progressed. Here we conducted a social media-based survey during the time of COVID-19 to assess the impact of anytime use of nicotine-based inhalant use on sleep quality in the setting of global stress. We recruited participants who either in the past or present had been e-cigarette vapers, conventional tobacco users, dual users of both e-cigarettes and conventional tobacco and never smokers/never vapers to take our University of California San Diego (UCSD) Inhalant Survey online. Our survey included detailed questions on sleep patterns and quality, at two separate time-points during the pandemic. We hypothesized that nicotine-based inhalant users would have worse sleep quality in the setting of the ongoing stress of the pandemic because they would not adapt as well as non-inhalant users. # Materials and methods This study was approved by the UCSD Institutional Review Board (protocol #160204). Inhalant habits involving both historical and active use were surveyed using our established UCSD Inhalant Questionnaire, while sleep quality was assessed via the Pittsburgh Sleep Quality Index (PSQI; scored from 0 to 21, with higher numbers indicating worse sleep quality). Specifically, types of inhalants and amount of vaping and smoking per day were both quantified. The years of tobacco use were included in the historical smoker questions. Participants were recruited through online advertisements posted to social media sites including Facebook, Craigslist, Reddit, and Twitter. All participants underwent informed consent prior to taking the survey. Participants (n = 554) were recruited in April 2020, and a subset (n = 217) retook the survey in June 2020. In the June retake survey, six questions were added to assess the subjects' current level of COVID concern. Specifically, these questions included: "How concerned about COVID are you right now? How much has COVID disrupted your life? Has the COVID-19 pandemic caused you anxiety? Since the pandemic began, how has your work-related stress changed? Have you contracted COVID? Have any family members, friends, or loved ones contracted COVID?" All participants were entered into a weekly lottery for a USD 250 Amazon gift card. Data were analyzed with GraphPad Prism (version 9.0.2, GraphPad software, San Diego, CA, USA). Descriptive analyses were performed for demographics and sleep quality outcome scores, with outcomes being summarized by both inhalant use groups and overall. Differences between inhalant users and non-inhalant users were analyzed by Welch's two-tailed t-test, and longitudinal changes were analyzed with paired t-tests. Differences across e-cigarette users, conventional smokers, dual users and non-inhalant users, both before and during COVID, were analyzed with 2-way ANOVA with Sidak for multiple comparisons. # Results ## Demographics A total of 217 participants completed the online survey. One hundred and fortynine were never smoker/never vapers, 39 were conventional tobacco smokers, 10 were e-cigarette or vaping device users, and 19 participants were dual users. The majority of participants identified as women (74%), with 25% men, and 1% non-binary. The majority of subjects (n = 139) self-identified as Caucasian. The ages of participants ranged from 18 to 75 years, with a considerable fraction of participants grouped between the ages of 21 to 30 (39%). Eighty-five percent of participants were located across the US with the remaining dispersed internationally including Australia, the United Kingdom, Canada, Israel, and Germany. ## Association between inhalant use and sleep quality In the setting of the global pandemic (June 2020), past or present inhalant users overall had higher PSQI scores, indicating worse sleep quality (6.221), relative to never smoker/never vapers (5.248, p = 0.012;. These data may suggest that any lifetime use of a nicotine containing inhalant (conventional tobacco, e-cigarettes, or both) is adversely associated with sleep quality, and that this association may be magnified in the presence of a continued stressor (in this study, the identified stressor is uncertainty and anxiety associated with an unresolved global pandemic). Even when controlling for age, gender and presence of any lung disease, inhalant use significantly was associated with a higher PSQI score. Using multivariate regression, inhalant use, age and lung disease were all independent predictors of the PSQI score. Advancing age predicted lower PSQI scores, with the parameter estimate for age being −0.030 per year (CI −0.008 to −0.053; p = 0.0087). PSQI score was also higher in people with lung disease, with the parameter estimate being 1.040 if present (CI 0.029 to 2.051; p = 0.0439). Finally, inhalant use predicted higher PSQI score, with a parameter estimate of 0.810 (CI 0.087 to 1.532; p = 0.0283). ## Association between inhalant use and sleep quality In the setting of the global pandemic (June 2020), past or present inhalant users overall had higher PSQI scores, indicating worse sleep quality (6.221), relative to never smoker/never vapers (5.248, p = 0.012;. These data may suggest that any lifetime use of a nicotine containing inhalant (conventional tobacco, e-cigarettes, or both) is adversely associated with sleep quality, and that this association may be magnified in the presence of a continued stressor (in this study, the identified stressor is uncertainty and anxiety associated with an unresolved global pandemic). Even when controlling for age, gender and presence of any lung disease, inhalant use significantly was associated with a higher PSQI score. Using multivariate regression, inhalant use, age and lung disease were all independent predictors of the PSQI score. Advancing age predicted lower PSQI scores, with the parameter estimate for age being −0.030 per year (CI −0.008 to −0.053; p = 0.0087). PSQI score was also higher in people with lung disease, with the parameter estimate being 1.040 if present (CI 0.029 to 2.051; p = 0.0439). Finally, inhalant use predicted higher PSQI score, with a parameter estimate of 0.810 (CI 0.087 to 1.532; p = 0.0283). ## Change in sleep quality during the pandemic When survey participants retook our survey two months later in June 2020, sleep quality for the group as a whole improved, as shown by lower PSQI scores (5.595), relative to the original survey in April 2020 (7.990; mean of differences (retake-original) = −2.395, SD ± 3.256, p < 0.0001;. This finding suggests that participants had improvement in sleep quality as the COVID pandemic progressed. However, when broken down by inhalant type, never smoker/never vapers were the only group that had a significant improvement in sleep quality (p < 0.0001;and. This suggests that noninhalant users were better able to adapt during the pandemic leading to improved sleep quality while nicotine inhalant users were less able to adapt and did not have improvements in sleep quality. ## Change in sleep quality during the pandemic When survey participants retook our survey two months later in June 2020, sleep quality for the group as a whole improved, as shown by lower PSQI scores (5.595), relative to the original survey in April 2020 (7.990; mean of differences (retake-original) = −2.395, SD ± 3.256, p < 0.0001;. This finding suggests that participants had improvement in sleep quality as the COVID pandemic progressed. However, when broken down by inhalant type, never smoker/never vapers were the only group that had a significant improvement in sleep quality (p < 0.0001;and. This suggests that noninhalant users were better able to adapt during the pandemic leading to improved sleep quality while nicotine inhalant users were less able to adapt and did not have improvements in sleep quality. To assess what factors might be contributing to sleep quality during the stress of the pandemic, six questions were included in the retake survey in June to assess for the subjects' mindsets related to COVID. The average response across inhalant groups on a scale of 1-10, with 10 representing survey respondents being extremely concerned about To assess what factors might be contributing to sleep quality during the stress of the pandemic, six questions were included in the retake survey in June to assess for the subjects' mindsets related to COVID. The average response across inhalant groups on a scale of 1-10, with 10 representing survey respondents being extremely concerned about COVID-19, was 6.28. The average response across inhalant groups with 10 representing survey respondents having their lives extremely disrupted by COVID-19, was 7.24. The largest portion of e-cigarette users felt that their work-related stress increased a little where the largest portion of dual users felt that their work stress increased a lot. Eighty-four percent of survey participants reported not having contracted COVID-19 at the time of the survey. Twenty-two percent of participants reported that a friend, family member, or loved one had contracted COVID-19 and tested positive. The pandemic caused 61.8% of our participants "a little" anxiety and 31.3% "a lot" of anxiety. # Discussion To our knowledge, these data are the first to find an adverse association of lifetime e-cigarette use on overall sleep quality. While we have run larger survey-based studies and have found adverse effects of dual use of e-cigarettes and conventional tobacco on certain aspects of sleep, the findings presented here suggest that inhalant users may be susceptible to overall diminished sleep quality in the setting of continued stressful life circumstances. In addition, our finding that non-inhalant using subjects had improvements in sleep quality as the pandemic went on, while none of the inhalant groups had similar improvements, lends support to the idea that nicotine use via any inhalant device may lead to poor sleep quality and a lack of adaptability in the time of stress. With the vast majority of the population either working from home during the pandemic or not working at all, we postulated that the improvement in PSQI scores in never smoker/never vapers from April to June 2020 may have been driven by lifestyle changes. Contributing factors to the improved PSQI scores could include extended sleep duration and decreased variability of bedtimes. The specific etiologies of the changes in sleep duration are an area of research that will require further investigation but could include decreased commute time, as well as decreased time spent in preparation for working outside of the home (i.e., grooming time, meal preparation). This study has several limitations. The sample size was small, with small numbers of ecigarette and dual users in particular. Larger studies are needed to confirm these findings as well as studying broader populations. Our cohort had significant age differences between the e-cigarette group and the other inhalant groups; however, multivariate analysis was able to define the impact of age as well as inhalant use on PSQI scores. As this survey-based study relied on participants' self-reporting, there may have been inaccuracy in responses. However, we anticipate such misclassification to be random and thus should bias towards the null hypothesis. A baseline, pre-pandemic PSQI score was not collected. Nonetheless, this study having multiple time points of data collection during the pandemic opens the door for eventual comparison to post-pandemic PSQI scores. Another limitation of this study is that it is only able to infer correlation between inhalant use and sleep quality, not causation. As such, we are only able to discuss the associations between inhalant use and sleep quality as the pandemic progressed, not impact or effects. The study assessed the relationship between the COVID pandemic and the participants lifestyle with a series of six questions that were added to the retake survey. Ideally, the study would have included more questions to evaluate the degree of limitations and emotional responses to the stressor of the global pandemic in more depth. However, in the interest of being able to recruit and retain participants, the number of questions within our survey were minimized. In addition, because of the multiple locations of study participants throughout the US and worldwide, it was not possible to define characteristics of the pandemic, including severity and evolution, at each location. Finally, as with any study that uses social media as the recruitment method, there is potential for sampling and participation bias. # Conclusions In this study, we found differences in sleep quality associated with history of inhalant use. As global events including the COVID-19 pandemic continue to occur, it is important for the general public to be informed about the possible adverse associations that nicotinebased inhalants could be having on sleep quality. As poor sleep quality is known to be detrimental to general health and wellbeing, we view measures to improve sleep and immune function as important strategies for disease prevention. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data will be made freely available to any interested parties.

Progress in the photodynamic therapy treatment of Leishmaniasis Leishmaniasis is a serious and endemic infectious disease that has been reported in more than 90 countries and territories. The classical treatment presents a series of problems ranging from difficulty in administration, development of resistance, and a series of side effects. Photodynamic therapy (PDT) has already shown great potential for use as a treatment for leishmaniasis that is effective and non-invasive, with very minor side effects. PDT can also be inexpensive and easy to administer. In this review, we will report the most recent developments in the field, starting with the chemical diversity of photosensitizers, highlighting important mechanistic aspects, and noting information that may assist in designing and developing new and promising photosensitizer molecules. # Introduction Leishmaniasis is a group of neglected diseases caused by parasites of the genus Leishmania. More than 20 species are found worldwide, while the main species. Transmission occurs through the bites of female sandflies, and the reservoirs of the parasites are humans, wild rodents, marsupials, and domestic dogs. There are three main forms of the disease: cutaneous, visceral or kala-azar, and mucocutaneous. It is estimated that one billion people live in endemic areas, and more than 80 economically vulnerable countries are affected by visceral leishmaniasis (VL). With one million new cases of cutaneous leishmaniasis (CL) annually, the most affected countries are Brazil, India, Ethiopia, Kenya, South Sudan, Somalia, and Sudan. The main symptoms of CL include single or multiple ulcerous or nodular lesions on the skin, which can become chronic or heal spontaneously six months after the sandfly bite. The condition is not fatal but it can be disabling and leave permanent scars. In VL or kala-azar, a systemic parasite infection occurs, with symptoms such as enlarged organs, especially the liver and spleen, as well as prolonged fever, weight loss, and anemia. VL can also affect the lymph nodes and bone marrow, leading to death when left untreated. A major problem with this form is the risk of co-infection, for example, with HIV. Such co-infections have already been reported in 35 endemic countries worldwide. Despite the toxicity, high cost, and difficult administration, treatment is still carried out with pentavalent antimonials, amphotericin B, pentamidines, and miltefosine. It is therefore necessary to find effective, safe, lowcost, and short-term treatment, of which photodynamic therapy (PDT) is a promising candidate. PDT is a technique used in the treatment of several diseases. It uses electronic excitation of a photosensitive compound (photosensitizer) to produce a variety of reactive oxidant species (ROS), including excited states, free radicals, and strong oxidants. It is one of the most promising strategies for local anti-microbial therapy and killing drug-resistant microorganisms. Compared with conventional therapies, PDT is a low-cost, minimally invasive technique with minor side effects. Since CL is a local infection, PDT has been studied by several researchers and clinical groups as a treatment alternative. PDT has three essential componentsthe photosensitizer, the light, and the molecular oxygen. The photosensitization processstarts with the absorption of light by the photosensitizer, raising it to the singlet excited state (S1), which can return to the ground state by losing heat or emitting light (fluorescence). S1 state may suffer changes in the electron spin (intersystem crossing), forming an excited triplet state (T1) with a longer lifetime. T1 has time to react and diffuse, either by transferring energy to molecular oxygen and forming singlet oxygen (Type II reaction) or by transferring electrons (or hydrogen) to a substrate (Type I reaction). The electron transfer reaction forms radicals and produces ROS after a subsequent reaction with oxygen, such as the superoxide radical anion, hydrogen peroxide, and the hydroxyl radical. Photosensitized oxidations, which are reactions triggered by the interaction of light with photosensitizer (PS) molecules, act by inducing damage in cytoplasmic or organelle membranes. These are key to modulating the mechanism and overall efficiency of regulated cell death. Type I reactions consist of the direct oxidation of biological targets (direct-contact reactions), while those of type II involve oxidation mediated by diffusing species (independent-contact reactions), mainly the singlet oxygen. In direct-contact reactions, the damage occurs at the exact point where the excited species are generated. In contrast, singlet oxygen or other diffusing species can carry oxidation potentials hundreds of nanometers or micrometers away from the point of light absorption. Nevertheless, the detailed molecular steps leading to biological injury remain largely uncharacterized, and the level of precision in the spatial damage caused by photosensitized oxidation reactions remains unclear. For a PS to fully compromise membrane function, it needs to engage in electron transfer reactions with either the lipid double bond or the lipid hydroperoxide. This process forms peroxyl and alkoxyl radicals within the membranes that undergo Beta-scission and generate lipid-truncated aldehydes, which cause membrane leakage. Therefore, cellular damage occurs precisely at the PS locus. This highlights the importance of finding molecule-specific oxidation-induced photodamage. Since the efficiency of membrane leakage results from an electron transfer reaction that usually causes photobleaching, PS regeneration should be exploited as an effective tool to develop improved PDT photosensitizers. Many series of PSs that are candidates for PDT are being tested as potential treatments for CL. Understanding how each class works will facilitate the design and development of the PS and improve PDT results. A growing body of literature has examined the use of various PSs in treating leishmaniasis, other infectious diseases, and cancer. In this review, we aim to describe and discuss the main achievements and challenges of using PDT to treat CL by examining recent results and a contemporary view of the mechanisms of the major series of photosensitizers, including phenothiazinium salts, delta-aminolevulinic acid (ALA) and ALA derivatives, phthalocyanine, porphyrin, and phenothiazinium. ## Ala-pdt Leishmania species are known to use tetrapyrroles to promote the growth of promastigotes and to transform amastigotes into promastigote forms. Tetrapyrroles are not acquired from heme biosynthesis since Leishmania spp. entirely lack or are deficient in seven of the eight enzymes in the heme biosynthetic pathway. A study involving Leishmania (L.) amazonensis showed that the parasites become sensitive to UV irradiation due to the presence of uroporphyrinogen I (URO), which is a byproduct of hydroxymethylbilane (heme group synthesis) inside the parasitic cells that causes a loss of motility in Leishmania parasites. These findings showed the potential of porphyrins as photosensitizers in Leishmania PDT. The first step in heme biosynthesis is the formation of ALA, which is caused by the condensation of glycine and succinyl-CoA in a process catalyzed by the enzyme ALA synthase (ALAS). The latter is a mitochondrial enzyme that undergoes negative feedback from heme as it can induce or inhibit the enzyme once present, interrupting the production of ALA. Although ALA is not a photosensitizer, it is the first precursor in the biosynthesis of the heme group and the universal precursor of tetrapyrroles like porphyrins. It became the target of PDT studies after it was discovered to be converted into protoporphyrin IX (PpIX) in heme synthesis, accumulating in some cells and acting as a photosensitizer. The synthesis of PpIX is determined by the amount of ALA produced, which is regulated in turn by the concentration of the free heme group. However, this feedback mechanism can be circumvented by the administration of exogenous ALA that induces the production and accumulation of PpIX or by certain mutations in the ALAS, which also cause PpIX accumulation. Akilov et al.performed a well-documented study on the action of PDT using ALA (termed ALA-PDT) in CL and a cellular assay. In the in vivo studies, the authors observed the formation of inflammation and necrosis, indicating damage to vascularized areas. Furthermore, ALA-PDT was found to decrease the parasitic burden 24-fold in the ear lesion. However, in the in vitro studies, the results showed that the amount of PpIX that L. (L.) major was able to obtain from the host cell was not sufficient to produce a photodynamic action (500-fold less than the amount needed to kill metacyclic forms). The results suggest that the success of the in vivo ALA-PDT is due to indirect action such as immune modulation since the PDT was not able to kill parasites. As mentioned above, Leishmania spp. needs a supplementary source of tetrapyrroles, meaning that producing PpIX from ALA may not be possible. In agreement with the results previously found by Akilov et al., another study was performed using ALA and the derivative MAL (methyl-5-aminolevulinate). Although these exhibited an internalization of the PS, no photodamage was observed in the promastigote forms, confirming the inability of Leishmania spp. in converting ALA to PpIX. However, exogenous PpIX was able to provoke phototoxicity in parasites. In contrast to results found for the promastigotes, infected cells produced PpIX but not in sufficient amounts to promote damage on intracellular amastigotes (35), consistent with previous findings. Despite MAL having an extra alkyl chain in the structure, no difference was found in the internalization or photoactivity between the two compounds. As previously seen, ALA-PDT may act through an immune modulation mechanism when applied directly to the lesion (34). This hypothesis was supported by some results that demonstrated a decrease in the parasitic load following an increase in interferon-gamma (INF-g) levels after ALA-PDT of infected mouse paws. The increase in INF-g levels is described as causing resistance to infection by L. (L.) major due to the development of protective immunity (37). Silva et al.studied macrophage modulation in mice infected by L. (V.) braziliensis, the action of ALA-PDT in the lesions, and whether ALA-PDT could alter heme oxygenase 1 (Hmox-1), an enzyme responsible for the persistence of infection and inflammatory response activation. As seen in the previous study, the parasite burden decreased significantly compared to the untreated group, and Hmox-1 levels were not significantly altered, indicating no association of the enzyme with the persistence of the infection or any decrease in the parasite load. However, a notable increase in iNOS (nitric oxide synthase) and iNOS/arginine ratio was observed. This could be associated with the ability of ALA-PDT to kill parasites since iNOS catalyzes the production of NO (nitric oxide), which is a leishmanicidal agent. Together with the earlier study, these findings show an association between ALA-PDT and the activation of macrophages that release leishmanicidal mediators. Taken together, these effects increase parasite mortality and decrease the rate of parasitism. ALA is not a PS and cannot inhibit the parasite itself since it is a prodrug, and the parasite is incapable of converting ALA to the photoactive product, porphyrin. However, it is an excellent option for use with PDT directly on the lesion as it causes an alteration in the immune response and decreases parasite load. Although ALA does not kill the parasite, the structure can be used to develop a new PS, exploiting the capacity of ALA to be converted into photosensitive porphyrins. Porphyrin can be used as a PS in PDT against Leishmania spp. successfully. ## Porphyrins Porphyrins have been described as an excellent choice for PSs since their effectiveness against Leishmania has already been demonstrated during PDT studies. One PDT study used exogenous PpIX as a positive control and tested against L. (L.) infantum and L. (V.) panamensis. Although PpIX was only used as a control, it showed promising results against the promastigote forms of L. (L.) infantum and L. (V.) panamensis. Before this, Bristow et al.studied four types of cationic porphyrin PSs as potential photodynamic anti-Leishmania agents. The cationic PSs were chosen due to the negative character of the membrane of Leishmania spp. and were tested against the promastigote forms of Leishmania (L.) major. Macrophages and keratinocytes were used to simulate amastigote intracellular forms of Leishmania and the healthy tissue around the skin lesion, respectively. PSs were tested and showed very different results, which was thought to be due to the differences in the membrane charge of the three cell lines studied. As noted, the cell membrane of the Leishmania spp. has an anionic character. Compounds 1 (phosphorous-centered cationic porphyrin) and 3 (nitrogen-centered cationic porphyrin) were photoactivated against the promastigote forms at a low concentration. However, compound 1 showed no activity while compound 3 inhibited macrophages with a similar LD 50 for L. (L.) major promastigotes. Meanwhile, compound 1 was active against keratinocytes at a concentration equivalent to almost half that needed to kill promastigotes, but compound 3 reached inhibition at a concentration 10 times higher than the one established for promastigotes. Since compound 3 had the best results, a dose adjustment was considered so that it could be used to kill promastigote and amastigote forms without causing damage in uninfected tissue. In 2018, Andrade et al.studied the effect of zinc porphyrin (ZnP), a cationic porphyrin, to verify the effect of the charge and the zinc on membranes. ZnP was active in both tested concentrations of about 65 and 90%. Furthermore, an analysis of the parasitic cells after 24 h of PDT showed that cells were incapable of replicating. These results were successful because the PS concentration was low, the incubation time was only 10 min, and the irradiation time was short. This treatment exhibited high permeability in the parasitic membrane. The microscopic alterations included a shortening and rounding of the parasitic cells, shrinkage of the plasmatic membrane, and vacuolization. ZnP was less cytotoxic than compounds 1 and 3 described by Bristow et al.Approximately 70% of human cells remained viable after ZnP PDT, while about 50% of cells showed viability after a cationic porphyrin assay. Both studieswere important demonstrations of the effectiveness of porphyrins in PDT against Leishmania. It was possible to observe that cationic porphyrin is a promising PS for use against Leishmania spp. However, the former studymade it clear that the positive charge is not the only factor to be considered. Meanwhile, the presence of the metal in the second studyindicates that this strategy may be relevant in structure activity studies. Carbaporphyrin ketals are porphyrin derivatives, called porphyrinoids, in which the pyrrole ring of porphyrin is replaced by a ketal-substituted indene ring. This class of compounds was tested in vitro and in vivo against Leishmania (L.) amazonensis, L. (L.) infantum, and L. (V.) panamensis. Such a study allows verification of whether the alteration in the activity is caused by the changes in compound structure. The carbaporphyrin dimethyl ketal (CKOMe) and carbaporphyrin diethyl ketal (CKOEt) had high activity levels against axenic and intracellular amastigotes of studied Leishmania. When used in a liposomal formulation, PDT had a stronger effect against L. (L.) amazonensis for both compounds. However, similarly to the case of cationic porphyrins, carbaporphyrins were toxic to human cells, although the toxicity of CKOMe to PMH cells (peritoneal macrophages obtained from hamsters) decreased when administered in a liposomal formulation. Even though CKOEt is more hydrophobic than CKOMe, the latter showed better results, even when solubilized in DMSO or a liposome, showing that something other than hydrophobicity is important. The size of the molecule may be important here, as CKOEt has two ethyl chains that could cause a steric effect that would affect activity. The study also shows that altering the porphyrin backbone is useful for increasing activity against Leishmania species. ## Phthalocyanine Phthalocyanine (Pc) is a synthetic dye that consists of four isoindole rings connected by four nitrogen atoms. Pc is an alternative to porphyrins since PSs do not absorb in the 400-600 nm range, having no phototoxicity to the skin. Unlike porphyrins, Pc has an absorption and fluorescence wavelengths in the range of 650-800 nm and high production of singlet oxygen. However, like porphyrins, Pc can form metal complexes that further increase the ability to produce singlet oxygen. When exposed to aluminum phthalocyanine chloride (AlPhCl) and light, promastigote forms of L. (L.) amazonensis were rapidly killed, while axenic amastigotes underwent structural alterations. Both groups exhibited loss of fluorescence, indicating cell lysis. It is important to note that neither the light nor AlPhCl was toxic when administered alone, and both the dose of light and the Pc concentration used were low. In addition, AlPhCl reduced the number of macrophages and amastigote forms in an infected culture. Escobar and colleagues (47) analyzed the photoactivity of aluminum chloride and zinc phthalocyanines (AlPc and ZnPc) against promastigote forms of L. (L.) chagasi and L. (V.) panamensis. AlPc had a dose-response activity and was more phototoxic to L. (L.) chagasi than L. (V.) panamensis (30-to 50-fold). AlPc also caused greater photosensitization than ZnPc in both parasitic species, and none of the PSs presented phototoxicity in the dark. The differences found between the two PSs could be related to the fact that ZnPc is very hydrophobic, which could make it difficult to enter the cell, unlike the amphiphilic AlPc. To facilitate entry into infected cells, decrease the formation of dimers, improve photoactivity, and reduce phototoxic effects, Hernández et al.reported the activity of ultradeformable liposomes containing chloroaluminum phthalocyanine (UDL-ClAlPc) and free ClAlPc against L. (L.) chagasi and L. (V.) panamensis promastigotes and amastigotes. UDL-ClAlPc was more phototoxic than free ClAlPc in both species and parasitic forms. As previously reported, ClAlPc showed no selectivity for the parasitic intracellular form compared to host cells. This result confirmed that the death of the parasite could occur due to a secondary mechanism. The use of genetically modified Leishmania to produce and accumulate URO has been successfully described. Therefore, Dutta and colleagues decided to use this technique along with AlPhCl to increase parasitic photodynamic efficiency. First of all, URO and AlPhCl alone showed a very different localization into cellular structures. As expected and described, the more hydrophobic AlPhCl showed a greater accumulation in structures such as the cell membrane, while the hydrophilic URO was more widespread in the cytoplasm. Promastigotes photosensitized with URO or AlPhCl alone showed a reduction in viability. Also, a total loss of cell viability was only possible after photosensitizing the cells with both PSs (URO and AlPhCl). This loss of viability was maintained after five days of culture, showing that the synergism technique may have photoinhibition applications. Notably, infected macrophages were not affected by this treatment, although all the parasitic intracellular burden was eliminated. The fact that the photosensitizers were used together and the host cells were not affected is an important outcome, as previous studiesshowed that PDT might have killed the parasites by a secondary route and not by directly acting on the parasite itself. evaluated the effectiveness of the PDT technique when associating the topical use of AlClPC (liposomes containing chloroaluminium phthalocyanine) with the drug miltefosine, already used to treat CL. After 20 days of treatment with miltefosine and PDT with AlClPC, they noted a reduction in the diameter of the infected paw accompanied by a considerable decrease in parasitic culture. A nanoemulsion containing ZnPc was tested to optimize PDT studies in the treatment of CL., free PS was toxic to macrophages with or without irradiation. Like free PS, the nanoemulsion also showed cytotoxicity but with a certain selectivity for parasites compared to host cells. A reduction in the parasitic burden of macrophages was also found (55). Escobar et al.built on these studies by analyzing the topical use of UDL-ClAlPc in BALB/c mice infected with L. (V.) braziliensis and in vitro activity in L. (V.) braziliensis promastigotes and amastigotes and mammalian cells. In the in vitro assay, the UDL-ClAlPc internalized both infected and uninfected cells, and was active in both parasitic and host cells after PDT, with no selectivity. UDL-ClAlPc also induced ROS generation in infected macrophages after PDT. Since no activity was found after treatment with UDL-ClAlPc and only low levels of ROS were produced without PDT, the authors suggested that ROS production after PDT may have been responsible for killing the parasites. Damage to the DNA was found with or without PDT, even in the empty UDL, but the mechanism behind the damage was not studied. The study did not find any effect in the BALB/c infected mice treated with topical UDL-ClAlPc and PDT, which was explained as possibly due to photobleaching or low penetration in the skin. These results contrast with those found by Ribeiro et al., who also used the PS within liposomes. The latter studycombined topical use of the liposome with miltefosine, which may have been responsible for reducing the lesions present in the animals. Another difference is in the type of formulation: the first study (56) used a lipid film while the second one (51) created a gel formulation. These differences may explain the effectiveness of the liposome on mouse lesions. According to the excellent results obtained previously in a study of the association of miltefosine with PDT using AlClPC as PS (51), Ribeiro et al.decided to evaluate the effectiveness of the association between AlClPC and PDT with the drug N-methyl glucamine (NMG) in mice infected with L. (L.) amazonensis. The standard recommendation of NMG for the CL treatment is 20 mg Sb V. kg -1. day -1 (58), so the researchers decided to test 20 mg Sb V /kg/day + PDT + AlPlPC (NMG20 + PDT) and 10 mg Sb V. kg -1. day -1 + PDT + AlPlPC (NMG10 + PDT). The latter concentration was used to verify whether the PDT can decrease the NMG dosage, which may diminish adverse effects. The treatment with NMG20 + PDT decreased the diameter of the animal's paw in 60 days after the end of treatment, a decrease similar to the negative control. It also showed negative results for amastigotes and parasitic cultures after 20 days of treatment and 60 days after the end of treatment. Cell viability was reduced after 10 and 20 days of treatment, even for NMG10 + PDT, although only NMG20 + PDT could maintain this reduction at 60 days after the end of the treatment. The results of NMG10 + PDT were similar to those of NMG20 alone (standard treatment), suggesting that it could be used in the future to minimize the adverse effects caused by the drug alone. It is important to note that this decrease is related to PDT + AlPlPC, indicating that AlClPC may be of interest as a PS. Phthalocyanines have proven to be an excellent PS for use in PDT against different species of Leishmania, although more in-depth studies should be carried out on the selectivity and toxicity in the host cells. However, studies involving liposomal formulations and the association with existing drugs for the disease have shown satisfactory results both in terms of reducing parasitic load and skin lesions, as well as reducing the usual concentration and thus achieving a possible improvement in adverse effects. ## Phenothiazinium salts Phenothiazine derivatives have been extensively studied, showing promising results against bacterial activity. Changes in structure, including the addition of methyl, nitro, and primary amine groups, the positioning of these groups, and the hydrophobicity of the molecule can improve the effects. Studies on the differences in chemical structure have shown that spatial constraints and the geometry of the phenothiazine derivatives are also important, for example, in aggregation. In addition to antibacterial activity, phenothiazine derivatives have also been studied for the treatment with PDT of other diseases such as Kaposi's sarcoma, herpes, and diabetic foot. Methylene blue (MB) is the most studied photosensitizer of the phenothiazine class against Leishmania species. MB is an interesting photosensitizer due to its high singlet oxygen quantum yield of around 0.5and its absorption band between 550 and 700 nm. MB is capable of forming dimers, although this characteristic depends on the concentration, the ionic strength, and the presence or not of charged interfaces. The absorption spectrum of monomers and dimers is different, with maximum absorption at 665 and 580 nm, respectively. performed in vitro and in vivo studies to assess the effectiveness of PDT and MB as a PS for treatment of CL. Although MB was effective without irradiation, the half inhibitory concentration (IC 50 ) value in the parasitic cells was reduced after irradiation in a dose-dependent manner. After the in vitro assay, one patient with three lesions caused by Leishmania (L.) amazonensis underwent treatment with a low concentration of pentavalent antimonial and PDT + MB, with one lesion treated with Sb V alone. The results showed that although the lesions were reduced and cured with Sb V treatment alone, PDT accelerated this process. Peloi et al.performed an in vivo PDT study using 10 nM of MB in a lotion or aqueous formulation in hamsters infected with L. (L.) amazonensis. MB alone could not decrease the animal's footpad size, while animals treated with MB and light showed a significant reduction in footpad size, with no differences between the formulations applied. Some 40% of the infected animals had ulcerated lesions, and after treatment with MB in lotion and water plus light, 40 and 50% of the lesions were cured, respectively, after 12 weeks of treatment. The analysis of the parasitic load in the spleen of infected and treated hamsters found no presence of the parasite regardless of the formulation used. However, parasites were found in lymph nodes of treated hamsters in a much lower percentage compared to untreated hamsters. To verify how PDT and MB can interfere in the interaction between macrophages and L. (V.) braziliensis, in vitro studies were performed. The parasitic load was decreased both in the group that received only MB and in the group that received MB + light, within the first 24 h of infection. After 48 h of infection, there was a significant 38% reduction in the group that received MB + light compared to the other groups. A 33% reduction in infectivity was observed within 24 h of treatment with MB and 58% when using MB + light. The infection rate of parasite macrophages was 71% lower in the group that received MB + light than the control. Also, when compared to the group that received only MB, the infection rate was 48% lower in the group that received MB + light after 24 h of treatment. The studies by Song et al.and de Oliveira et al.showed that, although MB has activity in parasitic cells, the use of PDT together with MB enhances its action, in addition to allowing the decrease in PS concentration. Furthermore, Song et al.and Peloi et al.found that the use of PDT and MB healed the wounds caused by L. (L.) amazonensis. Therefore, the use of PDT associated with the phenothiazine compound is effective in both in vitro and in vivo assays, helping to decrease the parasitic rate and infection rate, as well as reducing the concentration of drugs already used in current therapy. Pinto et al.carried out a study in which they evaluated the internalization and cell location of the MB along with cell viability and morphology after the application of PDT in the species L. (L.) major and L. (V.) braziliensis. The internalization results showed that PS does not accumulate in organelles but rather remains in the cytosol of the parasitic cell. The most intriguing results were demonstrated in the tests of cell viability and mitochondrial activity. Although mitochondrial activity was altered in the control groups, the trypan blue viability assay showed that MB is not toxic in any species without light exposure. Furthermore, after the application of PDT, viability was significantly reduced, showing that the variation in mitochondrial activity does not necessarily mean a change in cell viability. These results are in contrast to those reported by Song et al., who verified MB activity even in the dark after a mitochondria activity test using MTT. In their study, this mitochondrial activity did not always interfere with the cell viability itself since parasites were shown to be viable even with changes in the activity of the mitochondria. In addition to the above, PDT using MB as PS caused morphological changes in the promastigote forms of both species, suggesting interaction with the parasitic cell membrane. Sbeghen et al.conducted a study comparing the action of PDT + MB administered intradermally and topically in lesions caused by L. (V.) braziliensis on hamster footpads. The authors observed that the MB applied intradermally did not cure or decrease the lesion. However, topical treatment reduced and healed lesions in 30% of the animals after nine weeks. Similar to the findings of Peloi et al., the parasitic burden observed in the lymph nodes and spleen was low for animals treated with topical MB. Also, the treatment restored the lesion area and decreased inflammation. MB proved to be an interesting photosensitizer for the treatment of Leishmania along with the PDT technique. We found that the studies showed good results both in vitro and in vivo, leading to a decrease of parasite load, reduction of lesions in both animals and humans, as well as action on several Leishmania species. ## Concluding remarks PDT has proven to be a useful technique for the treatment of CL since it has a low cost, is non-invasive, and has low toxicity compared to conventional therapies. Although some adjustments are necessary, the PSs studied so far have shown promising results. In this review, a range of PSs and several methodologies were explored. These studies are important to assist in the search for increasingly efficient PSs against parasitic forms. We can highlight some of the aspects observed to be important in the development of a new PS, including the lipophilicity and amphiphilicity of compounds, the charges, the electrostatic interaction, and the presence or absence of metal . All of these factors interfered in some way during the studies. As seen, porphyrins are active against parasitic forms, and a prodrug such as ALA can be used. Although it is not active on its own, ALA can be transformed into porphyrin. Furthermore, cationic porphyrins interacted better with parasitic cells since the parasite has a negative charge on its membrane surface, improving the electrostatic interaction of cationic compounds. The presence of metal in the structure is another point to be considered, since an improvement in the activity of PSs has been observed. In addition, care should be taken when examining hydrophobicity, as highly hydrophobic molecules may not interact optimally. Like the amphiphilic molecules, they present better activity compared to hydrophobic molecules. Finally, the use of techniques such as adding PSs to liposomes seems interesting. As we have seen in some studies, this strategy improves the efficiency of PSs and decreases toxicity. Therefore, the study of PSs, especially in planning and development studies, should pay special attention to the efficiency in producing singlet oxygen while also adopting a molecular perspective, observing all the structural aspects important for the interaction between the PS and the target.

The Use of Penile Traction Devices for Peyronie's Disease: Position Statements from the European Society for Sexual Medicine Introduction: Penile traction therapy (PTT) aims to non-surgically reduce curvature, enhance girth, and recover lost length. Available clinical practice guidelines however lack clear recommendations regarding their use. Aim: To present a comprehensive review and recommendation regarding the available evidence to the use of PTT in Peyronie's disease (PD). Methods: A systematic literature search was performed on Pubmed and Medline for relevant studies from all times until 2019. Studies of PTT (monotherapy and in combination) in patients with PD with any documented degree of curvature and in either the acute or chronic phase of the disease were included. Full texts not published in English language were excluded. Main outcomes measures: Several scenarios, including preclinical data have been investigated. For each topic covered evidence was analyzed and expert opinion was stated. Results: The paucity of high-level studies precluded any strong recommendations, however, specific statements on this topic, summarizing the ESSM position, were provided. The available data about the use of PTT in PD are still poor, and the impact of this therapy for the treatment of PD has not been clearly stablished. Available data in the clinical setting are still poor, and the impact of these devices on PD evolution has not been clearly established. Conclusion: PTT seems to be a valid treatment option for PD, although there is not enough evidence to give any definitive recommendation in any clinical scenario. García-G omez B, Aversa A, Alonso-Isa M et al. # Introduction Penile traction therapy (PTT) represents an emergent therapeutic option for men with Peyronie's disease (PD). [bib_ref] Traction therapy for men with shortened penis prior to penile prosthesis implantation:..., Levine [/bib_ref] The accessibility for clinicians and patients in terms of acquisition and ease of use has increased their popularity in recent years. There are several penile traction devices easily available in the market with similar design, although most of them are not supported by any scientific background. The effect of very few has been specifically studied in the literature, as for instance FastSize Medical Extender (FastSize, Aliso Viejo, CA, USA) 2 , the Peni-Master Pro (MSC Concept, Berlin, Germany) [bib_ref] Penile traction therapy with the new device "Penimaster PRO" is effective and..., Moncada [/bib_ref] , or the RestoreX (PathRight Medical Inc, Plymouth, USA). [bib_ref] Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease:..., Ziegelmann [/bib_ref] The common stated clinical goals of all PTT are to non-surgically reduce curvature, enhance girth, and recover lost length, all of which are recognized concerns in patients suffering from PD. [bib_ref] Peyronie's disease and mechanotransduction: an in vitro analysis of the cellular changes..., Chung [/bib_ref] The American Urological Association Guideline on PD does not provide any recommendation related to PTT, acknowledging that the study samples were too small.The Evidence-Based Management Guidelines on PD, supported by the International Consultation on Sexual Medicine, however, stated that PTT may have some benefits in PD. [bib_ref] Evidence-Based Management Guidelines on Peyronie's Disease, Chung [/bib_ref] Accordingly, the Guidelines on Sexual and Reproductive Health by the European Association of Urology state that PTT seems to be effective and safe for patients with PD, but there is still lack of evidence to give any definitive recommendation in terms of monotherapy for PD.A recent review by Avant et al [bib_ref] Penile Traction Therapy and Vacuum Erection Devices in Peyronie's Disease, Avant [/bib_ref] concluded that PTT has a potential role as a primary lengthening therapy (modest improvements); in curvature correction prior to penile prosthesis insertion; and after surgical correction of PD as part of post-operative rehabilitation. Whereas pre-operative and postoperative PTT can result in length preservation after surgery for PD [bib_ref] Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease:..., Ziegelmann [/bib_ref] [bib_ref] A retrospective comparative study of traction therapy vs. no traction following tunica..., Rybak [/bib_ref] , the role of PTT in combination therapy with Clostridium Histolyticum (CCH) injections is still unclear. [bib_ref] Clinical Experience With Penile Traction Therapy Among Men Undergoing Collagenase Clostridium histolyticum..., Ziegelmann [/bib_ref] The aim of the present paper is to provide a detailed position statement of the European Society for Sexual Medicine (ESSM) on this topic, summarizing and emphasizing the current available evidence, any possible conflicting issues and the need of further clarifications. # Methods A literature search of full text English language articles on the use of PTT in PD was performed using PubMed and Medline on the 09/01/2019 with the following MeSH terms ((((Peyronie's disease or penile induration) and (traction)) "English" [Language]) AND ("All times" [date-publication]: "August 2019" [date-publication]). Inclusion criteria included original papers investigating the outcomes of traction therapy on PD. Exclusion criteria included posters or oral presentations, review articles, meta-analyses, expert opinions, and case reports. The scientific committee of the ESSM selected the authors, based on their long-standing clinical experience and scientific involvement in the specific area. Data acquisition was performed by two independent reviewers, and their results collated and reviewed by a third. Possible conflicts were resolved after a discussion among the authors. # Results We identified 53 abstracts that met our search criteria, of which 1 was a clinical report, 1 referred to an ocular syndrome, 1 was a guideline (European Association of Urology), 4 were letters in response to an original article, and 30 were reviews or expert opinions. In total 16 papers were included in the review [fig_ref] Figure 1: Flow chart diagram of the data acquisition [/fig_ref] summarizes the data acquisition. Due to limited evidence on clinical outcomes and the poor scientific quality of the identified studies, no recommendations based on the Oxford 2011 Levels of Evidence criteria were possible. However, specific statements on this topic are provided, which summarize the ESSM position, following a balanced ## Evidence Limited evidence has suggested that the use of mechanical traction, also known as mechanotransduction, results in the alteration of connective tissue by cellular proliferation and expansion of the extracellular matrix. 5, [bib_ref] Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins, Alenghat [/bib_ref] Chung et al 5 investigated the cellular changes of PD plaques following traction forces. In vitro analysis of PD cells cultured in a mechanical strained environment showed an alteration in collagen and tissue metalloproteinase (MMP) expression, highlighting MMP as one possible underlying mechanisms of action of PTT in PD remodeling. [bib_ref] FastSize Medical Extender for the treatment of Peyronie's disease, Levine [/bib_ref] Lin et al 14 investigated the influence of PTT or a vacuum erectile device (VED) in a rat model of PD. PD plaques were induced by intra-tunica albuginea injection of a plaque-inducing agent (human recombinant trans-forming growth factor-b1 [TGF-b1]).The authors noted that the PTT group resulted in less penile curvature than the VED and control groups. They postulated that the observed improvement might be related to anti-apoptosis, anti-fibrosis, and smooth muscle preservation. 11 ## Expert opinion It is important to recognize that the experimental conditions of the aforementioned studies do not resemble those present in a clinical scenario. In the study by Chung et al 5 , the strains applied to the model were not commensurate with those used in clinical practice (4-6 hours daily during a 6 months period). [bib_ref] Acute phase Peyronie's disease management with traction device: a nonrandomized prospective controlled..., Martínez-Salamanca [/bib_ref] Moreover, the findings from Lin et al 14 must be interpreted with caution since a rat model might not represent the actual human condition. Despite aforementioned limitations, the available results provide a preliminary understanding of the underlying mechanisms involved in PTT in PD. ## Clinical evidence The available evidence concerning the use of PPT in the treatment of PD with or without surgery will be separately discussed in the follow sections. ## Ptt as primary treatment for pd Statement 2. PTT shows promising results for patients with PD. Further stratification in terms of patient and disease characteristics are still required in order to identify those subjects most likely to benefit from PTT. The limited evidence prevents any definitive recommendation. ## Evidence Five studies assessed the outcomes of PTT as primary treatment for PD [fig_ref] Table 1: Results with PTT as a primary treatment PTT = penile traction therapy [/fig_ref]. Data interpretation was limited by the use of different devices (four studies used a conventional PTT device 15−18 , whereas one used a novel 'bending' mechanism 10 ). The recommended daily use ranged from 0.5 to 9 hours, and the follow-up period from 3 to 6 months. The latter trials identified described the use of PTT in different phases of PD: one included PD patients in the acute phase [bib_ref] Acute phase Peyronie's disease management with traction device: a nonrandomized prospective controlled..., Martínez-Salamanca [/bib_ref] , one in the chronic phase [bib_ref] Penile traction therapy with the new device "Penimaster PRO" is effective and..., Moncada [/bib_ref] , and the remaining three papers included mixed populations in both phases of the disease. [bib_ref] FastSize Medical Extender for the treatment of Peyronie's disease, Levine [/bib_ref] [bib_ref] Use of penile extender device in the treatment of penile curvature as..., Gontero [/bib_ref] [bib_ref] Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease:..., Ziegelmann [/bib_ref] The studies also differed in design, number of patients involved, and baseline mean penile curvature. The overall reduction in penile curvature ranged from 4°to 31.2°, corresponding to a relative improvement of 12.9-41.1%, most of which reached statistical significance [fig_ref] Table 1: Results with PTT as a primary treatment PTT = penile traction therapy [/fig_ref]. All studies claimed a gain in the stretched penile length (SPL), although defined measurement protocols were absent uniformly. Finally, a recent paper by Wymer et al [bib_ref] Comparative Cost-effectiveness of Surgery, Collagenase Clostridium Histolyticum, and Penile Traction Therapy in..., Wymer [/bib_ref] , compared the costeffectiveness of surgery, CCH and PTT for the treatment of PD, concluding that traction with the RestoreX (PathRight Medical Inc, Plymouth, USA) PTT device represented the most costeffective method for achieving ≥20% curvature improvement. As patients in this study were extracted from the Ziegelmann 10 series, their data were not described separately. ## Expert opinion Available data seem to support the use of PTT in either the acute or chronic phases of PD, resulting in improvement in penile curvature and SPLs. However, definitive conclusions were not possible due to the limited number of patients considered (267 in total), the heterogeneity in study design, and non-standardised inclusion and exclusion criteria. In addition, it is important to note that although the differences appreciated in the reduction of curvature may be statistically significant, they may not be clinically relevant. Patients as a consequence might need further treatment to ameliorate their curvature to achieve a satisfactory resolution of their functional deficit. Unfortunately, most studies did not include patients with calcified plaques, hourglass or hinge deformities, which are theoretically less likely to respond to PTT. Most of the studies failed to identify how the curvature characteristics were assessed (ideally, with a goniometer after induction of artificial erection with intra cavernous vasoactive agent, as describe in the paper by Ziegelmann [bib_ref] Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease:..., Ziegelmann [/bib_ref]. None of the studies used any tool to objectively measure the PTT time used by the patients. ## Ptt prior to pd surgery ## Evidence Only one study investigated the potential benefits of PTT prior to penile prosthesis implantation surgery in order to avoid postoperative length loss. [bib_ref] Traction therapy for men with shortened penis prior to penile prosthesis implantation:..., Levine [/bib_ref] Amongst the patients included, only 2 reported penile length loss due to PD (those who used the PTT 2 hours a day, for 2 months). Overall subjects experienced a penile length gain of 1.5 cm and 0.5 cm just before prosthesis implantation, with PTT and vacuum devices respectively. However, in only one of the patients the length gained persisted after prosthesis implantation, whereas the other reverted to pre-treatment length. ## Expert opinion Using PTT prior to any PD surgery may be theoretically beneficial to optimize penile length and reduce curvature. However, available data are too limited to support the use of PTT before PD surgery. Evidence PTT has been used as an adjuvant treatment following surgical correction (including penile shortening and lengthening procedures) for PD. Rybak et al 11 retrospectively compared the results of 134 patients who underwent PD surgery with or without PTT. PTT was initiated 3-4 weeks after surgery, and was applied for 2 or more hours a day, for 3 months. They found statistically significant improvement in SPL after both shortening and lengthening procedures (a mean gain of 0.85 cm in the shortening procedures + PTT vs a mean loss of 0.53 cm in shortening procedure only; and a mean gain of 1.48 cm in the lengthening procedures + PTT vs a mean gain of 0.24 cm in lengthening procedure only). [bib_ref] A retrospective comparative study of traction therapy vs. no traction following tunica..., Rybak [/bib_ref] Other reports have recommended the use of PPT (along with the use of a VED) in the immediate postoperative period in order to avoid retraction of the graft after a lengthening procedure. The results of these studies suggest that PTT can be beneficial in improving the outcomes of the PD surgery, in terms of ameliorating penile length loss and preventing curvature recurrence. ## Ptt after pd surgery ## Expert opinion PTT after a lengthening procedure may avoid retraction of the grafting material. However, it is important to recognize that the available evidence is limited and of poor quality. The results of Rybak et al 11 may be promising, but due to its retrospective nature it is not possible to give any definitive recommendation. Further studies are warranted to elucidate the role of PTT after shortening PD procedures and its timing. ## Ptt in combination with oral or intralesional treatments Statement 5. There is not enough data to recommend the use of PTT as a concomitant treatment with oral or intralesional therapy. ## Evidence The use of a PTT along with oral or intralesional therapies for PD became a popular approach especially after the approval of CCH for PD. Six studies evaluating the efficacy of PTT in combination with oral or intralesional treatments were identified [fig_ref] Table 2: Results of the PTT as a concomitant treatment [/fig_ref]. [bib_ref] Ten-year experience with multimodal treatment for acute phase Peyronie's disease: A real..., Gallo [/bib_ref] One paper addressed the use of multimodal therapy in the acute phase of PD and five in the chronic phase. draw any conclusions about the interaction of PTT itself in the results observed. ## Expert opinion PTT may enhance the efficacy of other PD treatments in reducing the penile curvature. However, it is not possible to give an evidence-based recommendation about its use, due to the inconsistence and heterogeneity of the available data. Ziegelmann et al 12 found that adding PTT to CCH treatment protocol worsened the curvature in PD patients. The lack of improvement could be attributed to the low number of patients or short duration of PTT, but worsening of the curvature requires further evaluations before recommending PTT for PD patients. Alom et al [bib_ref] Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile Traction Therapy in..., Alom [/bib_ref] found that only the use of the RestoreX device resulted in an improvement of the curvature, and not the use other PTT devices. This finding is consistent with the idea that this PTT device provokes a forced modeling that can be beneficial for CCH therapy. On the other hand, other studies found a general trend of SPL improvement with the use of the PTT. PTT SAFETY Statement 6. PTT adverse effects are mild and well tolerated. ## Evidence Not all the available studies describe adverse events related to the use of PTT. When reported, complications about the use of PTT are mild and well tolerated by the patients. Martínez-Salamanca et al 15 reported that 25.4% of PD patients experienced discomfort with the PTT (Andropeyronie -Andromecial SL, Madrid, Spain-) and there were 2 (3.6%) cases of erythema. When using the Penimaster PRO (MSP Concept GmBH&Co, Berlin, Germany) [bib_ref] Penile traction therapy with the new device "Penimaster PRO" is effective and..., Moncada [/bib_ref] , 43% of the patients complained of episodic glans numbness, local discomfort or glans edema; whereas PD patients who used the RestoreX (PathRight Medical Inc, Plymouth, USA) 10 , 45.2% complained of erythema and discoloration, 53.2% mild penile pain, 32% swelling and 1.6% a new lump. Although several studies instructed patients not to use PTT for more than 2 hours, and advised patients to include a resting period of 30 minutes between applications in order to avoid glans ischemia [bib_ref] Acute phase Peyronie's disease management with traction device: a nonrandomized prospective controlled..., Martínez-Salamanca [/bib_ref] [bib_ref] Outcomes of a Novel Penile Traction Device in Men with Peyronie's Disease:..., Ziegelmann [/bib_ref] , there was no published data about this adverse event. In the paper by Ziegelmann et al 10 , they even excluded patients with severe diabetes mellitus to avoid glans necrosis. ## Expert opinion Although the complications of the PTT are not systematically collected in every paper, they seem to be of mild importance, well tolerated by the patient and with no permanent consequences. Although there is no published report about glans ischemia, it seems reasonable to warn the patients about this risk. # Conclusions Data about the use of PTT in patients with PD is still limited. There are many aspects to that require clarification through well designed trials before evidence-based recommendations can be made. The ideal PD patient for PTT is still undefined and requires investigation. Moreover, the role of adjuvant PTT also requires further well-designed trials before strong recommendations can be made for its use. In summary, preliminary data suggests that PTT may be a promising treatment option for PD, although there is not enough evidence to give any definitive recommendation in any clinical scenario. Large well-designed and adequately powered RCTs are required to better clarify all these aspects. [fig] Figure 1: Flow chart diagram of the data acquisition. [/fig] [fig] Statement 3: Available data do not support the use of PPT for PD before surgery [/fig] [fig] Statement 4: Although promising results from preliminary studies, available data do not support the use of PTT after PD surgery. [/fig] [table] Table 1: Results with PTT as a primary treatment PTT = penile traction therapy; NRCT = non-randomized controlled trial; RCT = randomized controlled trial; SPL = stretched penile length. [/table] [table] Table 2: Results of the PTT as a concomitant treatment [/table]

Brain Changes Associated With Long-Term Ketamine Abuse, A Systematic Review Recently, the abuse of ketamine has soared. Therefore, it is of great importance to study its potential risks. The effects of prolonged ketamine on the brain can be observationally studied in chronic recreational users. We performed a systematic review of studies reporting functional and structural brain changes after repeated ketamine abuse. We searched the following electronic databases: Medline, Embase and PsycINFO We screened 11,438 records and 16 met inclusion criteria, totaling 440 chronic recreational ketamine users (2-9.7 years; mean use 2.4 g/day), 259 drug-free controls and 44 poly-drug controls. Long-term recreational ketamine use was associated with lower gray matter volume and less white matter integrity, lower functional thalamocortical and corticocortical connectivity. The observed differences in both structural and functional neuroanatomy between ketamine users and controls may explain some of its long-term cognitive and psychiatric side effects, such as memory impairment and executive functioning. Given the effect that long-term ketamine exposure may yield, an effort should be made to curb its abuse. # Introduction Ketamine is an anesthetic agent acting as an uncompetitive antagonist at the N-Methyl-D-Aspartate (NMDA) receptor. More recently, ketamine has emerged as a promising antidepressant [bib_ref] Antidepressant effects of ketamine in depressed patients, Berman [/bib_ref] [bib_ref] Replication of ketamine's antidepressant efficacy in bipolar depression: a randomized controlled add-on..., Zarate [/bib_ref] [bib_ref] The promise of ketamine for treatment-resistant depression: current evidence and future directions, Dewilde [/bib_ref] [bib_ref] NMDAR inhibition-independent antidepressant actions of ketamine metabolites, Albuquerque [/bib_ref] [bib_ref] A double-blind, randomized, placebo-controlled, dose-frequency study of intravenous ketamine in patients with..., Singh [/bib_ref] [bib_ref] Efficacy and safety of intranasal esketamine adjunctive to oral antidepressant therapy in..., Daly [/bib_ref]. In a meta-analysis including 201 patients with major depressive disorder (MDD), a single intravenous administration of ketamine was associated with a marked reduction in depression severity compared to placebo within 4 hours [bib_ref] Effects of low-dose and very low-dose ketamine among patients with major depression:..., Xu [/bib_ref] , and recently esketamine has been approved as a treatment for depression in both the United States and Europe . Despite the promising short-term effects, ketamine recently has been emerging as a drug of abuse. The prevalence of ketamine abuse was 1.7% in the United Kingdom in 2008/2009 (4% lifetime) [bib_ref] Emerging trends in the abuse of ketamine and its side effects on..., Zou [/bib_ref] , and around 1% in American college students [bib_ref] Party drugs: properties, prevalence, patterns, and problems, Maxwell [/bib_ref]. Chronic ketamine abuse in these countries was associated with long-term cognitive impairment, mood disorders, psychotic and dissociative symptoms, suggesting that prolonged ketamine use may indeed negatively affect brain structure and functioning. An alternative explanation is that primary depressive, psychotic or dissociative symptoms are reasons for ketamine self-medication rather than long-term sideeffects. It should be noted that recreational dosages are much higher than clinical dosages, both per dose and cumulatively. To date, the safety of prolonged ketamine administration has sparsely been investigated in humans in a prospective manner. The studies that have been done, have been conducted in clinical setting, with a much lower dose than the doses that are used recreationally. However, given the scarcity of research on the topic, these findings are worth mentioning. The most frequently reported side effects of short term ketamine (hours/days) are related to the nervous system, such as dissociation, sedation, headache, dizziness, blurred vision and memory impairment [bib_ref] Side-effects associated with ketamine use in depression: a systematic review, Short [/bib_ref]. Small case series of ketamine administration in various doses for up to one year in patients with MDD or chronic pain suggest that some of these neural side effects may remain with prolonged ketamine use [bib_ref] Side effects of ketamine in the long-term treatment of neuropathic pain, Cvrcek [/bib_ref] [bib_ref] A 12-month naturalistic observation of three patients receiving repeat intravenous ketamine infusions..., Szymkowicz [/bib_ref]. Contrastingly, another study suggests that prolonged add-on treatment with intranasal esketamine, which has recently been approved for treatment of treatment resistant depression (TRD) twice or thrice weekly did not worsen cognitive performance after 44 weeks of maintenance treatment compared to baseline in patients with treatment resistant depression (TRD) [bib_ref] Esketamine nasal spray plus oral antidepressant in patients with treatmentresistant depression: assessment..., Wajs [/bib_ref]. Despite the absence of prospective studies, some first insights might be gained from retrospective cohort studies in recreational ketamine users . We sought to systematically review all available studies measuring long-term structural and functional neuroanatomical differences between long-term recreational ketamine users and controls. The results of this review could provide some first insights into potential effects of prolonged recreational ketamine use which may inform clinicians better about the risks associated with ketamine abuse. # Methods ## Search strategy and selection criteria Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) guidelines were followed during the writing process [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref]. We formulated the following PICO: what neuroanatomical differences exist between longterm ketamine users compared to non-ketamine using controls. First, we performed a broad search to include all relevant studies investigating the effects of long-term ketamine use on all organ systems, for possible future reports. For this review, we focused on gray matter volume changes, loss of white matter integrity, differences in functional connectivity and activation patterns and receptor-binding after long-term recreational ketamine use in adults. The intervention of interest was repeatedly-dosed ketamine with a minimum duration of more than 14 days. The use of ketamine in recreational users was compared to non-drugusing controls or poly-drug users. Although we did not exclude studies in which subjects also used other drugs, we considered the limitation that differences other than ketamine use alone could exist between ketamine users and control subjects, including use of other drugs. A scoping search identified key articles and search concepts. All key articles had to be retrieved by the systematic search strategy. The search concept combination can be displayed as ketamine AND (chronic OR long term OR abuse OR dependence OR known long term use effects OR induced adverse effects). Medline, Embase and PsycINFO were systematically searched (JD JS, FD) from inception until February 2021 using the Ovid interface. The search strategy was designed by JD. A detailed search strategy can be obtained from the corresponding author. After removal of duplicates, 11,438 records were retrieved. The titles and abstracts of these records were independently screened by authors JS, CW and FD based on the selection criteria. Afterwards, conflicting results between the screeners were resolved by consensus. Original studies about recreative ketamine use in which neuroanatomical measurements were performed, either structural or functional, were included. To obtain the articles meeting this inclusion criterion we first excluded all articles that were not about ketamine. Then we excluded all articles that were not about the brain. Subsequently we excluded articles that were only about brain function and not about neuro-anatomical outcomes (e.g., performance on cognitive tests). Lastly, we excluded papers that were about animals or were no original investigations. Then, we sorted the articles levels of evidence. Of all remaining articles, full-texts and one congress abstract were read. Finally, we found our complete dataset consisting of 16 studies (see [fig_ref] FIGURE 1 |: Inclusion flowchart. [/fig_ref] for the inclusion flowchart. ## Data extraction Three of us (JS, FD, CW) independently extracted data from the retrieved articles: the N, characteristics of the ketamine consuming subjects, characteristics of the controls, significant differences between the ketamine consuming subjects compared to controls, the statistic measure and correlations [fig_ref] FIGURE 1 |: Inclusion flowchart. [/fig_ref]. The quality of the studies was assessed using the Sackett Scale and the Oxford CEBM levels of evidence scale [bib_ref] Rules of evidence and clinical recommendations on the use of antithrombotic agents, Sackett [/bib_ref]. # Analysis The results were subdivided into structural differences in gray and white matter, functional differences and effects on neurotransmission. Given the limited number of included studies and diversity of outcome measures in the studies, the data was deemed not suitable for meta-analysis. Therefore, we performed a conceptual synthesis of these heterogenous results. Because of these heterogenous results, we could not perform a quantitative bias analysis. Bias could play a role, since the 5 studies by [bib_ref] Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging..., Liao [/bib_ref] [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref] [bib_ref] Alterations in regional homogeneity of resting-state brain activity in ketamine addicts, Liao [/bib_ref] [bib_ref] Cue-induced brain activation in chronic ketamine-dependent subjects, cigarette smokers, and healthy controls:..., Liao [/bib_ref] were based on the same sample. For that reason, we compared the results if four of these five studies were left out of the analysis, to the situation in which all five studies were included. # Results We included 16 studies in our review, totaling 440 chronic ketamine users with a mean ketamine use of 2-9.7 years and 2.4 grams per day, compared to 259 drug-free controls and 44 poly-drug controls. Five studies were based on the same sample [bib_ref] Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging..., Liao [/bib_ref] [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref] [bib_ref] Alterations in regional homogeneity of resting-state brain activity in ketamine addicts, Liao [/bib_ref] [bib_ref] Cue-induced brain activation in chronic ketamine-dependent subjects, cigarette smokers, and healthy controls:..., Liao [/bib_ref]. The included studies described structural gray matter and white matter differences, differences in brain functionality and differences in neurotransmitter receptor binding. All retrieved studies were retrospective cohort studies, level IV on the Sackett scale or level 2b on the Oxford CEBM levels of evidence scale [bib_ref] Rules of evidence and clinical recommendations on the use of antithrombotic agents, Sackett [/bib_ref]. ## Structural differences: gray matter The results are shown in [fig_ref] TABLE 1 |: Structural differences [/fig_ref]. A structural MRI study in 41 chronic ketamine users and 44 drug-free controls, found smaller gray matter volume in the left superior frontal cortex and the right middle frontal cortex in the ketamine group compared to controls [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref]. Subjects in this study used on average 2 grams of ketamine per day for a mean duration of 3.4 years from the start of ketamine use until the subject was included in the study. Subjects consumed the ketamine by snorting ketamine powder. The duration of ketamine use was negatively correlated with the gray matter volume in the left superior frontal gyrus (SFG) and right middle frontal gyrus (MFG) [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref]. Also, estimated total lifetime consumption of ketamine was negatively correlated with gray matter volume in the left SFG, but not in the right MFG [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref]. In a second structural MRI study, smaller cortical thickness in several regions in the right frontal area was observed in 14 chronic ketamine users compared to 13 poly-drug controls [bib_ref] Translational neuroimaging reveals differential effects of chronic ketamine on brain macrostructure in..., Chesters [/bib_ref]. The route of ketamine administration was not mentioned. To assess the possible progression of brain changes following ketamine use over time, a third structural MRI study analyzed scans of 21 chronic ketamine users with varying durations of drug addiction, ranging from 0.5 to 12 years [bib_ref] Brain damages in ketamine addicts as revealed by magnetic resonance imaging, Wang [/bib_ref]. Changes were observed in both white and gray matter across the internal capsule, basal forebrain, cerebellum and diencephalon. Longer use of ketamine was associated with more extensive cortical atrophy in the parahippocampal gyrus and frontal, parietal and occipital cortex. Interestingly, subjects that had been addicted to ketamine for 3 years or less showed less atrophy than subjects that were addicted more than 3 years. For example, the cortex was not affected in subjects with a ketamine addiction of < 3 years, and the limbic system was not affected in subjects with an addiction of < 4 years. However, cortical atrophy occurred earlier than 3 years in a patient who had been using a high dose of 3 grams ketamine per day [bib_ref] Brain damages in ketamine addicts as revealed by magnetic resonance imaging, Wang [/bib_ref]. A structural MRI study with 124 ketamine chronic ketamine users (dose and duration of abuse not reported) found lower gray matter volume in the right orbitofrontal cortex (rOFC), the right medial prefrontal cortex (rmPFC), the left globus pallidus (lGP), the left hippocampus (lH) and the right nucleus accumbens (rNAC) in the ketamine group compared to 57 controls. Gray matter volumes in rOFC, rMPFC and rNAC were negatively correlated with ketamine dependence severity and gray matter volumes of the rOFC, rmPF, lCN, lGP, lH, and rNAC negatively correlated with cognitive performance. Different from other studies, this study also found higher gray matter volume in ketamine users compared to controls, i.e., in the left caudate nucleus. A study with 34 chronic ketamine users and 19 healthy controls found lower gray matter volume in the right insula, the left dorsolateral prefrontal cortex (DLPFC), the rOFC and the left inferior parietal cortex in ketamine users compared to controls [bib_ref] Effects of early ketamine exposure on cerebral gray matter volume and functional, Hung [/bib_ref]. Within the ketamine users group, adolescent onset users were compared to adult-onset users. Adolescentonset users showed a significantly smaller left precuneus volume than the adult-onset group and the healthy control group. ## Structural differences: white matter The results are shown in [fig_ref] TABLE 2 |: Structural differences [/fig_ref]. Using diffusion-weighted MRI scans, fractional anisotropy (FA) can be used for estimating white matter fiber density, myelination and axonal diameter. FA reductions were found in bilateral frontal and left temporoparietal white matter in 41 ketamine users with a mean use of 2 grams/day for 3.4 years, in comparison with 44 drug-free controls [bib_ref] Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging..., Liao [/bib_ref]. FA in the left and right Presence of psychiatric or somatic disorder was not mentioned. 1) Both primarily ketamine users and ketamine + polysubstance users had larger caudate nuclei than the non-drug controls. 2) Both ketamine groups had larger white matter volumes throughout the whole brain. 3) The K+polysubstance abuse showed even higher white matter volumes. White matter volume was measured as a percentage of total intracranial volume. frontal white matter was negatively correlated with the total lifetime consumption of ketamine. Diffusivity can be divided in axial and radial diffusivity. Axial diffusivity is thought to be a measure of axonal density and radial diffusivity is thought to be related to the degree of myelination [bib_ref] Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging..., Liao [/bib_ref]. In 16 ketamine users averaging 2.4 grams/day for 7.3 years, a lower level of axial diffusivity was found compared to 16 polydrug controls, especially in the frontal part of the right hemisphere (Edward [bib_ref] Abnormalities in white matter microstructure associated with chronic ketamine use, Roberts [/bib_ref]. Axial diffusivity was significantly lower in eight white matter clusters in the right hemisphere in the ketamine group compared to the control group, the three largest being located in the frontal cortex (Edward [bib_ref] Abnormalities in white matter microstructure associated with chronic ketamine use, Roberts [/bib_ref]. Also, probabilistic tractography was performed to investigate cortico-subcortical white matter connectivity profiles, which revealed that white matter connectivity between the caudate nucleus and the lateral prefrontal cortex was positively correlated with severity of long-term dissociative symptoms (Edward [bib_ref] Abnormalities in white matter microstructure associated with chronic ketamine use, Roberts [/bib_ref]. In a study by [bib_ref] Striatal and white matter volumes in chronic ketamine users with or without..., Liang [/bib_ref] , ketamine users had larger caudate volume and total white matter volume than non-drugs controls. Ketamine users that also used stimulants had even larger white matter structures, suggesting an additive effect of ketamine and these stimulants. Participants in this study were asked to refrain from drug use 4-7 days before imaging was performed [bib_ref] Striatal and white matter volumes in chronic ketamine users with or without..., Liang [/bib_ref]. The authors of this study hypothesize that the higher gray and white matter volumes reported in this study in ketamine users may not necessarily reflect neurotoxicity but rather represent positive compensatory changes as they were associated with less long-term cognitive impairments and depression. Opposingly, one could hypothesize that increased white matter volume may imply that an underlying low grade inflammatory edematous process exists, comparable to edema observed after brain injury [bib_ref] Acute cytotoxic and vasogenic edema after subarachnoid hemorrhage: a quantitative MRI study, Weimer [/bib_ref]. [formula] 1) (p = 0.030) 2) (p = 0.046) 3) (p = 0. [/formula] ## Functional differences The results are shown in [fig_ref] TABLE 3 |: Functional differences [/fig_ref]. Using resting-state functional MRI (fMRI), Liao al. investigated the functional connectivity between the thalamus and specific cortical regions in 40 chronic ketamine users with a mean use of 2 grams/day for 3.4 years, compared to 88 drug-free controls. Lower functional connectivity was found between the thalamus and the motor-, posterior parietal-and prefrontal cortex. Functional connectivity between the posterior parietal cortex and right lateral dorsal nucleus was significantly correlated to individual ketamine craving scores. Resting-state fMRI can also be used to measure regional homogeneity (ReHo). ReHo describes the summarized local functional connectivity between a voxel and its neighboring voxels. This is an index of network centrality, showing the importance of a voxel in a functional network. In 41 chronic ketamine users with a mean use of 2 grams/day for 3.4 years compared to 44 drug-free controls, lower ReHo in the right anterior cingulate cortex and higher ReHo in the left precentral frontal gyrus were found [bib_ref] Alterations in regional homogeneity of resting-state brain activity in ketamine addicts, Liao [/bib_ref]. The higher ReHo in the left precentral frontal gyrus was negatively correlated with estimated total lifetime ketamine consumption and ketamine craving [bib_ref] Alterations in regional homogeneity of resting-state brain activity in ketamine addicts, Liao [/bib_ref]. This may suggest that ReHo is initially increased more by ketamine use but that this increase eventually decreases with more prolonged and intensive use, which may alter functional organization in frontal networks. However, since subjects had to be abstinent from ketamine for only 48 hours, and since the direct effect of ketamine can last for more than 48 hours, the altered frontal network organization might also be a direct result of ketamine instead of a long-term side effect [bib_ref] Replication of ketamine's antidepressant efficacy in bipolar depression: a randomized controlled add-on..., Zarate [/bib_ref]. In the same sample, the authors compared smoking chronic ketamine users with non-ketamine smokers and with non-ketamine, non-smokers by performing fMRI. They found a higher activation in the anterior cingulate cortex (ACC) in response to ketamine cues. Also, ketamine subjects showed lower activation in the cerebellum and the middle temporal cortex in response to natural rewarding (sexual) cues [bib_ref] Cue-induced brain activation in chronic ketamine-dependent subjects, cigarette smokers, and healthy controls:..., Liao [/bib_ref]. [bib_ref] Depression in chronic ketamine users: Sex differences and neural bases, Li [/bib_ref] assessed resting-state functional connectivity of the subgenual anterior cingulate cortex (sgACC) in relation to depression scores of 36 chronic ketamine users with an average ketamine use of 4.9 years (dose not reported) compared to 20 drug-free controls. Overall, no difference in sgACC connectivity was found between groups, but in ketamine users higher depression scores correlated with lower sgACC connectivity to the right lateral and bilateral medial OFC. Further analysis revealed functional connectivity changes, with male and female ketamine users showing higher sgACC connectivity than controls to the bilateral superior temporal gyrus or dorsomedial prefrontal cortex (dmPFC), respectively [bib_ref] Depression in chronic ketamine users: Sex differences and neural bases, Li [/bib_ref]. Also, they found a correlation between higher sgACC connectivity with the dmPFC and higher depression scores in women, but not in men. Although ketamine has strong short-term antidepressant effects, the current data would suggest that chronic ketamine use may actually induce depression via sex-specific dysregulation of brain networks for positive and negative emotions. It remains unclear whether the altered connectivity patterns found in this study could be a direct result of ketamine. Being under the influence of ketamine was not an exclusion criterion for participation in this study. [bib_ref] Long-term heavy ketamine use is associated with spatial memory impairment and altered..., Morgan [/bib_ref] used fMRI to compare brain activity of 11 ketamine users with a mean use of 1.5 grams/day for 9.7 years, to 15 polydrug controls during a spatial memory task. Ketamine users showed lower activity in the right hippocampus and left parahippocampal gyrus compared to controls. Left caudate activity was greater in polydrug controls. These findings suggest an impact of long-term ketamine abuse on spatial memory processes associated with impaired (para)hippocampal activation. Lastly, in a small fMRI study using a motor task in which subjects had to flex and extend their upper limbs, three long-term ketamine users with a mean use of 1-2 grams/day for 2 years demonstrated less cerebellar activity compared to 3 drug-free controls [bib_ref] Downregulation in the human and mice cerebella after ketamine versus ketamine plus..., Chan [/bib_ref]. Being under the influence of ketamine was no exclusion criterion. Lin et al. used resting state fMRI to compare a group of chronic ketamine users, many of which also used other drugs like cannabis or cocaine, to healthy controls . They found lower functional connectivity of the default mode network in the orbital right inferior frontal gyrus, left anterior cingulate gyrus, paracingulate gyri, right superior temporal gyrus and bilateral vermic lobule VI of the cerebellum. In contrast, they found higher functional connectivity in the left middle occipital gyrus. In a study by [bib_ref] Effects of early ketamine exposure on cerebral gray matter volume and functional, Hung [/bib_ref] , chronic ketamine users compared to healthy controls showed higher functional connectivity between the left DLPFC and the right inferior frontal/superior temporal gyrus and the left OFC and the right insula/inferior temporal gyrus. Within the ketamine users group, adolescent onset users were compared to adult onset users. Both the adult and the adolescent groups had higher functional connectivity between the left and right precuneus [bib_ref] Effects of early ketamine exposure on cerebral gray matter volume and functional, Hung [/bib_ref]. In a pilot that studied white matter connectivity, chronic ketamine users showed higher connectivity between caudate nuclei and the dorsal anterior cingulate cortex (dACC). Ketamine users also showed a higher connectivity between the pallidum and the bilateral cerebellum. Furthermore, in ketamine users, the putamen showed higher connectivity to the OFC, which correlated with duration of ketamine use. Also, the ventral striatum (VS) showed lower connectivity with the right superior temporal sulcus (STS) and the left superior frontal gyrus (SFG) which was mediated by higher scores on the Barratt Impulsiveness Scale (BIS-11) [bib_ref] Striatal functional connectivity in chronic ketamine users: a pilot study, Hung [/bib_ref]. ## Dopamine d 1 receptors The results are shown in [fig_ref] TABLE 4 |: D 1 receptor. [/fig_ref]. One study investigated how long-term ketamine use affected neurotransmitter systems [bib_ref] Altered prefrontal dopaminergic function in chronic recreational ketamine users, Narendran [/bib_ref]. Dopamine D 1 binding potential was studied using positron emission tomography (PET) imaging after intravenously injecting the selective D 1 receptor radio ligand [ 11 C]NNC 112 in 14 ketamine users with a mean use of 0.75 gram/week for 4.1 years and 14 drug-free controls. D 1 receptor availability was significantly upregulated in the dorsolateral prefrontal cortex of ketamine users compared to controls, which could result from increased receptor density or affinity. D 1 binding potential correlated with the total amount of ketamine consumption [bib_ref] Altered prefrontal dopaminergic function in chronic recreational ketamine users, Narendran [/bib_ref]. # Discussion We systematically reviewed structural and functional brain changes after prolonged recreational ketamine use. Long-term ketamine abusers compared to controls displayed: (1) lower gray matter volume or cortical thickness in primarily the frontal, parietal and occipital lobes (2) lower white matter integrity in frontal and temporoparietal lobes (3) lower functional thalamocortical and corticocortical connectivity and abnormal frontal network organization (4) lower activity of brain regions for spatial memory and motor execution (5) higher functional connectivity between the DLPFC and the OFC and larger total white matter volume (6) higher dopamine D 1 binding potential in the dorsolateral prefrontal cortex. Including all five studies by Liao et al. could be a confound. Therefore, we also analyzed the results after excluding four of these five studies. As a result of this, finding (2) would change to "lower white matter integrity in right frontal and temporoparietal lobes" and/or finding (3) would not stand, depending on which articles were left out. Higher binding potential for D 1 receptor in dorsolateral prefrontal cortex. [ 11 C]NNC 112 binding potential ml/g Positive correlation between higher D 1 binding potential and amount of consumed ketamine (p = 0.005). ## Pet, positron-emitted tomography; dsm-iv, diagnostic and statistical manual of mental disorders, 4th edition. Many of the observed changes were correlated with the amount and duration of ketamine consumption, suggesting a possible dose dependent effect of prolonged ketamine on brain structure and function. Although the identified lower gray and white matter volumes or integrity could suggest direct neurotoxic effects of ketamine, the observed higher structural and functional connectivity and dopamine binding may suggest indirect compensatory effects. Together, these findings suggest that long-term intensive ketamine use may affect the structure and function of cortical gray and white matter, especially in frontoparietal regions. It must be noted that the reported changes were dependent on the dosage and duration of ketamine use which were substantially higher than for clinical use, so our findings cannot be translated to clinical ketamine use. All subjects in these studies used at least 0.2 grams of ketamine twice weekly, which is around two to four times the recommended clinical dosage (25-50 mg intravenously twice or thrice weekly, equivalent to 50-100 mg intranasal ketamine, the most common route of administration in recreational use), with most subjects even consuming more than 1 gram daily which is equivalent to 25-70 times the clinical dose [bib_ref] Antidepressant effects of ketamine in depressed patients, Berman [/bib_ref] [bib_ref] Effects of low-dose and very low-dose ketamine among patients with major depression:..., Xu [/bib_ref] [bib_ref] A consensus statement on the use of ketamine in the treatment of..., Sanacora [/bib_ref]. Nevertheless, this review does suggest that prolonged high dose ketamine use may have the potential to alter brain structure and function. Mechanistic support for such differences observed in long-term high dose ketamine comes from studies suggesting that exposure to ketamine induces apoptosis in adolescent primates and human brain cell cultures [bib_ref] Chronic ketamine exposure induces permanent impairment of brain functions in adolescent cynomolgus..., Sun [/bib_ref]. In several of these preclinical studies chronic dosing regimens have been comparable to the regimens of chronic ketamine abusers. For example, 6 months of daily 60 mg intraperitoneal ketamine in mice was associated with reduced expression of GluA1-containg AMPA receptors and memory impairments [bib_ref] Changes in hippocampal AMPA receptors and cognitive impairments in chronic ketamine addiction..., Ding [/bib_ref] and 28 days of daily 30 mg/kg intraperitoneal ketamine was associated with reduced expression of glutamatergic receptor units GluA1, GluA2, GluN2A, and GluN2B, as well as a lower expression of synaptic proteins Syn and PSD-95, deteriorated cognitive skills, lower spine density, impairments in long-term potentiation and hampered transmission in the hippocampal CA1 area [bib_ref] Chronic administration of ketamine induces cognitive deterioration by restraining synaptic signaling, Luo [/bib_ref]. Accordingly, in another study, the amount of parvalbumine positive interneurons in the CA1 region of mice hippocampi was reduced after 1 month of subcutaneous 16 mg/kg ketamine injections [bib_ref] Impaired hippocampal-dependent memory and reduced parvalbumin-positive interneurons in a ketamine mouse model..., Koh [/bib_ref]. These mice showed memory impairments, which is in line with a study in which parvalbumine positive interneurons are shown to play a role in hippocampal memory consolidation [bib_ref] Parvalbumin-expressing interneurons coordinate hippocampal network dynamics required for memory consolidation, Ognjanovski [/bib_ref]. Brains of mice and cynomolgus monkeys treated with 3 months of daily 30 mg/kg intraperitoneal ketamine (mice) or 1 mg/kg intravenous ketamine (monkeys) showed hyperphosphorylation of the tau protein, which could be interpreted as a neurotoxic effect since phosphorylation of tau protein is perceived as an aging marker [bib_ref] Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration..., Yeung [/bib_ref]. Mice treated with 3 or 6 months daily subanesthetic doses (30 or 60 mg/kg) of intraperitoneal ketamine showed hyperphosphorylation of tauprotein hampering trafficking of AMPA-receptors, which in turn worsened signal transduction [bib_ref] Long-term ketamine administration causes Tau protein phosphorylation and Tau proteindependent AMPA receptor..., Li [/bib_ref]. Another study suggested that impaired working memory after chronic exposure to ketamine in mice was mediated by upregulation of Gaba-5 subunit of the GABA A receptor in the prefrontal cortex [bib_ref] Gene expression changes in GABA(A) receptors and cognition following chronic ketamine administration..., Tan [/bib_ref]. The same group found that a 3-month ketamine treatment in mice was associated with upregulation of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholaminesynthesis. The upregulation of TH might in turn be caused by upregulation of BDNF, which was also found after long-term ketamine use. In conclusion, these animal studies may provide important clues for the potential neurotoxic effects of prolonged ketamine use. Prolonged ketamine may either up-or downregulate important regulatory neuronal proteins, potentially resulting in impaired neuronal functioning and cognitive performance. A possible mechanism for the white matter changes identified in the reviewed recreational ketamine studies could be AMPAreceptor mediated excitotoxicity. In rats, ketamine was found to acutely elevate presynaptic glutamate in the prefrontal cortex at AMPA/kainite receptors [bib_ref] Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway..., Moghaddam [/bib_ref] , and prolonged ketamine exposure may provoke cell death by regional glutamate-induced excitotoxicity. Excitation of AMPA receptors specifically induces axonal damage [bib_ref] Intracerebral injection of AMPA causes axonal damage in vivo, Fowler [/bib_ref] , which could provide a potential mechanism for the prominent white matter changes observed after sustained ketamine exposure in three of the reviewed studies. Also, white matter changes in one of these studies preceded more widespread cortical atrophy with longer ketamine use, supporting that axonal cells are most vulnerable for glutamate-induced excitotoxicity by ketamine. However, these observations are still based on comparison between subjects rather than longitudinal data. Our reviewed ketamine-associated brain changes might also explain some of the cognitive impairments and psychiatric symptoms observed in chronic ketamine users [bib_ref] Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and..., Morgan [/bib_ref]. Impairments of working-, semantic-, spatial-and episodic memory in chronic ketamine users [bib_ref] Acute and chronic effects of ketamine upon human memory: a review, Morgan [/bib_ref] [bib_ref] Effects of chronic ketamine use on frontal and medial temporal cognition, Chan [/bib_ref] may be related to the observed atrophy and impaired function of brain regions underlying these memory functions, including the hippocampal complex, prefrontal and temporoparietal cortex. Findings of impaired executive functioning in chronic ketamine users [bib_ref] Altered prefrontal dopaminergic function in chronic recreational ketamine users, Narendran [/bib_ref] [bib_ref] Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and..., Morgan [/bib_ref] align with the reviewed frontostriatal impairments. It needs to be considered however, that there may be a U-shaped dose-effect relation between ketamine and cognitive changes. In rats, different 5-7-days dosing regimens of ketamine yielded opposite effects on cognitive tasks in which the rats had to detect novel objects, or novel placement of objects. Whereas, low ketamine enhanced novelty detection compared to controls, higher doses impaired novelty detection [bib_ref] Different dosing regimens of repeated ketamine administration have opposite effects on novelty..., Schumacher [/bib_ref]. The observed upregulation of dorsolateral prefrontal D 1 receptors in ketamine users might be a compensatory mechanism for deficient prefrontal dopamine function underlying impaired working memory function [bib_ref] Altered prefrontal dopaminergic function in chronic recreational ketamine users, Narendran [/bib_ref]. Consistent with this, monkeys treated with another non-competitive NMDA antagonist, MK-801, showed lower performance on working memory tasks and lower prefrontal dopamine levels [bib_ref] Effect of MK801 on dopamine parameters in the monkey brain, Kakiuchi [/bib_ref]. Regarding psychotic symptoms, the observation of higher D 1 binding potential in the dorsal prefrontal cortex after chronic ketamine use, may mirror similar findings in schizophrenia, as a compensation mechanism for prefrontal dopamine dysfunction and potentially explaining the high incidence of psychosis in chronic ketamine users [bib_ref] Prefrontal dopamine D1 receptors and working memory in schizophrenia, Abi-Dargham [/bib_ref] [bib_ref] Altered prefrontal dopaminergic function in chronic recreational ketamine users, Narendran [/bib_ref] [bib_ref] Perceptual organization in ketamine users: preliminary evidence of deficits on night of..., Uhlhaas [/bib_ref] [bib_ref] Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and..., Morgan [/bib_ref] [bib_ref] Consequences of chronic ketamine self-administration upon neurocognitive function and psychological wellbeing: a..., Morgan [/bib_ref] , although it might also be an epiphenomenon of an inflammatory pathway induced by ketamine [bib_ref] Bimodal induction of dopaminemediated striatal neurotoxicity is mediated through both activation of..., Wersinger [/bib_ref] [bib_ref] Dopamine D1 receptors, regulation of gene expression in the brain, and neurodegeneration, Cadet [/bib_ref]. In addition, the observed hippocampal impairments in ketamine users may also have contributed to psychotic symptoms . Altered hippocampal functioning is associated with the transition from a prodromal psychotic state to acute psychosis and repeated ketamine administration was able to induce these hippocampal impairments during progression to psychosis in a mouse model [bib_ref] Imaging patients with psychosis and a mouse model establishes a spreading pattern..., Schobel [/bib_ref]. The dissociative symptoms after chronic ketamine use have been associated with differences in corticostriatal connectivity [bib_ref] Abnormalities in white matter microstructure associated with chronic ketamine use, Roberts [/bib_ref]. Finally, despite the short-term antidepressant effects of ketamine, chronic ketamine use is associated with depressive symptoms [bib_ref] Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and..., Morgan [/bib_ref] [bib_ref] Depression in chronic ketamine users: Sex differences and neural bases, Li [/bib_ref] , which correlates with altered functional connectivity between sgACC, OFC and ventromedial prefrontal cortex [bib_ref] Depression in chronic ketamine users: Sex differences and neural bases, Li [/bib_ref] , which are all areas involved in emotion regulation [bib_ref] Stereotactic cingulotomy with results of acute stimulation and serial psychological testing, Meyer [/bib_ref] [bib_ref] The cingulate gyrus and human behaviour, Talairach [/bib_ref]. Caution is required in deeming prolonged ketamine use causal to the observed brain differences for several reasons. The reviewed brain differences might have been pre-existing and may have predisposed subjects to ketamine dependence. This is plausible considering that many of the observed brain differences concerned prefrontal regions that are crucial for inhibiting addictive behaviors. On the other hand, prefrontal gray matter reductions may have been initiated by ketamine use, further impairing inhibition and facilitating ketamine dependence. Similar mechanisms have been demonstrated for prefrontal changes associated with other types of drug abuse, including MDMA and cocaine [bib_ref] Reduced cortical gray matter density in human MDMA (Ecstasy) users: a voxel-based..., Cowan [/bib_ref] [bib_ref] Brain macrostructural and microstructural abnormalities in cocaine dependence, Lim [/bib_ref]. Another finding that could explain transition to ketamine addiction in recreational users is lower thalamocortical connectivity, which may impair control over cognitive and emotional processes involved in drug-seeking behavior [bib_ref] Thalamocortical integration of instrumental learning and performance and their disintegration in addiction, Balleine [/bib_ref]. Although only a subset of recreational ketamine users are reported to develop dependence [bib_ref] Journey through the K-hole: phenomenological aspects of ketamine use, Muetzelfeldt [/bib_ref] and tolerance [bib_ref] Long-term ketamine self-injections in major depressive disorder: focus on tolerance in ketamine's..., Bonnet [/bib_ref] , the current review suggests several potential mechanisms for addiction that should be further explored to gain an understand of ketamine abuse. Even though this review shows associations between longterm ketamine and structural and functional neuroanatomical differences, it holds important limitations. The results have been obtained from recreational ketamine users for whom we do not precisely know what dose of ketamine they used, which type of ketamine (racemic or esketamine) and whether they consumed pure ketamine or ketamine contaminated with other substances. This review shows that several functional and structural changes appear to correlate with duration and dose of ketamine consumption and are most striking after more than 3 years of high doses. The results of this review are not translatable to clinical ketamine regimens, since therapeutic dosages may lead to significantly less brain changes than recreationally dosed ketamine, or that it may take much longer to develop brain changes with therapeutic regimens. However, studies on long-term brain effects of therapeutic ketamine are lacking. Nonetheless, evidence exists for opposite clinical effects of low dose vs. high dose ketamine. Low dose, twice weekly ketamine schedules have strong antidepressant effects in depressed patients [bib_ref] A double-blind, randomized, placebo-controlled, dose-frequency study of intravenous ketamine in patients with..., Singh [/bib_ref] , whereas high dose daily schedules have been associated with depression in ketamine abusers [bib_ref] Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and..., Morgan [/bib_ref]. Furthermore, low dose ketamine schedules in mice increased sprouting in the medial prefrontal cortex compared to saline injections , whereas chronic selfadministration of high dose ketamine in rats reduced glutamate receptor expression in the medial prefrontal cortex [bib_ref] Ketamine self-administration reduces the homeostasis of the glutamate synapse in the rat..., Caffino [/bib_ref]. This may be in line with brain volume loss and reduced connectivity after high dosing recreational ketamine schedules in humans [bib_ref] Frontal white matter abnormalities following chronic ketamine use: a diffusion tensor imaging..., Liao [/bib_ref] [bib_ref] Reduced dorsal prefrontal gray matter after chronic ketamine use, Liao [/bib_ref] [bib_ref] Alterations in regional homogeneity of resting-state brain activity in ketamine addicts, Liao [/bib_ref] [bib_ref] Brain damages in ketamine addicts as revealed by magnetic resonance imaging, Wang [/bib_ref] [bib_ref] Abnormalities in white matter microstructure associated with chronic ketamine use, Roberts [/bib_ref] [bib_ref] Translational neuroimaging reveals differential effects of chronic ketamine on brain macrostructure in..., Chesters [/bib_ref] , but enhanced brain connectivity after lower dose ketamine [bib_ref] Prefrontal connectivity and glutamate transmission: relevance to depression pathophysiology and ketamine treatment, Abdallah [/bib_ref]. Furthermore, long-term uncontrolled dosing may lead to ketamine-tolerance, as illustrated by a case report of a woman who became more depressed after administering herself increasing doses of ketamine [bib_ref] Long-term ketamine self-injections in major depressive disorder: focus on tolerance in ketamine's..., Bonnet [/bib_ref] , while short term controlled regimens in rats led to ketamine sensitization, as shown by increased locomotor activity after two ketamine injections compared to one injection [bib_ref] Differences between adolescents and adults in the acute effects of PCP and..., Rocha [/bib_ref]. Lastly, it should be noted that racemic ketamine and esketamine may have distinct toxicity profiles. Recreational users may mainly use racemic ketamine, whereas long-term treatment of depression mainly concerns esketamine which could be less toxic to the brain [bib_ref] Esketamine nasal spray plus oral antidepressant in patients with treatmentresistant depression: assessment..., Wajs [/bib_ref]. A second limitation of the reviewed studies is that use of other substances including tobacco was more prevalent among ketamine users compared to the drug-free controls, although several studies included polydrug users as a control group. Therefore, the observed brain changes cannot indisputably be ascribed to ketamine alone. In addition, street ketamine might not be pure ketamine but could be contaminated with other drugs, which would strengthen this confounding. Also, ketamine abuse itself might give rise to abuse of other substances. For example, ketamine has an effect on mu-opioid receptors and its antidepressant effect might even be partially mediated by this receptor [bib_ref] The interaction of ketamine with the opiate receptor, Smith [/bib_ref] [bib_ref] Potentiation of µ-opioid receptor-mediated signaling by ketamine, Gupta [/bib_ref] [bib_ref] Attenuation of Antidepressant Effects of Ketamine by Opioid Receptor Antagonism, Williams [/bib_ref]. This effect on the mu-opioid receptor might give rise to opioid abuse. In one of the included samples, 50% of ketamine users had also been using heroin, which could have contributed to the observed brain changes [bib_ref] Ketamine: promising path or false prophecy in the development of novel therapeutics..., Sanacora [/bib_ref] [bib_ref] Translational neuroimaging reveals differential effects of chronic ketamine on brain macrostructure in..., Chesters [/bib_ref]. This interplay between ketamine and muopioid receptors might also apply to other neurotransmitter systems and associated drugs of abuse. Third, since subjects were mostly recreational users, they might have used ketamine shortly before data were obtained. Therefore, the different functional connectivity patterns could in part be caused or influenced by the direct, short term effects of ketamine. Fourth, most of the included subjects were of Asian ethnicity, which might have influenced outcomes for instance through genetic differences in drug metabolism. However, it has been shown that frequencies of cytochrome P450 variants responsible for ketamine metabolism do not vary significantly between people with Asian or Caucasian ancestry [bib_ref] PM frequencies of major CYPs in Asians and Caucasians, Mizutani [/bib_ref] [bib_ref] Ketamine: A review of clinical pharmacokinetics and pharmacodynamics in anesthesia and pain..., Peltoniemi [/bib_ref]. We consider a few methodological limitations as well. The included studies followed a cross-sectional and retrospective design with considerable variability among studies in terms of subject age, ketamine type and dosage. The data was insufficient to perform a meta-analysis. Additionally, five of the 16 studies were based on the same sample. It should be noted that in some studies, ketamine users had a mood disorder and for many of the studies it was unclear whether the ketamine users were diagnosed with another substance use disorder or another psychiatric illness. Part of the structural and functional neuroanatomical differences could therefore be attributed to these concomitant conditions. Finally, we were unable to receive a few records containing potentially relevant data. In conclusion, prolonged high-dosed recreational ketamine use is associated with structural and functional brain differences. Why these differences exist, has not been established yet, but may follow patterns observed in animals, i.e., hyperphosphorylation of tau-protein and disrupting expression of several receptor subunits. Despite its limitations, this review underscores the need for further study of potential risks associated with repeated administration of ketamine. To elucidate causal associations between brain changes and prolonged ketamine use, prospective and longitudinal imaging studies with controlled lowdose administrations are needed, ideally combined with cognitive tasks. # Data availability statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. [fig] FIGURE 1 |: Inclusion flowchart. [/fig] [fig] -: sgACCOFC connectivity negatively correlated with CESD (p = 0.00001, r =0.66) in KU (M & F) sgACCSTG connectivity positively correlated with CESD both in KU (p = 0.00002, r = 0.74) and in HC (p = 0.009, r = 0.74), men only sgACCdmPFC connectivity positively correlated with the CESD in KU (p = 0.00003, r = 0.97), not in HC (p = 0.69, r = −0.16), female only Morgan et al. [/fig] [table] TABLE 1 |: Structural differences: gray matter. Participants had no major medical or neurological illness. [/table] [table] TABLE 2 |: Structural differences: white matter. Frontiers in Neuroanatomy | www.frontiersin.org [/table] [table] TABLE 3 |: Functional differences. [/table] [table] TABLE 4 |: D 1 receptor. [/table]

Does IRISIN Have a BRITE Future as a Therapeutic Agent in Humans? The epidemic of obesity has contributed to the rapid rise in comorbid conditions such as cardiovascular disease, type 2 diabetes, sleep apnea, and hypertension among others. Therefore, there is a critical need to develop therapeutic strategies to reduce the prevalence of the disease. Skeletal muscle cells secrete signaling cytokines/peptides (referred to as myokines) that act in autocrine, paracrine, and endocrine fashion. Myokines have been hypothesized to contribute to the immediate and chronic benefits of exercise and may thus serve as attractive therapeutic agents for the treatment of obesity. The recent discovery of the irisin, a proposed myokine, has gained much attention over the last two years as a potential therapeutic agent. Preliminary studies demonstrated that irisin has the potential to induce "browning" of white adipocytes in mice. If these findings in mice could be translated to humans, irisin could be a potential therapeutic agent for the treatment of obesity. Limitations with the available antibodies, however, have raised concerns regarding the detectability of irisin in circulation. Moreover, the gene encoding irisin, FNDC5, is expressed robustly not only in muscle but also in various white adipose tissues (WAT) in humans, raising the possibility for increased thermogenesis through autocrine mechanisms. Here we will discuss the browning of WAT, the discovery of irisin, and its potential role in improving metabolic health in humans. # Introduction Obesity has rapidly become a worldwide epidemic. In parallel, the prevalence of obesity-related comorbid conditions has also escalated, including insulin resistance, metabolic syndrome, type 2 diabetes, hypertension, chronic kidney disease, cardiovascular disease, heart failure, cancer, and dementia [bib_ref] Definition of metabolic syndrome: report of the National Heart, Lung, and Blood..., Grundy [/bib_ref] [bib_ref] Prevalence of obesity, diabetes, and obesity-related health risk factors, Mokdad [/bib_ref] [bib_ref] Heart disease and stroke statistics-2009 update: a report from the American Heart..., Carnethon [/bib_ref]. As expected, a recent meta-analysis from the US Centers for Disease Control and Prevention confirms that obesity is associated with increased all-cause mortality [bib_ref] Association of allcause mortality with overweight and obesity using standard body mass..., Flegal [/bib_ref]. The rapid increase in obesity and obesity-related comorbid conditions has coincided with the rapidly changing landscape of our obesogenic environment [bib_ref] Modern sedentary activities promote overconsumption of food in our current obesogenic environment, Chaput [/bib_ref]. In particular, it has coincided with the systemic reductions in total daily physical activity as well as reductions in vigorous physical activity [bib_ref] Modern sedentary activities promote overconsumption of food in our current obesogenic environment, Chaput [/bib_ref] [bib_ref] Trends over 5 decades in U.S. occupationrelated physical activity and their associations..., Church [/bib_ref]. Exercise has long been recognized for its pluripotent effects on body composition [bib_ref] Effect of exercise training intensity on abdominal visceral fat and body composition, Irving [/bib_ref] [bib_ref] Effect of exercise on total and intra-abdominal body fat in postmenopausal women:..., Irwin [/bib_ref] , metabolic health [bib_ref] Exercise training amount and intensity effects on metabolic syndrome (from Studies of..., Johnson [/bib_ref] , cardiovascular disease [bib_ref] Exercise and physical activity in the prevention and treatment of atherosclerotic cardiovascular..., Thompson [/bib_ref] [bib_ref] A prospective study of walking as compared with vigorous exercise in the..., Manson [/bib_ref] and mental health [bib_ref] Physical activity, including walking, and cognitive function in older women, Weuve [/bib_ref]. The underlying mechanism(s) for the clinical benefits of exercise remains to be fully elucidated. Over the past decade, it has become increasingly recognized that skeletal muscle cells secrete signaling cytokines/peptides that act in autocrine, paracrine, and endocrine fashion in response to skeletal muscle contraction (e.g., exercise) [bib_ref] Muscles, exercise and obesity: skeletal muscle as a secretory organ, Pedersen [/bib_ref]. The secreted cytokines/peptides, referred to as myokines, have been hypothesized to contribute to the immediate and chronic benefits of exercise [bib_ref] Muscles, exercise and obesity: skeletal muscle as a secretory organ, Pedersen [/bib_ref]. The recent discovery of irisin by Bostrom et al. , a putative exercise induced myokine, that is credited for improving metabolic health by its ability to brown white adipose tissue (WAT) in mice has received considerable attention over the last two years. Although it remains to be determined whether irisin has the ability to brown WAT and improve metabolic health in humans, it represents a potentially attractive therapeutic agent for treating obesity and metabolic disease in humans. Here we will briefly discuss the browning of WAT, the discovery of irisin, and the potential role that irisin may play in browning WAT and improving metabolic health in humans. ## Browning of white adipocytes Classically, adipose tissue is characterized as either WAT or brown adipose tissue (BAT). Adipocytes from WAT serve as the primary site for lipid storage; whereas adipocytes from BAT are highly specialized cells designed to produce heat through uncoupled respiration that leads to concomitant dissipation of energy [bib_ref] Beige adipocytes are a distinct type of thermogenic fat cell in mouse..., Wu [/bib_ref]. The physical, metabolic, and regulatory characteristics of WAT and BAT have been extensively reviewed elsewhere [bib_ref] White, brown, beige/brite: different adipose cells for different functions?, Giralt [/bib_ref] [bib_ref] Brown adipose tissue: function and physiological significance, Cannon [/bib_ref] [bib_ref] The adipose organ at a glance, Cinti [/bib_ref] [bib_ref] Developmental origin of fat: tracking obesity to its source, Gesta [/bib_ref] [bib_ref] Banting Lecture 2012: regulation of adipogenesis: toward new therapeutics for metabolic disease, Spiegelman [/bib_ref]. In brief, adipocytes from WAT have a unilocular lipid droplet, few mitochondria, and a relatively low metabolic rate [bib_ref] Banting Lecture 2012: regulation of adipogenesis: toward new therapeutics for metabolic disease, Spiegelman [/bib_ref]. In contrast, adipocytes from BAT have multilocular lipid droplets, many mitochondria, and a relatively high metabolic rate [bib_ref] Banting Lecture 2012: regulation of adipogenesis: toward new therapeutics for metabolic disease, Spiegelman [/bib_ref]. The relatively high metabolic rate observed in BAT compared to WAT is due to the presence of uncoupling protein 1 (UCP1), which is negligibly expressed in WAT [bib_ref] Banting Lecture 2012: regulation of adipogenesis: toward new therapeutics for metabolic disease, Spiegelman [/bib_ref]. The presence of brown adipocytes in WAT has been known for many years. Young et al. [bib_ref] Brown adipose tissue in the parametrial fat pad of the mouse, Young [/bib_ref] were the first to report the presence of brown adipocytes in WAT of female BALB/c mice following cold acclimatization. Subsequently, brown adipocytes were identified in multiple WAT fat pads in rats [bib_ref] Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological..., Cousin [/bib_ref]. Enrichment and activation of BAT represents an attractive therapeutic strategy to combat obesity and metabolic disease. The presence of UCP1 positive cells in WAT can also be pharmacologically enriched by β-adrenergic stimuli [bib_ref] Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological..., Cousin [/bib_ref] [bib_ref] PRDM16 controls a brown fat/skeletal muscle switch, Seale [/bib_ref] [bib_ref] Emergence of brown adipocytes in white fat in mice is under genetic..., Guerra [/bib_ref] as well as PPARγ agonist [bib_ref] Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte..., Petrovic [/bib_ref] [bib_ref] A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent..., Fukui [/bib_ref] [bib_ref] Peroxisome proliferator-activated receptor gamma agonism increases the capacity for sympathetically mediated thermogenesis..., Sell [/bib_ref]. Recent evidence has demonstrated that the brown adipocytes (i.e., UCP1 positive cells) found in WAT are actually a distinct sub-population of white adipocytes (referred to as brown-in-white (brite) or beige adipocytes) [bib_ref] Beige adipocytes are a distinct type of thermogenic fat cell in mouse..., Wu [/bib_ref] [bib_ref] Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte..., Petrovic [/bib_ref]. Co-culture experiments demonstrated that beige/brite adipocytes treated with rosiglitazone (a PPARγ agonist) can be induced to differentiate into adipocytes with thermogenic potential in the absence of classical brown adipocyte-specific markers (e.g., Zic2, Lhx8, Meox3, and PRDM16) [bib_ref] Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte..., Petrovic [/bib_ref]. In addition, these beige/brite adipocytes are also characterized as having the white adipocyte-specific marker Hoxc9 while lacking the white adipocyte-specific marker tcf21 [bib_ref] Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte..., Petrovic [/bib_ref]. Taken together, these results indicate that the beige/brite adipocytes are truly a distinct subtype of white adipocytes with potentially hidden capacity for higher metabolic rate [bib_ref] Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte..., Petrovic [/bib_ref]. In 2012, Wu et al. [bib_ref] Beige adipocytes are a distinct type of thermogenic fat cell in mouse..., Wu [/bib_ref] confirmed that the beige/brite adipocytes found in WAT depots in mice and humans are in fact distinct from classical brown adipocytes that are more abundant in mice as a specialized depot [bib_ref] Brown and beige fat: development, function and therapeutic potential, Harms [/bib_ref]. Specifically, for the first time they demonstrated that the beige/brite adipocytes emerge from non-myf-5 progenitor cells, in contrast to brown adipocytes, which are derived from myf-5 positive progenitor cells [bib_ref] Beige adipocytes are a distinct type of thermogenic fat cell in mouse..., Wu [/bib_ref]. The exquisite regulation of the "browning" of white adipocytes in response to environmental, hormonal, and metabolic stimuli is quite remarkable. Excellent reviews of the development and regulatory control of beige/brite adipocytes have recently been published [bib_ref] Brown and beige fat: development, function and therapeutic potential, Harms [/bib_ref]. Similar to BAT, enrichment and activation of beige/brite adipocytes represents an attractive therapeutic strategy to combat obesity and metabolic disease. The recent discovery of irisin and its potential to induce "browning" of white adipocytes has gained much attention over the last two years , which will be discussed in detail below. ## Discovery of irisin It is well accepted that exercise is the corner stone of a healthy lifestyle and the frontline defense for primary and secondary prevention of many metabolic and cardiovascular diseases. Chronic endurance training has also been shown to induce skeletal muscle mitochondrial biogenesis [bib_ref] Endurance exercise as a countermeasure for aging, Lanza [/bib_ref] [bib_ref] Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator..., Baar [/bib_ref] , which is regulated by the expression and activity of PPAR-γ coactivator-1α PGC1-α [bib_ref] Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator..., Baar [/bib_ref] [bib_ref] Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism, Handschin [/bib_ref] [bib_ref] Transcriptional regulatory circuits controlling mitochondrial biogenesis and function, Kelly [/bib_ref]. Indeed, the canonical PGC1-α (i.e., PGC1-α1 [bib_ref] A PGC-1alpha isoform induced by resistance training regulates skeletal muscle hypertrophy, Ruas [/bib_ref] has been shown to be the master regulator of mitochondrial biogenesis in multiple tissues [bib_ref] Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism, Handschin [/bib_ref] [bib_ref] Transcriptional regulatory circuits controlling mitochondrial biogenesis and function, Kelly [/bib_ref]. Likewise, skeletal muscle over-expression of PGC1-α1 mimics many of the protective effects of exercise on multiple tissues [bib_ref] Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging, Wenz [/bib_ref] [bib_ref] Elevated PGC-1alpha activity sustains mitochondrial biogenesis and muscle function without extending survival..., Cruz [/bib_ref]. Interestingly, the newly identified splice variant of PGC1-α, PGC1-α4, has also been shown to induce skeletal muscle hypertrophy when transgenically overexpressed in mice consistent with a resistance-trained phenotype [bib_ref] A PGC-1alpha isoform induced by resistance training regulates skeletal muscle hypertrophy, Ruas [/bib_ref]. As such, efforts were made to identify the mechanism by which the expression and activity of PGC1-α in skeletal muscle affects other tissues. These studies were conducted under the backdrop of recent interest in skeletal muscle secreted signaling peptides referred to as myokines [bib_ref] Muscle as an endocrine organ: focus on muscle-derived interleukin-6, Pedersen [/bib_ref]. Could the increased expression of PGC1-α in skeletal muscle result in the secretion of some known or unknown set of myokines that affect the function of other tissues? Bostrom et al. first examined the WAT depots of muscle specific PGC1-α transgenic mice to determine if there were molecular and/or metabolic differences in WAT compared to their wild-type littermates. They demonstrated that the inguinal WAT (subcutaneous WAT) consisted of a population of beige/brite adipocytes that had increased levels of UCP1 and Cidea in muscle specific PGC1-α transgenic mice compared to their wild-type littermates. Next, they demonstrated that wheel running and swimming substantially induced the expression of UCP1 in inguinal WAT (~25 fold, 65-fold, respectively) . Taken together, these findings indicate that increased expression of PGC1-α in murine skeletal muscle induces browning of inguinal WAT, similar to what is observed through traditional exercise regimens. To address whether this browning of the subcutaneous WAT was due directly to muscle-fat cell signaling (e.g., myokine stimulated browning of WAT) they next cultured primary murine subcutaneous adipocytes with conditioned media from PGC1-α over expressing murine myocytes. Again, they found that the conditioned media treated murine subcutaneous adipocytes had increased expression brown-fat-specific genes (e.g., UCP1 and Cidea) [bib_ref] Muscle as an endocrine organ: focus on muscle-derived interleukin-6, Pedersen [/bib_ref]. These findings suggest that PGC1-α overexpressing murine myocytes secrete one or more myokines capable of inducing a thermogenic program in murine subcutaneous adipocytes. Bostrom et al. - ] used a combination of gene array technology and advanced bioinformatic algorithms to predict potential proteins that could be secreted by skeletal muscle and induce browning of WAT. Fibronectin type III domaincontaining 5 (FNDC5) was one of five target genes of PGC1-α which could be secreted . Importantly, FNDC5 expression was increased in muscle from exercise trained mice as well as humans . Next, they demonstrated that the treatment of primary subcutaneous adipocytes during differentiation with recombinant-FNDC5 increased the expression of BAT genes (UCP1, Elovl3, Cox7a, and Otop1) Before the protein product of FNDC5 was termed "irisin" [14- - ], it had already been discovered by two independent groups and assigned the gene aliases "PeP" [bib_ref] Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development, Ferrer-Martinez [/bib_ref] and "frcp2" [bib_ref] Frcp1 and Frcp2, two novel fibronectin type III repeat containing genes, Teufel [/bib_ref]. PeP and frcp2 were both found to be expressed in skeletal muscle, heart, and brain of adult mice [bib_ref] Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development, Ferrer-Martinez [/bib_ref] [bib_ref] Frcp1 and Frcp2, two novel fibronectin type III repeat containing genes, Teufel [/bib_ref]. It should be noted, however, that PeP and frcp2 expression in adipose tissue was not assessed [bib_ref] Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development, Ferrer-Martinez [/bib_ref] [bib_ref] Frcp1 and Frcp2, two novel fibronectin type III repeat containing genes, Teufel [/bib_ref]. Bostrom et al. were the first to recognize and suggest that although the fulllength FNDC5 is a trans-membrane protein, its extracellular N-terminal portion of FNDC5 could potentially be cleaved by a yet to be identified protease. Identification of the FNDC5 fragment was initially determined by antibody binding, which was confirmed by mass spectrometry . Their analysis also revealed that the secreted form of FNDC5 was highly homologous between mouse and humans . They named this newly identified signaling peptide (myokine) irisin after the messenger goddess of ancient Hellenic mythology, Iris . Another potential mechanism that has been suggested for observing N-terminal (extracellular) fragments of FNDC5 (e.g., irisin) in cell culture media and/or plasma in vivo is through shedding of the extracellular fraction . Using an antibody against FNDC5 Bostrom et al. - ] were able to reduce the browning effect of media conditioned by skeletal muscle PGC1-α overexpressing murine myocytes on primary subcutaneous fat. Of note, the antibody used in these experiments was targeted at the c-terminus of FNDC5, which should not be present in irisin. They also indicated that irisin was present in both mouse and human plasma . Moreover, they demonstrate that plasma irisin concentrations were elevated in mice and older humans after short-term exercise training . Using adenoviral delivery of FNDC5 to the liver they were also able to increase plasma concentration of irisin which led to the browning of subcutaneous WAT while protecting against diet induced obesity and insulinresistance . The increased circulating levels of irisin were also associated with increased expression of mitochondrial genes in the subcutaneous WAT and a concomitant increase in oxygen consumption. Taken together these data indicate that irisin, which is secreted from active (murine) skeletal muscle has the potential to protect against obesity and insulin resistance. However, it remains to be demonstrated that irisin is secreted from human skeletal muscle, and if not so to determine whether the potential action of FNDC5 and/or irisin is derived from non-skeletal muscle tissue. ## Does irisin affect human white adipocytes Several manuscripts have demonstrated that irisin enhances the "browning" of white adipocytes in mice , particularly in white adipocytes that highly express CD137 [bib_ref] Beige adipocytes are a distinct type of thermogenic fat cell in mouse..., Wu [/bib_ref]. However, recent evidence has begun to question the physiological relevance of irisin in humans . Can the results obtained in mice be readily translated to humans? Specifically, is irisin synthesized and secreted from active human skeletal muscle? Is skeletal muscle the primary source of irisin and/or FNDC5? Can irisin induce browning of white adipocytes in humans? As previously discussed, Bostrom et al. reported that 10 weeks of combined endurance plus resistance exercise training increases circulating levels of irisin in older adults approximately two-fold, which was proportional to the increase in skeletal muscle mRNA expression of FNDC5. However, Timmons et al. demonstrated that neither endurance training nor resistance training increases skeletal muscle FNDC5 mRNA expression in healthy adults. Nevertheless, they did report that FNDC5 expression was elevated in a subset of exercise trained older adults but not younger adults compared to their sedentary counterparts, which is an interesting finding . Raschke et al. also failed to demonstrate an increase in FNDC5 expression using an in vitro model of endurance training (electrical pulse stimulation) in human myotubes as well as in response to aerobic interval training and strength training in sedentary males. Results of a recently published randomized clinical trial of (n=102) middle aged (30-60 years old) participants demonstrated that neither endurance training nor resistance training increase circulating irisin concentrations after 26 weeks of training compared to controls ]. An important observation from this study was that irisin is prone to storage-related degradation ]. Therefore, time related changes in circulating irisin concentrations in the absence of timed-matched controls should be interpreted with caution. In another study, it was demonstrated that an acute bout of endurance exercise, chronic endurance exercise, and chronic endurance combined with resistance exercise provide conflicting results with respect to skeletal muscle PGC1-α expression, FNDC5 expression, and circulating irisin . Twelve weeks of exercise training has also been reported to have little to no effect on genes expression in subcutaneous WAT for genes associated with browning of WAT (e.g., UCP1, PRDM16, TBX1, TMEM26, or CD137), despite significant increases in skeletal muscle FNDC5 . The human gene sequence of FNDC5 is also raising concerns regarding the translated product [bib_ref] Identification of evolutionarily conserved non-AUG-initiated Nterminal extensions in human coding sequences, Ivanov [/bib_ref]. Although the FNDC5 gene sequence is highly conserved across species , the human FNDC5 gene has a variation in its start codon that could essentially affect its translation [bib_ref] Identification of evolutionarily conserved non-AUG-initiated Nterminal extensions in human coding sequences, Ivanov [/bib_ref]. Specifically, the translational initiation "ATG" codon is mutated to "ATA" resulting in substantial reduction in the translational efficiency of FNDC5 and leading to the translation of only 1 % of full-length FNDC5 as reported . Furthermore, there is a truncated isoform of FNDC5 that is translated by a downstream "ATG" start codon that is lacking the signaling peptide . Consistent with this later finding, on September 5th 2012, the UniProt database was annotated to include a second, truncated, protein sequence for FNDC5 . Raschke et al. further demonstrated that neither recombinant-irisin nor recombinant-FNDC5 induces "browning" of human pre-adipocytes. In contrast, they demonstrated that BMP7 (i.e., the positive control) resulted in an activation of the genes regulating the "browning" of human preadipocytes characterized by elevations in PPARγ, UCP1, PGC-1β, as well as an elevation in mitochondrial protein content . Taken together, these findings indicate that the ability of endogenous irisin to stimulate "browning" of white adipocytes in humans remains to be proven. Surprisingly, little information is known about the expression and potential secretion of FNDC5/irisin from adipose tissue itself. A few recent papers, however, highlight the presence of FNDC5 in rat and human WAT . Using gene-array technology our research team recently demonstrated that FNDC5 is highly expressed in visceral adipose tissue, epigastric adipose tissue, and to a lesser extent subcutaneous adipose tissue of severely obese patients undergoing bariatric surgery . Likewise, PGC1-α was also abundantly expressed in these adipose tissue depots . Taken together, it appears that human WAT has some of the key components necessary for FNDC5-induced browning WAT in an autocrine fashion in humans. In another study, it was demonstrated by real-time PCR that FNDC5 gene expression was reduced in obese as well as patients with type 2 diabetes [bib_ref] Irisin is expressed and produced by human muscle and adipose tissue in..., Moreno-Navarrete [/bib_ref]. Moreover, FNDC5 gene expression in visceral and subcutaneous WAT was positively associated with brown adipose tissue markers (PRDM16 and UCP1) in humans [bib_ref] Irisin is expressed and produced by human muscle and adipose tissue in..., Moreno-Navarrete [/bib_ref]. Future investigations are warranted to examine the potential role of FNDC5 to induce browning of WAT in humans via autocrine mechanisms. However, it has been reported that the expression of FNDC5 in WAT is less than five percent of that observed in skeletal muscle in humans [bib_ref] Irisin is expressed and produced by human muscle and adipose tissue in..., Moreno-Navarrete [/bib_ref] [bib_ref] FNDC5 and irisin in humans: I. Predictors of circulating concentrations in serum..., Huh [/bib_ref]. Roca-Rivada et al. [bib_ref] FNDC5/irisin is not only a myokine but also an adipokine, Roca-Rivada [/bib_ref] recently reported that WAT explants secrete FNDC5/irisin, which is increased after 1 week of exercise in rats [bib_ref] FNDC5/irisin is not only a myokine but also an adipokine, Roca-Rivada [/bib_ref]. Interestingly, however, the secretion of FNDC5/ irisin from the WAT explants was reduced after 3 weeks of exercise training [bib_ref] FNDC5/irisin is not only a myokine but also an adipokine, Roca-Rivada [/bib_ref]. The secretion of FNDC5/irisin was based on quantification of the 25 kDa band (predicted MW of irisin~12 kDa), which was detected both by the Abcam and the Phoenix antibodies [bib_ref] FNDC5/irisin is not only a myokine but also an adipokine, Roca-Rivada [/bib_ref]. Moreover, the Abcam FNDC5 antibody recognizes the C-terminus of FNDC5 that is not supposed to be part of irisin . It should also be noted, the Abcam and the Phoenix antibodies do not share any sequence overlap . In summary, it appears that human WAT has some of the key components necessary for FNDC5 to act in an autocrine fashion to brown WAT in humans. However, future studies are needed using validated antibodies to determine whether human WAT secretes full-length FNDC5 and/or irisin. Likewise, experimental evidence demonstrating the ability of FNDC5/irisin to brown human WAT resulting in increased UCP1 expression and thermogenesis regardless of its source is currently lacking. # Conclusion The recent discovery of irisin has garnered much attention as a potential therapeutic agent for the treatment of obesity and its comorbid conditions. [fig_ref] Figure 1: Putative effects of FNDC5/irisin on browning of white adipocytes and improvements in... [/fig_ref] presents the putative effects of irisin on the browning of murine and human white adipocytes. Preliminary studies have indicated that recombinant FNDC5 and/or irisin can induce browning of murine WAT. However, there remains a dearth of evidence to indicate that recombinant FNDC5 and/or irisin can induce browning of human WAT. Moreover, recent studies in humans also indicate that neither acute nor chronic exercise consistently results in increased expression of endogenous FNDC5 and/or increased circulating concentrations of FNDC5/irisin in humans. These later findings may be due in part to the lack of available antibodies that are validated to detect FNDC5/irisin. In addition, care should be taken when extrapolating data derived from mouse studies to human physiology given the significant differences in the abundance of brown fat between humans and mice. Finally, the expression of FNDC5 in human WAT opens the door to the possibility that FNDC5 acts in an autocrine fashion to brown WAT in humans. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. [fig] Figure 1: Putative effects of FNDC5/irisin on browning of white adipocytes and improvements in energy expenditure, obesity, and metabolic health in mice and humans [/fig]

A Bioartificial Renal Tubule Device Embedding Human Renal Stem/Progenitor Cells We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs) was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na + K + ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (2065)% and (1365)%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative ''lab-on-a-chip'' platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses. # Introduction The human body is a heterogeneous and perfectly synchronized system, composed of different organs that are in turn made up of several, small and functionally autonomous units, called microorganoids, such as lobuli in the liver, nephrons in the kidney and alveoli in the lung. The behavior of a single micro-organoid is considered representative of the whole organ functionality [bib_ref] Design and prototyping of a chip-based multi-micro-organoid culture system for substance testing,..., Sonntag [/bib_ref]. For an in-depth understanding of human physiology and for promoting advances in medicine and toxicology, the availability of engineered platforms able to reproduce functional portions of living organs is challenging [bib_ref] Organs-on-chips: breaking the in vitro impasse, Van Der Meer [/bib_ref]. In this framework, a useful tool is offered by microfluidic techniques [bib_ref] Soft lithography in biology and biochemistry, Whitesides [/bib_ref] [bib_ref] Microfluidics: Fluid physics at the nanoliter scale, Squires [/bib_ref] [bib_ref] The origins and the future of microfluidics, Whitesides [/bib_ref] , namely of devices for cell culture that closely mimic physiological aspects of a well-organized biosystem at the same micro-scale as living cellular milieu [bib_ref] Development of microfluidic devices for biomedical and clinical application, Webster [/bib_ref] [bib_ref] Stem cells in microfluidics, Van Noort [/bib_ref] [bib_ref] Cell culture models in microfluidic systems, Meyvantsson [/bib_ref]. Differently from standard culture systems, microfluidic devices provide a tight control over flow conditions [bib_ref] The origins and the future of microfluidics, Whitesides [/bib_ref] [bib_ref] Microfluidic cell culture models for tissue engineering, Inamdar [/bib_ref] , and the distinctive possibility of maintaining constant fluid perfusion inside microchannels [bib_ref] Cell research with physically modified microfluidic channels: A review, Kim [/bib_ref] to induce a shear stress, which is advantageous for the functionality of many cells, including renal tubular cells [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] [bib_ref] Development of a renal microchip for in vitro distal tubule models, Baudoin [/bib_ref]. A recent advance enabled by the microfluidic approach consists in the fabrication of engineered ''organs-on-a-chip'' [bib_ref] Microengineered physiological biomimicry: organs-on-chips, Huh [/bib_ref] , re-creating in vitro microcompartments of blood vessels [bib_ref] Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane), Shin [/bib_ref] [bib_ref] Computercontrolled microcirculatory support system for endothelial cell culture and shearing, Song [/bib_ref] , liver [bib_ref] In vitro analysis of a hepatic device with intrinsic microvascular-based channels, Carraro [/bib_ref] [bib_ref] Microscale culture of human liver cells for drug development, Khetani [/bib_ref] , brain [bib_ref] Microfluidic culture platform for neuroscience research, Park [/bib_ref] , gut [bib_ref] Characterization of a gastrointestinal tract microscale cell culture analog used to predict..., Mahler [/bib_ref] and lung [bib_ref] Reconstituting organ-level lung functions on a chip, Huh [/bib_ref]. The aim of these studies is to reproduce the structural arrangements and biological functions of micro-organoids. A critical issue, in this context, is the cell source to be used in designing and developing organs-on-chip. Immortalized cell lines are very common and well characterized, but they show considerable phenotypic and genetic divergences if compared with in vivo human cells. Primary cell lines do not present this inconvenience but are scarcely available and difficult to culture over a long period of time. The use of adult stem cells extracted from patients would overcome these difficulties. In the kidney, resident adult renal stem/progenitor cells (ARPCs) have been identified [bib_ref] Stem-cell approaches for kidney repair: choosing the right cells, Sagrinati [/bib_ref] [bib_ref] Stem cells for kidney repair: useful tool for acute renal failure?, Yokoo [/bib_ref] , raising a lot of interest due to their potential therapeutic applications [bib_ref] Stem cell approaches for the treatment of renal failure, Brodie [/bib_ref] [bib_ref] Isolation of Renal Progenitor Cells from Adult Human Kidney, Bussolati [/bib_ref] [bib_ref] Isolation of renal progenitor cells from adult human kidney, Sagrinati [/bib_ref]. These cells, isolated both from the tubule interstitium [bib_ref] Isolation of Renal Progenitor Cells from Adult Human Kidney, Bussolati [/bib_ref] and Bowman's capsule [bib_ref] Isolation of renal progenitor cells from adult human kidney, Sagrinati [/bib_ref] , showed multipotent differentiation properties, by generating tubular epithelial-like, osteogenic-like, adipocyte-like and neuronal-like cells [bib_ref] Stem-cell approaches for kidney repair: choosing the right cells, Sagrinati [/bib_ref] [bib_ref] Stem cells for kidney repair: useful tool for acute renal failure?, Yokoo [/bib_ref] [bib_ref] TLR2 plays a role in the activation of human resident renal stem/progenitor..., Sallustio [/bib_ref]. In vitro, they can differentiate into epithelial cells expressing some markers of renal proximal and distal epithelium. In vivo, these cells can express some markers of both distal tubules such as the NaCl co-transporter and calbindin-D of proximal tubules [bib_ref] Isolation of renal progenitor cells from adult human kidney, Sagrinati [/bib_ref]. After the injection into mice with acute kidney injury, ARPCs showed an exceptional regenerating capacity for injured renal tubular cells and a marked resistance to apoptotic events [bib_ref] Isolation of Renal Progenitor Cells from Adult Human Kidney, Bussolati [/bib_ref] [bib_ref] Characterization of renal progenitors committed toward tubular lineage and their regenerative potential..., Angelotti [/bib_ref] [bib_ref] Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A..., Sallustio [/bib_ref]. For these peculiar abilities, ARPCs may be considered the future direction of renal regenerative medicine and a promising tool to treat acute and chronic renal injury [bib_ref] Characterization of renal progenitors committed toward tubular lineage and their regenerative potential..., Angelotti [/bib_ref] [bib_ref] Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A..., Sallustio [/bib_ref] [bib_ref] Contribution of stem cells to kidney repair, Bussolati [/bib_ref] [bib_ref] Regenerative potential of embryonic renal multipotent progenitors in acute renal failure, Lazzeri [/bib_ref]. Here we present a bioartificial proximal tubule-like structure based on a multi-layer microdevice embedding ARPCs. The device was composed of two overlapped elastomeric layers, sandwiching a porous polycarbonate (PC) membrane. The geometry was purposely designed to mimic the in vivo structure of a renal tubule, with the upper microchannel providing the lumen area, in which the apical portion of cells was exposed, and the lower microchannel simulating the interstitial area in contact with the basolateral membranes of cells. According to the design of a bioartificial renal tubule [bib_ref] Tissue engineering of a bioartificial renal tubule assist device: in vitro transport..., Humes [/bib_ref] , living cells were seeded on the polymeric membrane, which was water and solute permeable, to ensure the transport of solutes across it, and acted as scaffold for cell growth [bib_ref] Bioartificial kidney in the treatment of acute renal failure associated with sepsis, Tiranathanagul [/bib_ref]. Biochemical and physical parameters were optimized and used to promote the on-chip confluent growth of ARPCs, which were then exposed to physiological laminar fluid shear stress (FSS) and characterized for their recovery of urea and creatinine, analyzing the fluid outlets collected from the device. The induction of cell polarity in ARPCs was well characterized with apical and basolateral marker proteins, thus demonstrating that the feasibility of renal tubules-on-chip may open new perspectives also in view of the parallelization and adjuvant therapy for renal failure. . FITCconjugated anti-CD44 was from Instrumentation Laboratory (Milan, Italy). Rabbit anti-human Pax-2 pAb was from Covance (Princeton, NJ), mouse anti-human CD105 mAb and rabbit antihuman Oct-4 pAb were from Abcam (Cambridge, UK), mouse anti-human CD24 mAb was from Dako (Glostrup, Denmark), mouse anti-human CD44 mAb was from Chemicon (Temecula, CA), mouse anti-human Bmi1 mAb was from Upstate Biotechnology (Lake Placid, NY). The secondary antibodies Alexa Fluor 555 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG1 and the dye To-pro-3 were from Molecular Probes (Eugene, OR). The antibodies Na + /K + ATPase, aquaporin-2 (AQP2) and Zonula occludens 1 (ZO-1) were provided by Santa Cruz Biotechnology (Santa Cruz, CA). The Gel/Mount for immunofluorescent characterization was from Biomeda (Milan, Italy). Cells from human renal proximal tubule epithelial cell line (human kidney-2, HK-2) [bib_ref] HK-2: an immortalized proximal tubule epithelial cell line from normal adult human..., Ryan [/bib_ref] were provided by the CARSO consortium bio-bank chaired by Prof. F.P. Schena. Renal Proximal Tubule Epithelial Cells (RPTECs) were purchased from Lonza (Lonza, Basel, Switzerland). The creatinine and urea dosage kits were from SGM Italia (Rome, Italy). # Materials and methods # Materials ## Device fabrication and characterization The device core was a porous PC membrane sandwiched between two PDMS layers, each having an engraved microchannel with a width of 500 mm and a height of 120 mm. The master structures used for PDMS replicas were fabricated by photolithography (UV exposure for 10 s at 500 W) on SU-8 2100 photoresist. The resist was spin-cast (3000 rpm, 30 s) on Si/SiO 2 substrate and developed for 8 min after the bake processes (65uC for 5 min and 95uC for 30 min). The elastomeric layers were realized by replica molding through in situ polymerization (75uC for 15 min) of PDMS (10:1 w/w base: curing agent) on the master, peeled off and punched in correspondence to the inlet and outlet chambers of the microchannels. In lieu of performing oxygen plasma treatments [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] , the PC membrane was embedded between the two PDMS structured layers by introducing a thin mortar layer [bib_ref] Leakage-free bonding of porous membranes into layered microfluidic array systems, Chueh [/bib_ref] that was prepared by spin-coating a mixture of PDMS and toluene (40% w/v) on a clean glass cover slide (1500 rpm for 1 min). Then, the two PDMS elements were placed onto the glass slide coated with the adhesive mortar layer and allowed to stay in contact for 30 s. Finally, the PC membrane was placed onto one of the PDMS layers and pressed down into the thin adhesive film. This PDMS piece with the membrane attached was then aligned under a stereomicroscope and bonded with the second PDMS layer through overnight thermal curing at 35uC, which avoided shrinkage of the membrane. The membrane-integrating device was connected for fluid injection and outlet collection through plastic tubes that were fitted into the inlet and outlet ports. The extremities of the tubes were coupled to the stainless steel needles of 2.5 mL syringes (inlets), connected to an infusion pump (Harvard Apparatus, Holliston, MA) and to eppendorf tubes (outlets), respectively. Traps were inserted in the microfluidic circuit between needles and inlets to prevent air bubble formation in the perfusion cell culture. The bubble traps were constructed by bonding a flat and a textured layer of PDMS with oxygen plasma (50 W for 15 s). The structured element was fabricated by replica molding (75uC, 15 min) starting with an SU-8 master made up of two continuous straight channels (width 500 mm, height 120 mm), inlet and outlet, connected by a circular ridge (radius 4 mm, height 120 mm) on the top of which was placed a metal spacer (radius 4 mm, height 1 cm). For producing the flat elastomeric layer, the PDMS was spin-cast (1500 rpm for 30 min) on a glass substrate and cured (75uC, 15 min). When an air bubble approached the circular trapping chamber, it was captured and ruptured upon reaching the liquid-air interface. To analyze the device functionality in terms of diffusion of molecules through the porous membrane, preliminary diffusion tests were carried out by fluxing, in counter-current mode, a solution of rhodamine 6G (1 mg mL 21 ) and distilled water into the two microchannels, respectively, at a flow rate of 1 mL min 21 . Fluorescence variations were recorded with a microscope (Leica MZ16FA) equipped with a fluorescent lamp (100 W) and a video camera (Leica DFC490), and micrographs were analyzed by the ImageJ software. As control, rhodamine diffusion tests were also performed on a standard system, composed by the PC membrane placed in a CellCrown TM Cell Culture Insert and positioned in a 12-well polystyrene plate. The lower compartment was filled with distilled water and the upper compartment with rhodamine 6G solution (1 mg mL 21 ). The fluorescence variation in the lower compartment was recorded with an inverted microscope Eclipse Ti equipped by Nikon confocal A1 R MP system. 400 frames per second were analyzed by the Nikon NIS Element software. ## Hk-2 and rptec cell culture First, the device parameters were optimized to obtain an onchip confluent monolayer of human kidney HK-2 cells [bib_ref] HK-2: an immortalized proximal tubule epithelial cell line from normal adult human..., Ryan [/bib_ref] , used as the model cell type for renal tubule cells. To this aim, HK-2 cells were cultured in the presence of the DMEM/F12 medium supplemented with 10% FBS and 1% penicilline-streptomycine. RPTECs were cultured in the recommended medium REGM. Both cell lines were sub-cultured at least once a week and maintained at 37uC under a humidified atmosphere, constituted by 95% air and 5% CO 2 . ## Arpc isolation and cell culture ARPCs were obtained from fresh human renal cortical tissues harvested from patients with localized renal cell carcinoma that underwent nephrectomy, according to standard clinical protocols. At the time of radical nephrectomy, all patients gave signed consent for the use of part of removed tissues for research purposes. Portions of normal-appearing cortex were isolated surgically and histologically examined to exclude the presence of carcinoma. The CD133-positive ARPCs were isolated and characterized as previously described [bib_ref] TLR2 plays a role in the activation of human resident renal stem/progenitor..., Sallustio [/bib_ref] [bib_ref] Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A..., Sallustio [/bib_ref] [bib_ref] AQP5 is expressed in type-B intercalated cells in the collecting duct system..., Procino [/bib_ref] [bib_ref] BMP-2 induces a profibrotic phenotype in adult renal progenitor cells through Nox4..., Simone [/bib_ref]. Briefly, the cortex renal fractions were dissected by passage through a graded series of steel meshes sieves to remove the fibrous component. The cellular fraction was then passed through a 120-mesh sieve to isolate the capsulated glomeruli from the tubular fraction. After several washes, the two isolated fractions were cultured separately in the EGM-MV medium supplemented with 20% FBS. After 4-5 days, the cultures were washed twice with PBS buffer to remove non-adherent cells and after about 1 week in culture cell viability and number were checked. The CD133-positive cells were then isolated by magnetic cell separation technology (MACS) by means of CD133Ab-conjugated magnetic microbeads. The eluted cells were resuspended and maintained in the EGM-MV medium supplemented with 20% FBS and incubated at 37uC with 5.0% CO 2 . The ARPCs markers were checked by cytofluorimetric determination and by cell immunofluorescence microscopy. Cytofluori-metric assays were performed using a Partec Flow-Max cytofluorimeter (Munster, Germany) and the following primary human antibodies: anti-CD133/2 (293C3), anti-CD34, anti-CD45, anti-CD105, anti-CD24 and anti-CD44. FITC-conjugated mouse IgG1 was used as an isotype control. Non-specific sites were blocked with the FcR blocking reagent. Each determination was performed on 10 5 cells. For immunofluorescence experiments, ARPCs were fixed in 4% paraformaldehyde. The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with monoclonal or polyclonal primary antibodies overnight at 4uC or for 2 h at room temperature, respectively. The following primary human antibodies were used: anti-CD133/1 mAb, anti-CD133/2 mAb, anti-Pax-2 pAb, anti-CD105 mAb, anti-CD24 mAb, anti-CD44 mAb, anti-Bmi1 mAb, anti-Oct-4 pAb and ZO-1. The immune complexes were identified after the incubation of the cells with the specific secondary antibodies for 1 h at room temperature. The cells were washed in PBS after each step, counterstained with To-pro-3, mounted in Gel/Mount, and sealed with nail varnish. The stained cells were viewed under the Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope. ## Membrane surface treatment Before seeding cells in the device, extensive tests were carried out to choose the most suitable extracellular matrix (ECM) protein for the biofunctionalization of the PC membrane. Three different surface treatments were analyzed: fibronectin (10 mg/mL in PBS), laminin (10 mg/mL in PBS) and Matrigel (a dilution 1:5 in complete DMEM/F12 culture medium). The PC membranes, placed in the 6-well polystyrene plates, were covered with a volume of 0.5 mL of each protein solution, and incubated at 4uC for 2 h. Confluent HK-2 cells in the flask were then washed with PBS, removed with a trypsin/EDTA solution and seeded on functionalized membranes at a concentration of 1610 5 cells mL [bib_ref] Stem-cell approaches for kidney repair: choosing the right cells, Sagrinati [/bib_ref]. As controls, cells were also seeded on untreated membranes and polystyrene wells. Cell viability on all tested substrates was evaluated after 2 days by MTS assay, by exploiting the conversion of tetrazolium salt to soluble formazan dye due to metabolically active cells. Experiments on each set of samples were repeated three times. The apparent static water contact angle (WCA, h) of the bare and treated membrane was also investigated, by means of a KSVCAM200 instrument (KSV, Finland). Distilled water droplets with a typical volume of about 2 mL were dispensed onto the surfaces by a syringe, connected to the contact angle measuring system. ## Cell seeding Before each experiment, the chip and all the fluidic connectors were sterilized by UV germicidal irradiation (8 W lamp, G30T8, Sankyo Denki) in a laminar flow hood for 1 h. For cell seeding, the same procedure was carried out for HK-2, RPTECs and ARPCs. Briefly, confluent cells in the flask were washed with HBSS, removed with a trypsin/EDTA solution and seeded in the fibronectin-coated device at typical concentrations of 0.5 and 1.5610 6 cells mL 21 . After 24 h, non-attached or dead cells were removed by rinsing in the cell culture medium. For static culture devices, both HK-2 and ARPCs were grown to confluence on fibronectin-coated membranes over 4 days after seeding in a CO 2 incubator by entirely changing the culture medium every 8 h. In this way the supply of the fresh cell medium and the removal of the metabolic waste were both ensured. In order to investigate the ARPC morphology in macroscopic cultures, cells were seeded on standard systems composed by the porous PC membrane placed in a cell culture insert. In detail, after coating the surface of the membrane with fibronectin (10 mg/mL in PBS), a concentration of 1.5610 6 cells mL 21 was transferred onto the membrane. Also in these experiments, cells were grown over 4 days after seeding in a CO 2 incubator by changing the culture medium every 8 h. As negative controls, microfluidic devices without cells were stored in a CO 2 incubator under identical conditions and undergoing the same medium changes. Experiments on each set of samples were repeated three times, on different devices. ## Flow tests Once cell confluence onto the membrane surface was reached, the lumen microchannel was perfused with the complete culture medium supplemented with urea (25 mg dL 21 ), creatinine (1 mg dL 21 ) and glucose (0.01 mg dL 21 ); the interstitial microchannel was instead fluxed with the complete culture medium without urea and creatinine and glucose in counter-current with respect to the lumen flow. Cells were exposed to a volumetric flux of 1 mL min 21 for 6 h at 37uC. Outlet samples from both the lumen and interstitial microchannels were taken and the recovery of urea, creatinine and glucose across the porous membrane colonized by cells was evaluated, following well-established colorimetric methods. Microchips without cells were analyzed as negative controls. Experiments on each set of samples were repeated three times on different devices. ## On-chip immunofluorescence assays After completing flow measurements, immunocytochemistry experiments were performed in order to assess the morphology and growth of renal tubule cells within the device. Staining was carried out directly in the chip, rinsing with PBS, fixing cells grown on the PC membrane by a solution of 4% paraformaldehyde in PBS for 20 min, and washing 3 times with PBS for 5 min each. Cell membranes were permeabilized by incubation with 0.1% (v/ v) Triton-X100 in PBS for 10 min, followed by incubation in 1% BSA in PBS for 30 min to reduce nonspecific background staining. For investigating cell morphology, the device was incubated for 40 min with phalloidin-FITC/TRITC (25 mg/mL), washed with PBS and stained with DAPI (3 mg/mL) for 10 min. To visualize the markers AQP2, Na + K + ATPase, before phalloidin and DAPI staining, chips were incubated for 2 h in the primary antibody (anti-AQP2 1:50 in BSA; anti-Na + K + ATPase 1:50 in BSA), . The bio-inspired renal microdevice. The multi-layered chip, resembling the in vivo structure of a proximal kidney tubule, was composed by two overlapped PDMS layers (A), with engraved microchannels. The two microchannels simulated the lumen area in which the apical portion of cells is exposed (upper channel) and the interstitial area in contact with basolateral membranes (lower channel). The channels were physically separated by a microporous PC membrane and were continuously fed in counter-current modality. Once assembled (B), the device presented two inlet and two outlet ports, each connected to plastic tubes and syringe pumps for fluid control. washed with PBS and incubated with the secondary antibody (anti goat-FITC 1:100 for AQP2 staining; anti mouse-FITC 1:400 for Na + K + ATPase staining) for 1 h. Finally, the membrane, with cell attached, was taken apart from the device and visualized by inverted microscopy Eclipse Ti equipped by confocal A1 R MP system (Nikon, Melville, NY). Experiments were repeated three times on different devices. # Results ## Design and characterization of the microfluidic device In our bioartificial proximal tubule, shown in -C, both the top and the bottom layers were made of PDMS, chosen for its well-established oxygen permeability, ease of use and optical transparency [bib_ref] Microfluidic cell culture models for tissue engineering, Inamdar [/bib_ref] [bib_ref] Reconstituting organ-level lung functions on a chip, Huh [/bib_ref]. While PDMS has been used in many bioengineered microfluidic devices [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] , in our report each PDMS layer contained an engraved microchannel coupled to external flows. This architecture supported two different fluid streams, separated by a porous membrane colonized by cells. The PC membrane is highly compatible with proteins and cells, and exhibits an optimal ultrafiltration capacity and a very low risk of solute back diffusion [bib_ref] Complications of dialysis, Pereira [/bib_ref]. The inlet and outlet ports of the device were connected to plastic tubes and syringe pumps, for controlling the fluid passage in each microchannel independently . Finally, the device was connected with two bubble traps to prevent the entry of air bubbles and ensuring the viability of cells cultured in the microchip. In fact, when an air bubble approaches the cells, these can be stretched by forces exerted at the liquid-air interface, which may lead to the rupture of the cell membranes [bib_ref] Prevention of air bubble formation in a microfluidic perfusion cell culture system..., Sung [/bib_ref]. Firstly, the microfluidic device was characterized by dyediffusion through the PC membrane. Rhodamine is a good dye system in this respect for its bright fluorescence and because its hydrodynamic radius (7.7610 210 m) is comparable to those of solutes involved in kidney functions such as urea (1.8610 210 m) and creatinine (2.6610 210 m). Experiments were carried out by fluxing, in counter-current mode, a rhodamine 6G solution (1 mg mL 21 ) and distilled water into the two microchannels, respectively. The resulting temporal curve represented the mean of three independent experiments, carried out on different devices. As shown in [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref] , the fluorescence intensity grew with time at the outlet of the lower channel, corresponding to an increase of rhodamine 6G concentration. The diffusion process stabilized in a time, t S , of 100 s, necessary to bring the device to the stationary conditions to be cleared [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. The temporal behaviour of the increase of the fluorescence intensity was found to be basically the same as in a system where two compartments were separated by the same membrane used in the microfluidic device, and under static conditions of the liquids (superimposed line in [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. This indicated that diffusion was indeed the dominant mechanism leading molecules to pass through the pores, instead of leakages and of possible contributions from convection, which in principle could occur in counter-flow modality due to the presence of pressure differences in the microchannels on the two sides of the membranes [bib_ref] Size selective DNA transport through a nanoporous membrane in a PDMS microfluidic..., Sheng [/bib_ref]. In addition, the apparent diffusion coefficient across the membrane, D m > d 2 /t S , where d is the membrane thickness, was of 16 mm 2 /s lower than the diffusion coefficient of rhodamine in free water (414 mm 2 /s) [bib_ref] Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy, Müller [/bib_ref]. The corresponding membrane permeability or mass transfer coefficient, K = D m /d, was about 0.40 mm/s for the here used fluorescent compound (molecular weight of 480 Da), in good agreement with the value (0.34 mm/s) found for other rhodamine species (sulforhodamine, 607 Da) moving across a similar, 6 mm-thick PC membrane [bib_ref] Glucose recovery in a microfluidic microdialysis biochip, Hsieh [/bib_ref]. ## Functional renal biochip To choose the most suitable ECM protein for the functionalization of the PC membrane, the proliferation of HK-2 cells on PC membranes coated with either fibronectin, laminin or Matrigel was compared. Untreated membranes and polystyrene substrates were included as control [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. The number of metabolically active cells on fibronectin-coated membranes (, 10 5 cells) was about 1.4 and 1.6 times higher than that obtained by membranes functionalized with laminin and Matrigel, respectively. Untreated membranes and polystyrene substrates showed a proliferation rate about two times lower. In particular, we noticed that even the untreated membranes were a more suitable scaffold than standard polystyrene surfaces, due to the microporous structure that mimics the tridimensional microarchitecture of native ECM [bib_ref] Protein adsorption, attachment, growth and activity of primary rat osteoblasts on polylactide..., Gugala [/bib_ref]. The wetting properties of membranes were also investigated by contact angle measurements. The functionalization by fibronectin led to a decrease of the WCA by about 20u [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref] , thus enhancing surface wettability, which is known to favour cell adhesion, spreading and growth [bib_ref] Wettability of substrata controls cellsubstrate and cell-cell adhesions, Tzoneva [/bib_ref]. The on-chip investigation by immunostaining of the fibronectin coating finally demonstrated a roughly uniform distribution of the protein along the microchannel [fig_ref] Figure S1: Immunostaining of FN coating along the microchannel [/fig_ref]. Then we optimized the culture conditions on-chip by using HK-2 cells as model. An initial concentration of 1.5610 6 cells mL 21 was found to effectively lead to cell confluence onto the membrane [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. Furthermore, dynamic and static flow conditions were compared [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. In the first case, the system was continuously fed for 4 days at a constant flow rate of 1 mL min 21 after cell seeding. However, the flux might have interfered with the cell adhesion onto the membrane since a continuous monolayer of cell was not observed [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. On the other hand, the confluence was achieved [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref] when, after seeding, the cells grew under a static culture environment for about 24 h, followed by changes of the culture medium twice a day. Once the device architecture and working conditions were established, we realized the functional renal tubule biochip. After 4 days of cell culture, the lumen microchannel was perfused with the complete culture medium containing urea (25 mg dL 21 ) and creatinine (1 mg dL 21 ) and the same solution (but without urea and creatinine) was injected into the interstitial microchannel in counter-current mode. Flow rates were chosen to mimic physiological FSS to which renal cells are exposed, ranging between 0.2 culture medium containing urea (UR) and creatinine (CR) and the medium without UR and CR was injected in counter-current into the lower, interstitial microchannel. and 20 dyn cm 22 [bib_ref] The use of controlled surface topography and flow-induced shear stress to influence..., Frohlich [/bib_ref] [bib_ref] Fluid-shearstress-induced translocation of aquaporin-2 and reorganization of actin cytoskeleton in renal tubular..., Jang [/bib_ref]. The FSS produced at the microchannel walls can be estimated by t = 6 mQ/bh 2 , where m is the medium kinetic viscosity (dynes cm 22 ), Q is the volumetric flow rate, b is the channel width, and h is the channel height [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] [bib_ref] The use of controlled surface topography and flow-induced shear stress to influence..., Frohlich [/bib_ref] [bib_ref] Fluid-shearstress-induced translocation of aquaporin-2 and reorganization of actin cytoskeleton in renal tubular..., Jang [/bib_ref] [bib_ref] Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional..., Duan [/bib_ref]. In our case, a volumetric flux of 1 mL min 21 corresponded to a FSS of about 0.2 dyn cm 22 , applied for 6 h after waiting about 100 sec to reach the stationary conditions. The use of FSS has been demonstrated to be important for the functional behaviour of these cells [bib_ref] Mechanical strains induced by tubular flow affect the phenotype of proximal tubular..., Essig [/bib_ref] , since it can regulate the formation of tight junctions, the regulation of ion movements and the homeostasis of water [bib_ref] The use of controlled surface topography and flow-induced shear stress to influence..., Frohlich [/bib_ref] [bib_ref] Fluid-shearstress-induced translocation of aquaporin-2 and reorganization of actin cytoskeleton in renal tubular..., Jang [/bib_ref]. Outlet samples from both lumen and interstitial microchannels were collected to quantify the percentage recovery of urea and creatinine, calculated as: [formula] R(%)~(C I out C L in )|100ð1Þ [/formula] where c out I is the output solute concentration collected from the interstitial channel and c in L is the input solute concentration in the lumen channel. In a counter-current arrangement, the two solutions flow in opposite directions, and at any coordinate nearby the membrane the solute concentration in the interstitial channel is lower than that in the lumen channel [bib_ref] Size selective DNA transport through a nanoporous membrane in a PDMS microfluidic..., Sheng [/bib_ref]. Hence, the recovery can be higher than 50%. Our device employed a counter-flow arrangement, as in renal tubules in vivo, with equal flow rates in the two channels. The results, expressed as (mean 6 standard error), were obtained from three independent experiments performed on different devices. For devices with cells, we obtained a recovery of (1665)% for urea and (1865)% for creatinine. In control experiments without cells, performed on fibronectin-coated membranes, the values increased to (6467)% and (4567)%, respectively, indicating a higher overall permeability in absence of cellular coverage on the membrane surface [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. For sake of comparison, we recall that a recovery up to about 80% has been reported for glucose diffusing through a PC membrane separating a static reservoir and a channel supporting a 1.5 mL min 21 flow [bib_ref] Glucose recovery in a microfluidic microdialysis biochip, Hsieh [/bib_ref]. Here, the presence of a highly uniform layer of HK-2 cells in the bioartificial device was also confirmed by immunocytochemistry assays performed to stain nuclei labeled with DAPI (blue fluorescence) [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref] and actin filaments with phalloidin-TRITC (red fluorescence) [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. ## Stem cell isolation To reproduce the essential functions of a living kidney, it is necessary to replace standard immortalized cell lines with primary renal human cells. A possible strategy could be, in principle, the use of primary cells which are phenotypically and physiologically similar to in vivo human cells. For instance, RPTECs, which are primary human renal proximal tubule epithelial cells, are commonly used as model system for kidney cell biology [bib_ref] hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their..., Wieser [/bib_ref]. However, we found that RPTECs did not colonize the microchip appreciably, regardless of the seeding concentration [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref]. This result, attributable to the much higher sensitivity of these cells to culture conditions, with respect to immortalized cell lines, strengthens the importance of the use of stem cells in kidneymimicking bioartificial chips. green) (D) and Na + K + ATPase pump (I). X-Z section confocal images for AQP2 (apical marker protein) (E) and Na + K + ATPase pump (basolateral marker protein) (J) following FSS in the chip. Scale bars = 15 mm. The section along the longitudinal axis of the microchannel is indicated by the white line in D and I, respectively. X-Z section confocal images were also collected for AQP2 (F) and Na + K + ATPase pump (K) in statically cultured ARPCs. Scale bars = 15 mm. doi:10.1371/journal.pone.0087496.g005 Differently from RPTECs, ARPCs indeed retain a greater plasticity [bib_ref] Adult stem cell plasticity, Poulsom [/bib_ref] [bib_ref] Plasticity of adult stem cells, Wagers [/bib_ref] and exceptional adaptability to various microenvironments. We firstly extracted ARPCs from the tubular portion of the cortex renal fraction, in order to realize a reliable bioartificial proximal tubule-like microdevice based on a multilayer microdevice [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref]. Therefore, we characterized tubular ARPCs for renal stem cell markers by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Our recovered populations were homogeneously positive for stem cell markers CD133 and CD24 and for the renal transcription factor PAX-2 [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref]. However, the CD34, CD105, and CD45 markers of mesenchymal and hematopoietic stem cells were not detectable (data not shown), thus demonstrating that we were dealing with ARPCs resident in the kidney. Moreover, these CD133+ cells expressed the hyaluronic acid receptor CD44, the blastocyst stem cell marker Oct-4, and the adult stem cell marker BMI-1 [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref] , all of which are typically expressed in ARPCs [bib_ref] Isolation of renal progenitor cells from adult human kidney, Sagrinati [/bib_ref] [bib_ref] TLR2 plays a role in the activation of human resident renal stem/progenitor..., Sallustio [/bib_ref]. CD133+ CD24+ ARPCs can be maintained in culture without losing CD133 expression for up to 7-9 passages [bib_ref] Isolation of Renal Progenitor Cells from Adult Human Kidney, Bussolati [/bib_ref] , giving rise to homogenous clonal populations [bib_ref] TLR2 plays a role in the activation of human resident renal stem/progenitor..., Sallustio [/bib_ref]. Moreover, renal progenitors can grow continuously for 60 to 90 population doublings, depending on the donor, during a period of 4 months and when assessed at 50 population doublings, cells exhibited diploid DNA content [bib_ref] Isolation of renal progenitor cells from adult human kidney, Sagrinati [/bib_ref]. ## Stem cell growth and polarization on chip ARPCs in the chip were cultured under the conditions previously described for HK-2 cells. After 4 days, solute diffusion experiments evidenced a recovery of urea and creatinine of (2065)% and (1365)%, respectively, which were comparable with values obtained by HK-2 cultures. This was in agreement with the formation of a layer of stem cells on the microporous membrane [bib_ref] Evaluation of long-term transport ability of a bioartificial renal tubule device using..., Ozgen [/bib_ref] [bib_ref] Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular..., Sanechika [/bib_ref]. We also measured the recovery of glucose, which was found to be of (5265)% in the ARPC-based device. In order to assess the morphology and the cytoskeleton organization of stem cells in the bioartificial device, we performed immunocytochemistry assays by staining actin filaments labeled with phalloidin-TRITC (red fluorescence), and nuclei with DAPI (blue fluorescence), which confirmed that the polymeric membranes were uniformly covered [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref]. Cells grew strictly confined along the tubule-like microchannel without penetrating its external, watertight sealed borders. Differently from HK-2 cells [fig_ref] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells [/fig_ref] , ARPCs showed a distinctive morphology which evidenced their plasticity, namely a clearly elongated aspect and a partially oriented arrangement of actin filaments [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref]. This elongated configuration is often observed in ARPCs, even if they are differentiated in epithelial cells [fig_ref] Figure 4: Characterization of ARPC morphology [/fig_ref]. In addition, the formation of junctions between adjacent ARPCs in a layer was visualized by ZO-1 immunostaining [fig_ref] Figure 4: Characterization of ARPC morphology [/fig_ref]. Finally, the functional response of ARPCs was investigated, as induced by the exposure to the FSS [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref]. For this aim, a triple staining was carried out for visualizing nuclei (DAPI, [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] and 5G), actin filaments (phalloidin-TRITC, [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] , and the markers AQP2 or Na + K + ATPase pump (by their relative primary and secondary antibodies, green fluorescence), which are an apical and a basolateral protein, respectively [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref]. As shown in [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] , AQP2 was localized at the apical region of cells, whereas the Na + K + ATPase pump [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] was observed in the basolateral portion of cells, thus demonstrating a perfectly organized cell polarization. On the contrary, in statically cultured ARPCs, AQP2 [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] and Na + K + ATPase pump [fig_ref] Figure 5: Polarization of ARPCs following FSS in the chip [/fig_ref] did not localize at the extremities of cells but showed a diffuse cytoplasmic distribution, as typical of an unpolarized phenotype. The absence of a polarized morphology was also evidenced in macroscopic cultures, performed in standard two-compartment systems with static liquid conditions, as shown in the [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref] of Supplementary Information. # Discussion The kidney is still one of the most difficult organs to be successfully mimicked and studied in vitro, since it presents a high structural and functional complexity, along with a distinctive cellular variety. The understanding of both physiological and pathological aspects of the different portions of the kidney may significantly benefit from the realization of miniaturized organ-onchip devices, which combine biological and engineering approaches [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] [bib_ref] Development of a renal microchip for in vitro distal tubule models, Baudoin [/bib_ref] [bib_ref] Contribution of stem cells to kidney repair, Bussolati [/bib_ref] [bib_ref] Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment, Jang [/bib_ref]. In the present microsystem, the in vivo tubule-like environment was closely resembled, since the upper microchannel provided a lumen area, in which the apical portion of cells was exposed, while the lower microchannel simulated the interstitial area, in contact with the basolateral membranes of cells. Moreover, this geometry was suitable to induce the functional polarization of epithelial cell lines [bib_ref] Reconstituting organ-level lung functions on a chip, Huh [/bib_ref]. To mimic the characteristic kidney tubule microenvironment, it is important to consider the presence of the basement membrane, composed by several ECM proteins and acting as structural and functional meshwork for tubule epithelial and endothelial cells [bib_ref] The use of controlled surface topography and flow-induced shear stress to influence..., Frohlich [/bib_ref]. We found that fibronectin promoted an enhanced cell proliferation compared to other proteins. Consistently with this result, the positive reaction to fibronectin is considered a key phenotype indicator of well-differentiated proximal tubular cells [bib_ref] HK-2: an immortalized proximal tubule epithelial cell line from normal adult human..., Ryan [/bib_ref] [bib_ref] The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their..., Lechleitner [/bib_ref]. The next fundamental step to reproduce physiologically meaningful functions at device level was to understand the conditions by which renal tubule cells would cover to confluence the membrane surface. This is still one of the major drawbacks in the realization of a fully functional renal biochip [bib_ref] Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular..., Sanechika [/bib_ref]. In this work, the culture conditions on-chip were optimized by using HK-2 cells as model. We found that the confluence was achieved by seeding an initial concentration of 1.5610 6 cells mL 21 and letting cells grow under a static culture environment for about 24 h and then replacing the culture medium twice a day. Once defined the optimal working conditions, we realized a functional biochip with renal tubule cells. After 4 days of cell culture, we perfused the lumen microchannel with the complete culture medium containing urea and creatinine and the interstitial microchannel with the same solution without urea and creatinine in a counter-current mode. Then, we collected the outlet samples from both lumen and interstitial microchannels to quantify the recovery of urea and creatinine. Creatinine and urea are ideal molecular probes since they are partially reabsorbed or secreted along the proximal tubule portion, therefore it is expected that the presence of a continuous layer of cells determines a variation of their permeability across the microporous membrane. Indeed, we obtained a lower recovery for urea and creatinine for devices with cells [(1665)% and (1865)%, respectively] with respect to the controls without cells [(6467)% and (4567)%]. This result clearly indicated a lower permeability in the presence of a highly uniform layer of HK-2 cells on the membrane surface, also confirmed by immunocytochemistry assays. Previous bioartificial kidney devices have been realized by involving cell lines that grow easily in culture, such as cancerporcine [bib_ref] Evaluation of long-term transport ability of a bioartificial renal tubule device using..., Ozgen [/bib_ref] , canine [bib_ref] Development of a renal microchip for in vitro distal tubule models, Baudoin [/bib_ref] , rat cells [bib_ref] A multi-layer microfluidic device for efficient culture and analysis of renal tubular..., Jang [/bib_ref] and, among human cell lines, renal proximal tubular epithelial cells directly isolated [bib_ref] Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment, Jang [/bib_ref] [bib_ref] The use of fibrous, supramolecular membranes and human tubular cells for renal..., Dankers [/bib_ref] or genetically modified to extend their lifespan [bib_ref] Development of bioartificial renal tubule devices with lifespan-extended human renal proximal tubular..., Sanechika [/bib_ref] [bib_ref] The use of controlled surface topography and flow-induced shear stress to influence..., Frohlich [/bib_ref]. However, these devices are poorly predictive and lack the ability to reconstitute the real structural and functional features of human living kidney. Here we extracted ARPCs from the tubular portion of the cortex renal fraction and characterized them for renal stem cell markers (CD133, CD24, PAX-2, CD44, Oct-4, and BMI-1). Then, we cultured ARPCs in the device under the conditions previously optimized with HK-2 cells. After 4 days, we measured a recovery of urea and creatinine [(2065)% and (1365)%, respectively]. We also measured the recovery of glucose, which was found to be of (5265)% in the ARPC-based device. This value could be further increased by exploiting more mature phenotypes of ARPCs compared to those used in this work, namely cells fully differentiated into epithelial cells and hence fully exhibiting transepithelial transport processes such as those involving glucose, or bicarbonate, and the associated net fluid reabsorption [bib_ref] The relationship between renal metabolism and proximal tubule transport during ontogeny, Barac-Nieto [/bib_ref]. Finally, a polarization of ARPCs was induced by the FSS in the chip. AQP2 was localized at the apical region of cells, whereas the Na + K + ATPase pump was observed in the basolateral portion of cells, thus demonstrating a perfectly organized cell polarization. On the contrary, in statically cultured ARPCs, AQP2 and Na + K + ATPase pump did not localize at the extremities of cells but showed a diffuse distribution, as typical of an unpolarized phenotype. Overall, the bioartificial proximal tubule-like, ARPC-embedding renal microdevice developed in this work provides an important proof of principle, since it integrates the topological, structural, chemical and biological features proper of the living kidney. On one hand, it provides a biomimetic platform to efficiently culture and analyze the physiological and pathological response of renal tubule cells. The culture of ARPCs, used for the first time in a miniaturized chip, demonstrates that it is possible to induce a well-defined polarization as highlighted by the apical and basolateral marker proteins. On the other hand, while this ''microorganoid-on-a-chip'' device will need further characterization and validation, it opens new perspectives to recapitulate physiological functions. For this reason, the scale up and parallelization of such a single bioartificial renal tubule to multiple integrated microsystems, and ultimately its integration with other functional modules, might drive the development of new adjuvant for replacement therapy in kidney disease. [fig_ref] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs [/fig_ref] Polarization of ARPCs in a static macroscopic system. X-Z section confocal images for AQP2 (apical marker protein) (A) and Na + K + ATPase pump (basolateral marker protein) (B) in ARPCs cultured in a culturing system with two compartments separated by the polycarbonate membrane and using static conditions of liquids. (TIF) ## Supporting information Information S1 Immunofluorescent staining of FN in the microchannel. ## (docx) Author Contributions [fig] Figure 2: On-chip formation of a confluent monolayer of renal tubule cells. (A) Device fluidic characterization by diffusion test: a solution of rhodamine 6G was fed in the upper channel while distilled water was fed in the lower channel (interstitial). The increase in the fluorescence intensity with time at the outlet of the lower channel was correlated to the rhodamine diffusion into the channel. The superimposed line is the corresponding data in a standard cell culture insert using the same porous membrane. Inset: device and counter-current flow schematics. (B) HK-2 cell proliferation described by MTS assay after 2 days of culture on the membranes functionalized with fibronectin (FN), laminin (LN) and Matrigel (M), compared to the untreated membranes (Untr.) and the positive controls of polystyrene dishes (PS). Results are expressed as (mean 6 standard deviation) of three independent experiments. Bars show statistically significant differences (P,0.05). (inset) Optical micrographs of water droplets on membranes with or without FN, and corresponding WCA value. (C-F) Optimization of HK-2 cell growth in the device. A starting concentration of 5610 5 cells mL 21 was insufficient for a successful colonization of the membrane by cells (C), while a concentration $ 1.5610 6 cells mL 21 led to a confluent growth (D). Culturing cells for 4 days under a constant flux (1 mL min 21 ) of cell medium did not allow the formation of a continuous monolayer (E). Cell confluence was instead achieved by letting seeded cells in a static fluid environment for 24 h and culturing them over 4 days by changing the complete growth medium twice a day (F). Scale bars = 100 mm. (G) Scheme of the resulting cell confluent monolayer on the membrane. Confluent living cells were stained with DAPI (blue) (H) and TRITC-phalloidin (red) (I). Scale bars = 100 mm. doi:10.1371/journal.pone.0087496.g002 [/fig] [fig] Figure 3: Fulfillment of a bioartificial proximal tubule on-a-chip embedding ARPCs. (A) Scheme of the glomerulus and the proximal tubule structure in a human kidney nephron. Here tubular ARPCs were seeded into the device, whose cross section illustrates a confluent layer of ARPCs within the lumen microchannel and adherent to the membrane. After 4 days of culture, the lumen microchannel was perfused with the complete Renal Proximal Tubule On-Chip with Stem Cells PLOS ONE | www.plosone.org [/fig] [fig] Figure 4: Characterization of ARPC morphology. (A) Light microscopy image of ARPCs. (B) ARPCs can be differentiated in renal tubular cells, and form junctions as shown by ZO-1 immunostaining (rabbit anti-human ZO-1 polyclonal Ab, green). To-pro-3 counterstains nuclei (blue). Scale bar = 40 mm. doi:10.1371/journal.pone.0087496.g004 [/fig] [fig] Figure 5: Polarization of ARPCs following FSS in the chip. (A) Cell model of proximal tubule cells with transporters. The scheme shows the AQP2 transporter localized in the apical membrane and the Na + K + ATPase present in the basolateral membrane. (B-J) Cellular arrangement of ARPCs grown into the microfluidic device after 6 hs of FSS at 0.2 dyn/cm 2 . Two different chips were stained in (B-E) and in (G-J), respectively. Immunofluorescence images of stained DNA (DAPI, blue) (B, G), actin (TRITC-phalloidin, red) (C, H), AQP2 (FITC-anti goat, [/fig] [fig] Figure S1: Immunostaining of FN coating along the microchannel. (A) The assay was performed directly inside the microchip, by using sequentially a primary anti-Fibronectin antibody and the secondary antibody labeled with Fluorescein Isothiocyanate. (B) shows the negative control represented by the staining of a device not functionalized with fibronectin. (TIF) Figure S2 Investigation of RPTEC growth on-chip. (A) Optical micrograph demonstrating the standard growth of RPTECs in a conventional polystyrene flask. (B) RPTECs unsuccessful growth into the microfluidic device. Scale bar: 100 mm. (TIF) [/fig]

Healthy community‐living older men differ from women in associations between myostatin levels and skeletal muscle mass Background Myostatin is a negative regulator of muscle growth but the relationship between serum myostatin levels and muscle mass is unclear. This study investigated the association between serum myostatin levels and skeletal muscle mass among healthy older community residents in Taiwan, to evaluate the potential of serum myostatin as a biomarker for diagnosing sarcopenia and/or evaluating the effect of its treatment. Methods Study data were excerpted from a random subsample of the I-Lan Longitudinal Aging Study population. Serum myostatin levels were determined and categorized into tertiles (low, medium, high). Relative appendicular skeletal muscle mass (RASM) was calculated as appendicular lean body mass by dual-energy X-ray absorptiometry divided by height squared (kg/m 2 ). Low muscle mass was defined as recommended by the Asian Working Group for Sarcopenia. Results The analytic study sample comprised 463 adults (mean age: 69.1 years; 49.5% men). Compared with subjects with normal RASM, those with lower RASM were older and frailer, with significantly higher prevalence of malnutrition, lower serum dehydroepiandrosterone (DHEA) levels, and were more likely to have low serum myostatin status. Multivariable logistic regression analysis showed that male sex (OR 3.60, 95% CI 1.30-9.92), malnutrition (OR 4.39, 95% CI 1.56-12.36), DHEA (OR 0.99, 95% CI 0.99-1.00), and low myostatin (OR 3.23, 95% CI 1.49-7.01) were all independent risk factors for low RASM (all P < 0.05). In men, DHEA (OR 0.99, 95% CI 0.98-1.00) and low myostatin (OR 4.89, 95% CI 1.79-13.37) were significantly associated with low RASM (both P < 0.05); however, only malnutrition was associated with low RASM in women (OR 13.59,, P < 0.05). Conclusions Among healthy community-living older adults, low serum myostatin levels were associated with low skeletal muscle mass in men, but not in women. Our results do not support using serum myostatin levels to diagnose sarcopenia, or to monitor how it responds to treatments. Further research is needed to understand why men apparently differ from women in the interrelationship between their myostatin levels and muscle mass. # Introduction An important characteristic of aging is temporal changes in body composition between bone, muscle, and fat mass. From age 30, people may lose skeletal muscle mass at 3-8% per decade and at a considerably accelerated rate as they become older. [bib_ref] The biology of aging, Holloszy [/bib_ref] [bib_ref] Epidemiology of sarcopenia, Melton Lj 3rd [/bib_ref] Loss of muscle mass may also lead to diminished muscle strength and physical performance, which heighten the risk of gait unsteadiness and falls, functional impairment, disability, and mortality [bib_ref] Strength is a major factor in balance, gait, and the occurrence of..., Wolfson [/bib_ref] [bib_ref] Leg muscle mass and composition in relation to lower extremity performance in..., Visser [/bib_ref] [bib_ref] The role of muscle mass and body fat on disability among older..., Tyrovolas [/bib_ref] [bib_ref] Muscle mass index as a predictor of longevity in older adults, Srikanthan [/bib_ref] ; the contemporary definition of sarcopenia encompasses the aforementioned clinical characteristics. The aetiology and pathophysiology of sarcopenia are complex and multifactorial, involving physical inactivity, nutritional status, loss of muscle fibres and motor units, and hormonal changes, among others. [bib_ref] Molecular mechanisms involved in muscle wasting in cancer and ageing: cachexia versus..., Argiles [/bib_ref] The balance between myostatin, activin, and follistatin in the sarcopenia nexus has garnered extensive research interest, and several potential pharmacotherapeutic agents based on the action of myostatin have been developed. The growth/differentiation factor 8 gene (GDF8) was discovered in 1997 8 ; its product, myostatin, is a member of the transforming growth factor-beta superfamily that is secreted primarily by myocytes in skeletal muscle, and was the first negative regulator of skeletal muscle growth to be identified. [bib_ref] Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily..., Mcpherron [/bib_ref] [bib_ref] Powerful genes-myostatin regulation of human muscle mass, Mcnally [/bib_ref] [bib_ref] Induction of cachexia in mice by systemically administered myostatin, Zimmers [/bib_ref] [bib_ref] Role of serum myostatin during the lactation period, Hosoyama [/bib_ref] Knockout mice had muscular hypertrophy due to increased numbers of myocytes, myofiber size, and overall muscle size [bib_ref] Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily..., Mcpherron [/bib_ref] and rats with lower myostatin levels had accelerated myogenesis, [bib_ref] Role of serum myostatin during the lactation period, Hosoyama [/bib_ref] whereas transgenic mice with higher serum myostatin had cachexia. [bib_ref] Induction of cachexia in mice by systemically administered myostatin, Zimmers [/bib_ref] Congruently, a child with a GDF8 loss-of-function mutation had significant muscle hypertrophy. [bib_ref] Myostatin mutation associated with gross muscle hypertrophy in a child, Schuelke [/bib_ref] Given its action on skeletal muscle, serum levels of myostatin have hypothetical potential as a biomarker for sarcopenia. [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] However, the results of studies evaluating the relationship between serum myostatin and skeletal muscle mass are inconsistent, besides mostly deriving from subjects with particular diseases rather than healthy individuals. Inverse correlations between serum myostatinimmunoreactive protein concentration and height-corrected fat-free mass and muscle mass suggest that myostatin may be a biomarker of age-associated sarcopenia. [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] Serum myostatin was also negatively associated with skeletal muscle mass in male patients with chronic obstructive pulmonary disease (COPD). 14 Conversely, decreased plasma myostatin levels in heart-failure patients with cachexia suggest that lower myostatin may prevent loss of skeletal muscle and progression of cachexia. [bib_ref] Serum myostatin levels are independently associated with skeletal muscle wasting in patients..., Furihata [/bib_ref] Besides these inconsistencies, little is known about the relationship between serum myostatin and skeletal muscle mass among healthy individuals. Therefore, we investigated the association between serum myostatin and skeletal muscle mass among healthy older community residents. # Methods ## Study design and subjects Study subjects were a random subsample of the I-Lan Longitudinal Aging Study (ILAS), which is a population-based research cohort of 1839 adults aged 53-92 years, residing in I-Lan (Yilan) County, Taiwan, who were randomly sampled through household registration records; individuals were invited to participate via mail or telephone and those who accepted were enrolled after giving written informed consent. ILAS excluded subjects who (i) were unable to cooperate or communicate with study investigators; (ii) declined or were unable to grant consent; (iii) were currently institutionalized; (iv) were known to have active diseases (cancer, sepsis, heart failure, COPD, etc.) or functional dependence, or had life-expectancy <6 months; and (v) were planning to leave Yilan County. This study analysed serum myostatin, activin, and follistatin measurements taken from a random subsample of 463 ILAS subjects. The Institutional Review Boards of National Yang Ming University and Taipei Veterans General Hospital approved the study protocol. ## Demographic characteristics and laboratory data Research nurses collected participants' demographic and medical details and took anthropometric measurements. Alcohol consumption was categorized as drinking or non-drinking, and tobacco usage as currently smoking or non-smoking. All participants gave peripheral blood samples at 7-9 AM, after a 10 h overnight fast. Biochemistry measurements included serum albumin, alanine aminotransferase, uric acid, total cholesterol, serum creatinine, high-sensitivity C-reactive protein, homocysteine, testosterone, insulin-like growth factor-1, dehydroepiandrosterone (DHEA), and vitamin D3. The homeostasis model assessment of insulin resistance index was calculated for insulin resistance. Serum levels of myostatin, activin, and follistatin were measured by sandwich enzyme immunoassay kits (R&D systems, Inc., Minneapolis, USA). Low serum myostatin was defined as the lower tertile of subjects' collective serum myostatin levels. ## Functional assessment and physical performance Research nurses performed comprehensive functional assessments for all participants. Nutrition status was evaluated by Mini Nutritional Assessment, [bib_ref] The Mini Nutritional Assessment (MNA) review of the literature-What does it tell..., Guigoz [/bib_ref] and cognitive impairment defined by a Mini-Mental State Examination score <24. [bib_ref] A practical method for grading the cognitive state of patients for the..., Folstein [/bib_ref] Mood was evaluated by the five-item geriatric depression scale, with the five-item geriatric depression scale ≥2 denoting depressive symptoms. [bib_ref] Development and testing of a five-item version of the geriatric depression scale, Hoyl [/bib_ref] Physical activity was assessed by International Physical Activity Questionnaire,the severity of underlying medical conditions by the Charlson Comorbidity Index, [bib_ref] A new method of classifying prognostic comorbidity in longitudinal studies: development and..., Charlson [/bib_ref] and frailty status was determined according to the Fried criteria, which comprise weight loss, physical inactivity, weakness, slowness, and exhaustion 21 ; frailty was defined as having ≥3 items and pre-frailty was defined as 1 or 2 itemsthose who met no Fried criteria were considered robust. Gait speed was measured by a timed 6 m walk at the participant's usual pace, and handgrip strength of the dominant hand in an upright position was measured with a digital dynamometer (Smedlay's Dynamo Meter; TTM, Tokyo, Japan). ## Body composition Total fat mass, fat-free lean body mass, and bone mineral density were calculated from whole-body dual-energy X-ray absorptiometry scan data. Appendicular skeletal mass was defined as the total fat-free lean body mass from four limbs, and the relative appendicular skeletal muscle mass index (RASM) was derived from appendicular skeletal mass divided by height squared (kg/m 2 ). Total body fat percentage was calculated as total fat mass divided by total body mass times 100. ## Diagnosis of sarcopenia and related measurements This study defined sarcopenia, according to recommendations by the Asian Working Group for Sarcopenia (AWGS), [bib_ref] Sarcopenia in Asia: consensus report of the Asian Working Group for Sarcopenia, Chen [/bib_ref] as low muscle mass plus low muscle strength and/or low physical performance; the respective cut-off values were RASM: 7.0 kg/m 2 for men and 5.4 kg/m 2 for women based on dualenergy X-ray absorptiometry; handgrip strength: < 26 kg for men and <18 kg for women; gait speed: less than 0.8 m/s. # Statistical analysis Categorical variables are expressed by percentage, and continuous data as mean plus/minus standard deviation. Categorical variables were compared by Chi-square test, and continuous variables by Student's t-test, as appropriate. Multivariate logistic regression was used to determine independent risk factors of RASM below the AWGS lower reference limit compared with normal RASM, by entering all variables with P < 0.1 in univariate analysis as covariates. All statistical analyses were performed using SPSS Statistics Version 18.0 for Microsoft Windows XP (SPSS Inc., Chicago, IL, USA). A two-tailed P-value of <0.05 was considered statistically significant. # Results The 463 enrolled subjects had mean age of 69.1 ± 9.2 years and 49.5% were men. Based on the AWGS cut-offs, 54 subjects (11.7%) had low RASM; however, the overall prevalence of sarcopenia was low, only 4.1% overall. [fig_ref] Table 1: Demographic characteristics of subjects with low vs [/fig_ref] summarizes the functional, cognitive and clinical characteristics, and health behaviour of groups with low RASM and normal RASM. Low RASM was significantly more prevalent in men than in women, and men with low RASM had a significantly lower total body fat percentage than those with normal RASM. Compared with participants with normal RASM, the group with low RASM had lower mean body mass index, and had a higher proportion who were current smokers, and a lower proportion who consumed alcohol. Among the functional domains, a significantly higher proportion of subjects with low RASM were malnourished, but there were no significant differences in cognitive function, depressive symptoms, or walking speed. Among known risk factors for sarcopenia-specifically, the inflammatory marker C-reactive protein and hormonal profiles-only serum DHEA levels were significantly lower among subjects with low vs. normal RASM. Although there were no significant between-group differences in serum activin or follistatin levels, the low RASM group serum tended to have lower myostatin levels, and subjects with low RASM were significantly more likely to have serum myostatin in the lowest tertile. [fig_ref] Table 2: Independent predictors of low muscle mass among otherwise healthy community-living older adults [/fig_ref] summarizes the logistic regression results. In Model 4 (adjusted for age, sex, smoking status, alcohol consumption, Charlson Comorbidity Index score, total body fat percentage, malnutrition, DHEA, and myostatin status), male sex, malnutrition, DHEA, and low myostatin status were all independent risk factors for low RASM. Due to different muscle mass between the sexes, we performed separate logistic regression analyses in men and women [fig_ref] Table 3: Independent predictors of low muscle mass in men and women aged ≥53... [/fig_ref] ; lower DHEA level and low myostatin status were significantly associated with low RASM in men, whereas only malnutrition was directly related to low RASM in women. In post hoc analyses, there was no significant difference in serum myostatin levels between subjects aged 53-70 vs. ≥70 years (P = 0.085) or significant associations in either sex between serum myostatin and follistatin (men: P = 0.921; women P = 0.410); however, an association between myostatin and total body fat percentage was evident in men (men: P = 0.041; women: P = 0.704). # Discussion To the best of our knowledge, this is the first report of the association between serum myostatin and lower skeletal muscle mass among healthy community-living older adults; lower serum DHEA levels and low serum myostatin status were independent risk factors of low RASM in men. Although the muscle-wasting effect of myostatin is wellestablished, [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] [bib_ref] Serum myostatin levels and skeletal muscle wasting in chronic obstructive pulmonary disease, Ju [/bib_ref] the relationship between serum myostatin and skeletal muscle mass in humans is complex and remains controversial; moreover, reported associations were observed in patients with COPD or heart failure. [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] [bib_ref] Serum myostatin levels are independently associated with skeletal muscle wasting in patients..., Furihata [/bib_ref] [bib_ref] Prevalence of cachexia in chronic heart failure and characteristics of body composition..., Christensen [/bib_ref] [bib_ref] Exercise training leads to a reduction of elevated myostatin levels in patients..., Lenk [/bib_ref] Age-related loss of muscle mass was associated inversely with serum myostatin among frail older adults, [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] and circulating myostatin in male patients with COPD also negatively correlated with skeletal mass calculated using a validated formula. 14 Conversely, others have reported skeletal muscle wasting associated with lower myostatin levels, especially in heart failure patients with compensatory status, cachexia, or undertaking exercise training. [bib_ref] Serum myostatin levels are independently associated with skeletal muscle wasting in patients..., Furihata [/bib_ref] [bib_ref] Prevalence of cachexia in chronic heart failure and characteristics of body composition..., Christensen [/bib_ref] [bib_ref] Exercise training leads to a reduction of elevated myostatin levels in patients..., Lenk [/bib_ref] This inconsistent evidence makes it difficult to assess the potential of serum myostatin as a biomarker for sarcopenia. Our finding that low serum myostatin was an independent risk factor for lower RASM in healthy older adults appears contradictory to current understanding. One explanation may relate to the myostatin splice variant protein, which binds to myostatin and antagonizes canonical signalling 25 ; Jeanplong et al. reported that an energy-restricted diet reduced the semitendinosus muscle mass of young ewes and that myostatin activity was inhibited by increased myostatin splice variant expression, implying an important influence of nutritional status on the action of myostatin in skeletal muscle development. [bib_ref] Undernutrition regulates the expression of a novel splice variant of myostatin and..., Jeanplong [/bib_ref] Another reason could be differences in the age distributions of study cohorts. Yarasheski et al. observed that serum myostatin was higher among women aged 76-92 years old than younger ones, [bib_ref] Serum myostatin-immunoreactive protein is increased in 60-92 year old women and men..., Yarasheski [/bib_ref] indicating that age might influence myostatin expression; however, we found no significant difference in serum myostatin between subjects aged 53-70 vs. ≥70 years. Follistatin is a myostatin antagonist that maintains tissue homeostasis. Interestingly, we found no significant association between serum follistatin and myostatin in either sex, perhaps because the liver usually secretes follistatin immediately after exercise and our participants' blood samples were all taken early in the morning, before daily activities. Myostatin also inhibits or promotes adipogenesis, depending on the circumstances 26 ; although its action is chiefly inhibitory, myostatin may also have an adipogenic effect, by promoting mesenchymal stem cell differentiation. [bib_ref] Myostatin inhibits myogenesis and promotes adipogenesis in C3H 10T(1/2) mesenchymal multipotent cells, Artaza [/bib_ref] In our study, we found an association between myostatin and adipose tissue in men, but not in women. The pathophysiology of myostatin in adipose tissue is very complex and incompletely understood; it is thought to be influenced by insulin resistance, [bib_ref] Inhibition of myostatin in mice improves insulin sensitivity via irisinmediated cross talk..., Dong [/bib_ref] insulin-like growth factor-1, 29 and the sex steroid precursor DHEA, [bib_ref] Association of dehydroepiandrosterone sulfate, body composition, and physical fitness in independent community-dwelling..., Abbasi [/bib_ref] [bib_ref] Dehydroepiandrosterone exerts antiglucocorticoid action on human preadipocyte proliferation, differentiation, and glucose uptake, Mcnelis [/bib_ref] which was associated with low muscle mass among male participants in our study. Given these complicated relationships, a longitudinal study is needed to clarify whether low serum myostatin is a cause or a consequence of low muscle mass. The risk of becoming malnourished increases with advancing age, due to decreased appetite, poor digestion, polypharmacy, cognitive impairment, or depressed mood, as well as acute or chronic medical conditions and socioeconomic factors. Although only 6.3% of ILAS subjects were malnourished, malnutrition and lower skeletal muscle mass were strongly interrelated, consistent with other studies in older men or women. [bib_ref] Prevalence of sarcopenia and its association with functional and nutritional status among..., Bahat [/bib_ref] [bib_ref] Lean mass loss is associated with low protein intake during dietary-induced weight..., Bopp [/bib_ref] [bib_ref] Nutritional status and its effects on muscle wasting in patients with chronic..., Saitoh [/bib_ref] [bib_ref] The relationship between sarcopenia,undernutrition, physical mobility and basic activities of daily living..., Velázquez Alva Mdel [/bib_ref] Among people with heart failure, malnutrition plays a prominent role in muscle loss and sarcopenia, as well as disease severity, [bib_ref] Muscle wasting in heart failure. The role of nutrition, Saitoh [/bib_ref] and in a prospective cohort, lower Mini Nutritional Assessment score independently predicted muscle wasting and mortality. [bib_ref] Nutritional status and its effects on muscle wasting in patients with chronic..., Saitoh [/bib_ref] Among postmenopausal women receiving weight-loss intervention, higher protein intake prevented loss of lean body mass. [bib_ref] Lean mass loss is associated with low protein intake during dietary-induced weight..., Bopp [/bib_ref] Hence, nutritional factors should be taken into account when evaluating serum myostatin as a sarcopenia biomarker. Among ILAS subjects, female sex was not a risk factor for low RASM or the association between serum myostatin and low muscle mass. Sex is an important determinant of reduced muscle strength and age-related diminution of muscle mass. [bib_ref] Muscle distribution: variations with body weight, gender, and age, Gallagher [/bib_ref] Men generally start losing muscle mass when serum testosterone drops after age 40. [bib_ref] Testosterone, body composition and aging, Vermeulen [/bib_ref] Women may gradually lose 10-15% of their muscle mass between age 25 and menopause onset, rising to 2% annually thereafter. [bib_ref] Aging of skeletal muscle: a 12-yr longitudinal study, Frontera [/bib_ref] The prevalence of sarcopenia among community-dwelling adults in Minnesota, USA, as determined by dual-energy Xray absorptiometry, was 10% for men and 8% for women aged 60-69 years, and 40% for men and 18% for women aged over 80 years, 2 and whole body magnetic resonance imaging has shown that men lose more skeletal muscle mass with advancing age than do women. [bib_ref] Skeletal muscle mass and distribution in 468 men and women aged 18-88..., Janssen [/bib_ref] Men and women in a longitudinal study all lost significant total and leg lean muscle mass over 3 years, with greater proportional and absolute lean mass diminution in men. [bib_ref] The loss of skeletal muscle strength, mass, and quality in older adults:..., Goodpaster [/bib_ref] This evidence suggests that male sex is a potential risk factor for low muscle mass, possibly through mechanisms associated with age-related decline of endocrine factors, such as androgens, growth hormone, and insulin-like growth factor-1. [bib_ref] Skeletal muscle mass and distribution in 468 men and women aged 18-88..., Janssen [/bib_ref] Serum DHEA peaks during puberty declines to 20% of the maximal value in later life, and falls to only 5% after age 85. [bib_ref] Changes in serum concentrations of conjugated and unconjugated steroids in 40-to 80-yearold..., Bélanger [/bib_ref] [bib_ref] Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood, Orentreich [/bib_ref] [bib_ref] Long-term longitudinal measurements of plasma dehydroepiandrosterone sulfate in normal men, Orentreich [/bib_ref] The waning DHEA level parallels other age-related changes, such as muscle wasting. [bib_ref] Skeletal muscle mass and distribution in 468 men and women aged 18-88..., Janssen [/bib_ref] Consistent with other studies, 30,46,47 we found serum DHEA to be significantly associated with lower RASM, with a stronger association in older men than among women. Balagopa et al. reported that myosin heavy-chain synthesis declined progressively with age, and that serum DHEA was significantly associated with the fractional synthesis rate. [bib_ref] Effects of aging on in vivo synthesis of skeletal muscle myosin heavychain..., Balagopal [/bib_ref] Others found that DHEA was positively correlated with lean body mass in men aged over 60 years, [bib_ref] Association of dehydroepiandrosterone sulfate, body composition, and physical fitness in independent community-dwelling..., Abbasi [/bib_ref] and have reported that serum DHEA level was an independent associated factor for calf-muscle area. [bib_ref] Effect of DHEAS on skeletal muscle over the life span: the InCHIANTI..., Valenti [/bib_ref] However, data supporting an effect of DHEA supplementation in preventing sarcopenia and frailty are equivocal. [bib_ref] DHEA in elderly women and DHEA or testosterone in elderly men, Nair [/bib_ref] [bib_ref] Effects of DHEA replacement on bone mineral density and body composition in..., Villareal [/bib_ref] [bib_ref] Dehydroepiandrosterone supplementation in elderly men: a meta-analysis study of placebo-controlled trials, Corona [/bib_ref] ## Study strengths and limitations As the first investigation on this specific question, our results have important implications for further research. Moreover, we have made comprehensive adjustments for major factors related to skeletal muscle mass (high-sensitivity C-reactive protein, hormone profiles, vitamin D, nutritional status, cognitive function, physical activity), which previous studies did not take into account. Nevertheless, there are several noteworthy limitations. First, due to the cross-sectional design, the causality of relationships between the biomarkers assayed and muscle mass could not be established; low muscle mass may either cause or result from low serum myostatin. Second, the dynamic effect of myostatin-activinfollistatin interaction on skeletal muscle mass is uncertain. Third, low serum myostatin might result from low muscle mass among otherwise healthy older adults not currently experiencing rapid muscle loss. # Conclusions Male sex, malnutrition, lower serum DHEA, and low serum myostatin status were significantly associated with low muscle mass among otherwise healthy community-living older adults in Taiwan; however, the associations with low serum myostatin and low skeletal muscle mass were observed only in men, not women. Although myostatin signalling is a potential pharmaceutical target for sarcopenia, our results do not support using serum myostatin levels to diagnose sarcopenia nor to monitor its response to treatments. Further research to clarify the potential of myostatin as a biomarker is needed. [table] Table 1: Demographic characteristics of subjects with low vs. normal relative appendicular skeletal muscle mass Data show number (%) or mean ± standard deviation [/table] [table] Table 3: Independent predictors of low muscle mass in men and women aged ≥53 years [/table] [table] Table 2: Independent predictors of low muscle mass among otherwise healthy community-living older adults [/table]

CRISPR-Based Genetic Switches and Other Complex Circuits: Research and Application Citation: Du, P.; Lou, C.; Zhao, X.; Wang, Q.; Ji, X.; Wei, W. CRISPR-Based Genetic Switches and OtherAbstract: CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and endoribonuclease have served in genetic switches for activating or repressing gene expression, at both transcriptional and translational levels. With these genetic switches, more complex circuits have been assembled to achieve sophisticated functions including inducible switches, non-linear response and logical biocomputation. As more CRISPR enzymes continue to be excavated, CRISPR-based genetic switches will be used in a much wider range of applications. # Introduction As a fast-growing multidisciplinary field, synthetic biology has aimed to revolutionize biological research with the concept of engineering since its earliest days. In two decades, numerous synthetic parts with increasing complexity have emerged with the capability of mimicking the function of a number of basic electric circuits, including biological bistable switches [bib_ref] Construction of a genetic toggle switch in Escherichia coli, Gardner [/bib_ref] , oscillators [bib_ref] A sensing array of radically coupled genetic 'biopixels', Prindle [/bib_ref] , spatial pattern formation [bib_ref] Sequential establishment of stripe patterns in an expanding cell population, Liu [/bib_ref] [bib_ref] A unified design space of synthetic stripe-forming networks, Schaerli [/bib_ref] , logic gates [bib_ref] Robust multicellular computing using genetically encoded NOR gates and chemical 'wires', Tamsir [/bib_ref] [bib_ref] Programmable full-adder computations in communicating three-dimensional cell cultures, Auslander [/bib_ref] [bib_ref] Genomic mining of prokaryotic repressors for orthogonal logic gates, Stanton [/bib_ref] , memory devices [bib_ref] Genomically encoded analog memory with precise in vivo DNA writing in living..., Farzadfard [/bib_ref] [bib_ref] Rewritable digital data storage in live cells via engineered control of recombination..., Bonnet [/bib_ref] , intercellular communication [bib_ref] De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication..., Du [/bib_ref] [bib_ref] A scalable peptide-GPCR language for engineering multicellular communication, Billerbeck [/bib_ref] [bib_ref] Synthetic two-way communication between mammalian cells, Bacchus [/bib_ref] [bib_ref] Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis..., Chen [/bib_ref] and so on. Built upon these parts, synthetic biological circuits have been introduced into a wide range of applications including autonomous metabolic engineering [bib_ref] Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing..., Gupta [/bib_ref] [bib_ref] Self-induced metabolic state switching by a tunable cell density sensor for microbial..., Soma [/bib_ref] [bib_ref] Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon..., Tsao [/bib_ref] [bib_ref] Bacterial co-culture with cell signaling translator and growth controller modules for autonomously..., Stephens [/bib_ref] , cell-based therapies [bib_ref] Programming gene and engineered-cell therapies with synthetic biology, Kitada [/bib_ref] , antibiotic-free pathogen control [bib_ref] Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms, Sedlmayer [/bib_ref] [bib_ref] Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen, Saeidi [/bib_ref] and so on. These circuits operate on all levels of life including transcription, translation and post-translation levels. Nevertheless, at their core, the essence of synthetic circuits is to exert precise spatial and temporal control of gene expression as output signals in response to input signals. Compared to the traditional gene regulation elements such as inducible promoters, synthetic circuits are far superior because of their complexity that enables a sensitive or adaptative response, multiple inputs/outputs and logical signal computation. However, attempts to further increase the complexity of genetic circuits have been hindered by the inherent properties of basic synthetic parts. For example, transcription factors (TFs), the most widely adopted gene expression regulator in circuits, are known to be challenging from an engineering perspective. Specifically, the TFs offer important characteristics such as a high dynamic range and ultrasensitive response to the regulation of gene expression, but engineering a TF with specific parameters is a difficult task because of (1) poor designability, ## Crispr-based genetic switches in transcription level The first widely used CRISPR-based genetic switches functioning at transcription level are the CRISPR/dCas9 systems designed based on the Cas9 endonuclease, an RNAguided DNA endonuclease originating from subtype II of the class 2 CRISPR system [bib_ref] DNA targeting specificity of RNA-guided Cas9 nucleases, Hsu [/bib_ref]. As a CRISPR endonuclease, Cas9 identifies its target through a short PAM (protospacer adjacent motif, NGG in Streptococcus pyogenes), binds to the adjacent 20 nt dsDNA sequence through two RNA duplexes and cleaves at a specific location [bib_ref] CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III, Deltcheva [/bib_ref]. Such specificity and programmability make Cas9 an ideal candidate as a tool for gene editing and gene expression regulation. For simplicity and convenience, the two RNA duplexes for Cas9 were combined as a single guide RNA (sgRNA) [bib_ref] A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Jinek [/bib_ref]. In addition, although the typical length of dsDNA binding region within a sgRNA is 20 nt, shortening such a length to 17-18 nt could benefit the on-target cleavage efficiency of Cas9 [bib_ref] Improving CRISPR-Cas nuclease specificity using truncated guide RNAs, Fu [/bib_ref]. To engineer endonucleases as a part of genetic switches, the endonuclease activity of these enzymes was deactivated, leaving only the DNA binding activity. Take Cas9 as an example. Two mutations (D10A and H840A) were introduced to the RuvC and HNH endonuclease domain of SpCas9 (Cas9 from Streptococcus pyogenes), which abolished its ability to cleave dsDNA. The resulting deactivated Cas9, with the capability to tightly bind to DNA, was named dCas9 and often used as a programmable "roadblock" of transcription in genetic switches [bib_ref] Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Qi [/bib_ref] [fig_ref] Figure 1: Schematic representation of CRISPR-based genetic switches in transcription level [/fig_ref] , left). Such a "roadblock" can serve as a transcriptional repressor, which is often referred to as CRISPR interference (CRISPRi). Therefore, genetic switches based on CRISPRi would function as a NOT gate, which generates an output decreased signal as the input signal increases. In recent years, dCas9 has been successfully demonstrated as CRISPRi alone in bacteria [bib_ref] Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Qi [/bib_ref] [bib_ref] Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas..., Bikard [/bib_ref] , alone or with fused additional Mxi1 domain in yeast [bib_ref] CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Gilbert [/bib_ref] , and a fused KRAB (Krüppel associated box) [bib_ref] Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas, Farzadfard [/bib_ref] [bib_ref] CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Gilbert [/bib_ref] [bib_ref] Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory..., Thakore [/bib_ref] [bib_ref] CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs, Mandegar [/bib_ref] or SRDX (EARrepression) [bib_ref] A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation, Lowder [/bib_ref] domain in mammalian cells and plants, respectively [fig_ref] Figure 1: Schematic representation of CRISPR-based genetic switches in transcription level [/fig_ref] , right). On the other hand, dCas9 can also be engineered as a transcriptional activator (CRISPRa), by incorporating an activation domain by either direct fusion, RNA-scaffold recruitment, or the combination of both approaches [fig_ref] Figure 1: Schematic representation of CRISPR-based genetic switches in transcription level [/fig_ref]. For prokaryotic cells, the activation domains available to be directly fused to dCas9 include the phage activator AsiA and the RNAP ω (omega) subunit [bib_ref] Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas..., Bikard [/bib_ref] [bib_ref] Programmable CRISPR-Cas transcriptional activation in bacteria, Ho [/bib_ref]. Some other activation domains, including SoxS and PspF, can be fused to RNA binding domains and indirectly recruited by dCas9 through binding with the RNA scaffolds connected to sgRNA [bib_ref] Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria, Dong [/bib_ref] [bib_ref] Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target..., Fontana [/bib_ref] [bib_ref] Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria, Liu [/bib_ref]. Interestingly, the output of transcription regulation can be tuned by manipulating the DNA binding region of sgRNA by introducing mismatches [bib_ref] Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria, Liu [/bib_ref]. In eukaryotic cells, all the reported dCas9-based transcriptional activators are designed by direct fusion of an activation domain, including multiple copies of Herpes simplex viral protein 16 domains (VP64) [bib_ref] Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas, Farzadfard [/bib_ref] [bib_ref] CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes, Gilbert [/bib_ref] [bib_ref] A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation, Lowder [/bib_ref] [bib_ref] RNA-guided gene activation by CRISPR-Cas9-based transcription factors, Perez-Pinera [/bib_ref] [bib_ref] CRISPR RNA-guided activation of endogenous human genes, Maeder [/bib_ref] , the p65 domain [bib_ref] Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas, Farzadfard [/bib_ref] , the combined VPR (VP64-p65-RTA) [bib_ref] Highly efficient Cas9-mediated transcriptional programming, Chavez [/bib_ref] and VTR3 [bib_ref] Rational Design of Mini-Cas9 for Transcriptional Activation, Ma [/bib_ref] domains, the multimeric peptide array (SunTag) [bib_ref] A protein-tagging system for signal amplification in gene expression and fluorescence imaging, Tanenbaum [/bib_ref] and the synergistic activation mediator (SAM) [bib_ref] Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Konermann [/bib_ref] [fig_ref] Figure 1: Schematic representation of CRISPR-based genetic switches in transcription level [/fig_ref]. To push the CRISPR technology forward, new CRISPR endonucleases besides Cas9 have been continuously excavated in the past decade. One subtype V endonuclease of the class 2 CRISPR system, Cas12a (previously known as Cpf1), has drawn more attention than others for its unique dual functionality to process its own pre-crRNA before cleaving dsDNA [bib_ref] CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Chen [/bib_ref]. In the same way as Cas9, Cas12a has also been mutated to serve as transcription regulators. Multiple versions of DNase deactivated Cas12a (dCas12a), some of which have shown great potential for replacing dCas9 in genetic switches [bib_ref] Systematically investigating the key features of the DNase deactivated Cpf1 for tunable..., Miao [/bib_ref] [bib_ref] A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli, Kim [/bib_ref] [bib_ref] Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors, Liu [/bib_ref]. For example, dCas12a has been used alone or fused with KRAB or SRDX domains as CRISPRi, and functions as CRISPRa after fusing with VP64, p65 or VTR domain [bib_ref] Systematically investigating the key features of the DNase deactivated Cpf1 for tunable..., Miao [/bib_ref] [bib_ref] A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli, Kim [/bib_ref] [bib_ref] Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors, Liu [/bib_ref] [bib_ref] Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum, Li [/bib_ref] [bib_ref] Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts, Campa [/bib_ref] [bib_ref] Gene repression via multiplex gRNA strategy in Y. lipolytica. Microb, Zhang [/bib_ref] [bib_ref] Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors, Tak [/bib_ref]. As with transcriptional factors, the CRISPR-based systems are also not perfect. The CRISPR/Cas9 system has been troubled by (1) the toxicity from off-target effects [bib_ref] Off-target Effects in CRISPR/Cas9-mediated Genome Engineering, Zhang [/bib_ref] [bib_ref] High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells, Fu [/bib_ref] [bib_ref] High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity, Pattanayak [/bib_ref] , (2) the large size of Cas9/dCas9 and (3) the lack of non-linearity [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [bib_ref] Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits, Menn [/bib_ref]. First, the toxicity of Cas9 has severely limited its application potential, particularly in gene therapy where safety is the top concern. Meanwhile, in a complex circuit with multiple dCas9-based switches, the off-target binding of dCas9 could also affect the orthogonality among these genetic switches. Second, the relatively large size of dCas9 makes it easy to cause physiological burden to the host, which limits its use in instances that require a high expression level of dCas9. For example, the limitation of dCas9 expression level could cap the maximum activation signal in CRISPRa or increase the background signal in CRISPRi, both conditions that pose a negative impact on the performance of dCas9-based genetic switches by reducing their dynamic range. Notably, the search for endonuclease smaller than Cas9 has led to the discovery of more compact endonucleases, including Cas12a [bib_ref] CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Chen [/bib_ref] , Cas12b [bib_ref] Repurposing CRISPR-Cas12b for mammalian genome engineering, Teng [/bib_ref] , CasΦ [bib_ref] CRISPR-CasPhi from huge phages is a hypercompact genome editor, Pausch [/bib_ref] and CasX [bib_ref] CasX enzymes comprise a distinct family of RNA-guided genome editors, Liu [/bib_ref] , which greatly promoted the delivery of CRISPR endonuclease in gene therapy. Third, the lack of non-linear behavior (e.g., ultrasensitivity) of CRISPR-based genetic switches is determined by the binding mechanism of CRISPR endonuclease, which functions by a monomeric endonuclease binding to DNA via a single binding site [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref]. Achieving nonlinear functions with CRISPR-based systems requires the cooperative activity of multimers or multiple binding sites [bib_ref] CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli, Ji [/bib_ref] , as in the TF-based genetic switches [bib_ref] Engineering the Ultrasensitive Transcription Factors by Fusing a Modular Oligomerization Domain, Hou [/bib_ref] [bib_ref] Rational Design of an Ultrasensitive Quorum-Sensing Switch, Zeng [/bib_ref]. Therefore, to achieve non-linear functions with CRISPR-based switches, a more complex design is required. To push the CRISPR technology forward, new CRISPR endonucleases besides Cas9 have been continuously excavated in the past decade. One subtype V endonuclease of the class 2 CRISPR system, Cas12a (previously known as Cpf1), has drawn more attention than others for its unique dual functionality to process its own pre-crRNA before cleaving dsDNA [bib_ref] CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Chen [/bib_ref]. In the same way as Cas9, Cas12a has also been mutated to serve as transcription regulators. Multiple versions of DNase deactivated Cas12a (dCas12a), some of which have shown great potential for replacing dCas9 in genetic switches [bib_ref] Systematically investigating the key features of the DNase deactivated Cpf1 for tunable..., Miao [/bib_ref] [bib_ref] A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli, Kim [/bib_ref] [bib_ref] Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors, Liu [/bib_ref]. For example, dCas12a has been used alone or fused with KRAB or SRDX domains as CRISPRi, and functions as CRISPRa after fusing with VP64, p65 or VTR domain [bib_ref] Systematically investigating the key features of the DNase deactivated Cpf1 for tunable..., Miao [/bib_ref] [bib_ref] A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli, Kim [/bib_ref] [bib_ref] Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors, Liu [/bib_ref] [bib_ref] Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum, Li [/bib_ref] [bib_ref] Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts, Campa [/bib_ref] [bib_ref] Gene repression via multiplex gRNA strategy in Y. lipolytica. Microb, Zhang [/bib_ref] [bib_ref] Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors, Tak [/bib_ref]. As with transcriptional factors, the CRISPR-based systems are also not perfect. The CRISPR/Cas9 system has been troubled by (1) the toxicity from off-target effects [bib_ref] Off-target Effects in CRISPR/Cas9-mediated Genome Engineering, Zhang [/bib_ref] [bib_ref] High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells, Fu [/bib_ref] [bib_ref] High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity, Pattanayak [/bib_ref] , (2) the large size of Cas9/dCas9 and (3) the lack of non-linearity [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [bib_ref] Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits, Menn [/bib_ref]. First, the toxicity of Cas9 has severely limited its application potential, particularly in gene therapy where safety is the top concern. Meanwhile, in a complex circuit with multiple dCas9-based ## Crispr-based genetic switches in translation level Besides DNA endonuclease, CRISPR systems also contain endoribonuclease that recognize specific RNA sequences and structures on the repeats of pre-crRNA and cleaves at a certain location [bib_ref] Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering, Wright [/bib_ref]. The genetic switches designed based on these endoribonucleases operate at the level of translation, which can work alongside the switches at the transcription level in genetic circuits [bib_ref] RNA processing enables predictable programming of gene expression, Qi [/bib_ref]. The first widely used CRISPR endoribonuclease in a genetic switch is Csy4, a type I CRISPR endoribonuclease discovered in Pseudomonas aeruginosa that recognizes a 28-nt RNA hairpin structure and precisely cleaves between the 20G and 21C [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref] [bib_ref] Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR..., Haurwitz [/bib_ref] [fig_ref] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level [/fig_ref]. Such precision enables Csy4 to control translation via cleaving of its target RNA hairpin inserted at the 5 capping or 3 UTR region of an mRNA, which could severely reduce mRNA stability and, consequently, translation rate [bib_ref] Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4, Borchardt [/bib_ref] [fig_ref] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level [/fig_ref]. Furthermore, Csy4 has been demonstrated to control translation in a more sophisticated way for better performance and programmability. For example, a Csy4-based translational activator has been engineered by inserting the Csy4 target RNA hairpin between an RBS (Ribosome Binding Site) and a cis-repressive element [bib_ref] Engineering Translational Activators with CRISPR-Cas System, Du [/bib_ref]. As a result, mRNA translation is inhibited as the cis-repressive element binds to RBS in a complementary manner, and will be restarted after Csy4 cleavage that releases the cis-repressive element from the RBS [fig_ref] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level [/fig_ref]. As an endoribonuclease, Csy4 has also been used in many multiplexed gene regulations at translation level in yeast and mammalian cells [bib_ref] Multiplexed and programmable regulation of gene networks with an integrated RNA and..., Nissim [/bib_ref] [bib_ref] Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Zalatan [/bib_ref] [bib_ref] Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast, Mccarty [/bib_ref] [bib_ref] Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system, Lian [/bib_ref]. By simultaneously processing multiple gRNAs with different target sequences for directing dCas9, Csy4, along with other similar endoribonuclease such as Cas5d, Cas6a and Cse3, can build complex logic gates for precisely regulating gene expression [bib_ref] Engineering Translational Activators with CRISPR-Cas System, Du [/bib_ref] [fig_ref] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level [/fig_ref]. Moreover, Cas12a, with the dual functionality of cleaving dsDNA and processing its own gRNA, is also valuable in multiplexed gene regulation because it allows simultaneous control of multiple genes using a single enzyme pairing with different gRNAs, which is much simpler than the Csy4/dCas9 systems [bib_ref] Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum, Li [/bib_ref] [bib_ref] Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts, Campa [/bib_ref] [bib_ref] Gene repression via multiplex gRNA strategy in Y. lipolytica. Microb, Zhang [/bib_ref] [bib_ref] Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a, Ciurkot [/bib_ref] [bib_ref] Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors, Tak [/bib_ref] [fig_ref] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level [/fig_ref]. Besides RNA endoribonuclease, another category of CRISPR enzyme, the RNA-guided RNA endonuclease, have also emerged in recent years with the potential as genetic switches at translational level, namely the Cas13a and Cas7-11 endonuclease [bib_ref] Programmable RNA targeting with the single-protein CRISPR effector Cas7-11, Ozcan [/bib_ref] [bib_ref] C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Abudayyeh [/bib_ref] [bib_ref] RNA targeting with CRISPR-Cas13, Abudayyeh [/bib_ref]. These enzymes possess two distinct RNase activities: (1) processing their own pre-crRNA in a way similar to Csy4 [bib_ref] Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection, East-Seletsky [/bib_ref] ; (2) targeting specific RNA sequence for cleavage. Such dual functionality makes them particularly ideal for the knockdown of RNA transcripts in vivo. Specifically, Cas13a, a subtype VI effector in class 2 CRISPR-Cas systems, previously known as C2c2, has been used for gene silencing in E. coli, plants and human cells [bib_ref] RNA targeting with CRISPR-Cas13, Abudayyeh [/bib_ref] [bib_ref] Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors..., Liao [/bib_ref]. Cas13a possesses a unique "collateral" cleavage effect, which means, upon recognition of its target, Cas13a engages cleavage of nearby non-target RNA [bib_ref] C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Abudayyeh [/bib_ref]. Such a cleavage pattern of Cas13a has been harnessed in a nucleic acid detection method known as SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), in which the target nucleotide sequence is incorporated in the gRNA, and an RNA probe is designed to be fluorescent after being cleaved via "collateral" cleavage. Cas7-11, on the other hand, is a subtype III effector in the class 1 CRISPR-Cas system recently reported to have a similar function as Cas13a [bib_ref] Programmable RNA targeting with the single-protein CRISPR effector Cas7-11, Ozcan [/bib_ref]. Currently, although these endonucleases are primarily being employed for gene silencing, it is apparent that they have great potential to be engineered as multiplexed genetic switches similar to Csy4 and Cas12a. ## Application of crispr-based switches in genetic circuits With the individual genetic switches, larger circuits can be built to offer more sophisticated functions and provide greater potential for a wider range of applications. Thus far, a number of CRISPR-based genetic circuits have been reported, which primarily incorporate CRISPR-based genetic switches in three different ways: (1) Ligand/Light-inducible genetic switches, (2) genetic switches with certain non-linear behavior or feedback patterns, (3) multi-input biocomputation circuits [fig_ref] Table 2: Applications of CRISPR-based genetic switches in circuits [/fig_ref]. The first refers to CRISPR-based genetic switches capable of sensing input signals (e.g., chemical ligands) and generates certain output signals; the second mainly includes the circuits that still function as a genetic switch, but offer non-linear functions such as ultrasensitivity, biostability, oscillation or IFFL (incoherent feed-forward loop); the third implies the circuits capable of computing Boolean logics including AND, NOT, NOR and XOR, etc. Ligand/Light-inducible genetic switches are powerful tools for understanding the spatial and temporal pattern of gene expression because they offer the crucial capability of controlling genetic switches with external or internal input signals, such as chemicals or the light of specific wavelengths [fig_ref] Figure 3: Schematic representation of genetic switches inducible by light, chemical ligands and nucleotide... [/fig_ref]. Typically, such inducibility is achieved by endonuclease (mainly dCas9) fused with chemical-induced dimerizing domains (CID) or optogenetically inducible dimerizing domains (OID) that sense chemicals or light signals, respectively. Specifically, the two parts of dimerization domains are separately fused with endonucleases (dCas9 or dCas12a) and the effector domain. Then, dimerization occurs when certain chemical ligands or lights of certain wavelengths are detected, which completes the recruitment of the effector domain to the endonuclease binding sites on DNA. Thus far, the reported CIDs include FKBP-FRB [bib_ref] Orthogonal Genetic Regulation in Human Cells Using Chemically Induced CRISPR/Cas9 Activators, Bao [/bib_ref] [bib_ref] A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche [/bib_ref] and DmrA-DmrC [bib_ref] Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors, Tak [/bib_ref] induced by rapamycin, ABI-PYL1 induced by abscisic acid (ABA) [bib_ref] Orthogonal Genetic Regulation in Human Cells Using Chemically Induced CRISPR/Cas9 Activators, Bao [/bib_ref] [bib_ref] Complex transcriptional modulation with orthogonal and inducible dCas9 regulators, Gao [/bib_ref] and GID1-GAI24 induced by gibberellin (GA) [bib_ref] Complex transcriptional modulation with orthogonal and inducible dCas9 regulators, Gao [/bib_ref]. Meanwhile, various OIDs have also been reported, including PhyB-PIF induced by red light [bib_ref] Spatiotemporal control of cell signalling using a light-switchable protein interaction, Levskaya [/bib_ref] , and pMag-nMag and CRY2-CIB1 induced by blue light [bib_ref] CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation, Nihongaki [/bib_ref] [bib_ref] Photoactivatable CRISPR-Cas9 for optogenetic genome editing, Nihongaki [/bib_ref] [bib_ref] A light-inducible CRISPR-Cas9 system for control of endogenous gene activation, Polstein [/bib_ref] [bib_ref] CRISPR-Cas9-based photoactivatable transcription system, Nihongaki [/bib_ref]. Alternatively, genetic switches can also be constructed based on split dCas9 [fig_ref] Figure 3: Schematic representation of genetic switches inducible by light, chemical ligands and nucleotide... [/fig_ref]. In this case, dCas9 is split into two pieces and each piece is fused with part of a heterodimer [bib_ref] A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche [/bib_ref] [bib_ref] Photoactivatable CRISPR-Cas9 for optogenetic genome editing, Nihongaki [/bib_ref]. Upon ligand induction, heterodimerization leads to the restoration of dCas9 enzymatic activity and subsequent DNA binding, which allows dCas9 to act as a transcriptional repressor [bib_ref] A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche [/bib_ref]. Similarly, Cas9 can also be split and restored as an inducible genome editor [bib_ref] Photoactivatable CRISPR-Cas9 for optogenetic genome editing, Nihongaki [/bib_ref]. GA: gibberellin; ABA: abscisic acid; DAPG: 2,4-Diacetylphloroglucinol; Ara: Arabinose; AHL: acyl homoserine lactone; aTc: Anhydrotetracycline. Besides chemicals and light signals, oligonucleotide sequences can also be a form of input signal that activates inducible CRISPR-based genetic switches. In other words, CRISPR endonuclease (e.g., dCas9 or dCasd12a) recognizes specific DNA or RNA sequences as input signals, and activates subsequent signals accordingly. Such switches are particularly suitable for nucleotide detection, which makes them quite popular as IVD (in vitro diagnostic) methods. For example, several toolkits for detecting SARS-CoV-2 have been developed based on Cas12a or Cas13a, which functions by detecting specific RNA and cleaving complementary DNA or RNA probes as output signals, respectively [bib_ref] CRISPR-Cas12-based detection of SARS-CoV-2, Broughton [/bib_ref] [bib_ref] Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay, Ding [/bib_ref] [bib_ref] An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid..., Ooi [/bib_ref] [bib_ref] Rapid detection of SARS-CoV-2 with CRISPR-Cas12a, Xiong [/bib_ref] [bib_ref] Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy, Fozouni [/bib_ref] [bib_ref] Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA, Patchsung [/bib_ref] [fig_ref] Figure 3: Schematic representation of genetic switches inducible by light, chemical ligands and nucleotide... [/fig_ref]. Additionally, IVD methods using dCas9 have also been reported, in which the two parts of a split luciferase are fused with two dCas9 molecules. Positive signals are generated when the enzymatic activity of luciferase is restored upon proper placement of the two dCas9 on the target DNA [bib_ref] Paired Design of dCas9 as a Systematic Platform for the Detection of..., Zhang [/bib_ref] [fig_ref] Figure 3: Schematic representation of genetic switches inducible by light, chemical ligands and nucleotide... [/fig_ref] , right). As described above, one of the problems of CRISPR-based genetic switches is the lack of non-linear function. However, such a shortcoming can be overcome with a more complex design of CRISPR-based circuits that offers ultrasensitive, bistable and oscillatory signals. For example, toggle switches with ultrasensitive and bistable signals have been built with dCas9 in E. coli that achieved bistable toggle between two states [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Moreover, oscillators based on Cas9 and Cas12a have also emerged recently with a robust oscillatory state in microfluid chambers [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] [bib_ref] Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas, Kuo [/bib_ref] [bib_ref] Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback..., Henningsen [/bib_ref]. Additionally, circuits that exhibit spatial-temporal behaviors can also be constructed with CRISPR-based genetic switches. By combining CRISPRi and CRISPRa, IFFL circuits have been constructed in E. coli and mammalian cells that display pulse-generating and stripe-forming patterns [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] [bib_ref] An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells, Jones [/bib_ref] [bib_ref] Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells, Nakamura [/bib_ref]. Biocomputation with logic gates is an important task of genetic circuits for signal integration, processing and logic computation. The foundation of biocomputation is various types of logic gates with robust performance. Many Boolean logic gates, such as AND, NOT and NOR gates, have been constructed with CRISPR-based circuits that functions in E. coli, yeast and human cells. For example, one-input logic gate, such as a NOT gate, can simply be a dCas9-based repressor, which converts "1" to "0" [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Likewise, more layer of repressors (or activators) can be connected in a consecutive manner, which will further convert the signal back to "1" and then jump back and forth between "0" and "1" [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [bib_ref] Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits, Menn [/bib_ref] [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] [bib_ref] Digital logic circuits in yeast with CRISPR-dCas9 NOR gates, Gander [/bib_ref] [bib_ref] A Framework for the Modular and Combinatorial Assembly of Synthetic Gene Circuits, Santos-Moreno [/bib_ref] [bib_ref] CRISPR transcriptional repression devices and layered circuits in mammalian cells, Kiani [/bib_ref] [bib_ref] Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9, Didovyk [/bib_ref] [bib_ref] A CRISPR/Cas9-based central processing unit to program complex logic computation in human..., Kim [/bib_ref]. Thus far, the input signal can be converted up to seven times with NOT gates [bib_ref] Digital logic circuits in yeast with CRISPR-dCas9 NOR gates, Gander [/bib_ref]. On the other hand, the circuit designs to integrate two signals with a single logic gate are more complex and diverse. A straightforward design strategy is to control the expression of two orthogonal sgRNAs and their corresponding endonucleases with two different inducers that serve as input signals [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref]. In this case, the type of logic gate is determined by the design of the two endonucleases. Specifically, two transcriptional activators will make the circuit an AND gate [bib_ref] A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast, Hofmann [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref] , while two transcriptional repressors will result in a NOR gate [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [bib_ref] Digital logic circuits in yeast with CRISPR-dCas9 NOR gates, Gander [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Alternatively, an AND gate can be built with other methods such as heterodimerization induced by two different ligands [bib_ref] A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche [/bib_ref] [bib_ref] Complex transcriptional modulation with orthogonal and inducible dCas9 regulators, Gao [/bib_ref] [bib_ref] Photoactivatable CRISPR-Cas9 for optogenetic genome editing, Nihongaki [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Notably, a NOR gate is known to be "Boolean-complete", which means all types of logic gates can be built by combining NOR gates in different ways. For example, converting the output signal of a NOR gate with a NOT gate creates an OR gate [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Similarly, by converting the two input signals of a NOR gate with two NOT gates beforehand, the combination of the three gates (two NOT gates and one NOR gate) equals to an AND gate [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref]. As described above, one of the problems of CRISPR-based genetic switches is th of non-linear function. However, such a shortcoming can be overcome with a more plex design of CRISPR-based circuits that offers ultrasensitive, bistable and oscil signals. For example, toggle switches with ultrasensitive and bistable signals have built with dCas9 in E. coli that achieved bistable toggle between two states [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] (F 4A). Moreover, oscillators based on Cas9 and Cas12a have also emerged recently w robust oscillatory state in microfluid chambers [bib_ref] Multistable and dynamic CRISPRi-based synthetic circuits, Santos-Moreno [/bib_ref] [bib_ref] Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas, Kuo [/bib_ref] [bib_ref] Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback..., Henningsen [/bib_ref]. Additionally, circuits that e AND gate can be built with other methods such as heterodimerization induced by two different ligands [bib_ref] A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche [/bib_ref] [bib_ref] Complex transcriptional modulation with orthogonal and inducible dCas9 regulators, Gao [/bib_ref] [bib_ref] Photoactivatable CRISPR-Cas9 for optogenetic genome editing, Nihongaki [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Notably, a NOR gate is known to be "Boolean-complete", which means all types of logic gates can be built by combining NOR gates in different ways. For example, converting the output signal of a NOR gate with a NOT gate creates an OR gate [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref] [fig_ref] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits [/fig_ref]. Similarly, by converting the two input signals of a NOR gate with two NOT gates beforehand, the combination of the three gates (two NOT gates and one NOR gate) equals to an AND gate [bib_ref] Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks, Nielsen [/bib_ref]. # Conclusions The enzymes discovered from CRISPR systems, with their abundance, diversity and unrivaled programmability, have shown remarkable value and potential in the construction of genetic switches operating in both transcription and translation levels. The prevalence of their existence in nature has also been a great gift that has bestowed Cas9, Cas12a, Cas13, Csy4, and Cas7-11 and much more to come. The advantages of genetic switches constructed with these enzymes have attracted the interest of many circuit designers who have reported a number of circuits with diverse functions in return. It is apparent that CRISPR-based genetic switches will continue to stay in the spotlight of the research of synthetic biology and make their potential into reality in various applications. Author Contributions: Conceptualization, C.L. and P.D.; resources, P.D., X.Z., X.J. and W.W.; writing-original draft preparation, P.D.; writing-review and editing, P.D., C.L., X.Z. and Q.W. All authors have read and agreed to the published version of the manuscript. ## Conflicts of interest: The authors declare no conflict of interest. [fig] Figure 1: Schematic representation of CRISPR-based genetic switches in transcription level. (A) Schematic of dCas9 or dCas12a function as a transcriptional repressor by blocking RNAP by itself (Left), with a fused repressor such as Mxi1, KRAB or SRDX domain (Right). (B) Schematic of dCas9 or dCas12a function as a transcription activator by fusing with an activation domain such as AsiA and the RNAP ω (omega) subunit, VP64, VPR, etc. (left), or by recruiting an activator through the aptamer fused with sgRNA such as SoxS and PspF (Right). (C) Schematic of dCas9 or dCas12a function as a transcriptional activator in combination with various types of activation domains. Red light indicates repression, green light indicates activation. [/fig] [fig] Figure 2: Schematic representation of CRISPR-based genetic switches in translational level. (A) Csy4 cleavage of the RNA transcript in the processing of gRNA. (B) Csy4 function as a translational repressor by decreasing mRNA stability through cleavage of the 3′ end. (C) Csy4 function as a translational activator. (D) Csy4 and dCas9 in multiplexed gene regulation. Different color of sgRNA indicates different sgRNA that directs dCas9 to the corresponding target genes. (E) Multiplexed gene regulation based on the dual functionality of Cas12a. Different color of sgRNA indicates different sgRNA that directs dCas12a to the corresponding target genes. Red light indicates repression, green light indicates activation.Besides RNA endoribonuclease, another category of CRISPR enzyme, the RNAguided RNA endonuclease, have also emerged in recent years with the potential as genetic switches at translational level, namely the Cas13a and Cas7-11 endonuclease[78][79][80]. These enzymes possess two distinct RNase activities: (1) processing their own pre-crRNA in a way similar to Csy4 [81]; (2) targeting specific RNA sequence for cleavage. Such dual functionality makes them particularly ideal for the knockdown of RNA transcripts in vivo. [/fig] [fig] Life 2021 ,: 11, 1255 [/fig] [fig] Figure 3: Schematic representation of genetic switches inducible by light, chemical ligands and nucleotide sequences. (A Genetic switches activated by light-(left) or chemical ligand-(middle) induced dimerization that connects CRISPR endo nuclease (dCas9 or dCas12) and activation/repression domain, as well as dimerization of split dCas9 induced by chemica inducer (right). Activation or repression depends on the type of the domains recruited after dimerization. Red light ind cates repression, green light indicates activation. (B) Genetic switches activated by nucleotide sequences identified b Cas12a (left), Cas13 (middle) and dCas9 (right). Glows indicate fluorescence on probes (left and middle), or luminescenc stimulated by luciferase. [/fig] [fig] Figure 4: Schematic representation of CRISPR-based non-linear and Boolean logic circuits. (A) Toggle switch built by dCas9 with two different gRNA controlled by two different input signals. (B-E) Boolean logic circuits including NOT gate (B), AND gate (C), NOR gate (D) and OR gate (E). [/fig] [fig] Funding: This research was funded by the National Key Research and Development Program of China grant number [No. 2020YFA0907102]; the Chinese Academy of Sciences grant number [No. QYZDB-SSW-SMC050, No. XDPB1801] and Shenzhen Institute of Synthetic Biology grant number [No. JCHZ20200005]. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. [/fig] [table] Table 1: CRISPR-based genetic switches. [/table] [table] Table 2: Applications of CRISPR-based genetic switches in circuits. [/table]

Social distancing is a social dilemma game played by every individual against his/her population function ,pop_stg_eq_2] = test_run_1 () % % Simulation of collective behavior of social distancing % % (Small, multi-populations) % % m --# total individuals in population, m = 100 % % m1 --#individuals in subpop 1 % m2 --#individuals in subpop 2 % % A --connectivity matrix of social activities, n x n, n = 85 % w --contact weights assigned to social activities, n x 1 % % ind_stg_in_1 --initial strategies of individuals in subpop 1, n x m1 % pop_stg_in_1 --initial strategy of subpop 1, n x 1 % % ind_stg_in_2 --initial strategies of individuals in subpop 2, n x m2 % pop_stg_in_2 --initial strategy of subpop 2, n x 1 % % ind_stg_eq_1 --equilibrium strategies of individuals in subpop 1, n x m1 % pop_stg_eq_1 --equilibrium strategy of subpop 1, n x 1 % % ind_stg_eq_2 --equilibrium strategies of individuals in subpop 2, n x m2 % pop_stg_eq_2 --equilibrium strategy of subpop 2, n x 1 % % ret_info --1 --suceeds, 0 --fails % % Zhijun Wu, 12/20/2020, Math Dept, Iowa State University % % Load contact matrix and contact weights A = load('A.dat','-ascii'); w = load('w.dat','-ascii'); k = size(A,1); % Obtain # strategies and # individuals n = k; m = 10*n; m1 = m/2; m2 = m -m1; % activity sets for subpop 1 and sub pop 2 % % Subpopulation activity sets 1 % h1 = [1,1,1,1,1,1,1,0,0,0]'; h2 = [1,1,1,1,0,0,0,1,1,1]'; % Subpopulation activity sets 2 % % h1 = [1,1,1,1,1,1,1,0,1,0]'; % h2 = [1,1,1,1,1,0,0,1,0,1]'; ind_stg_in_1 = zeros(n,m1); pop_stg_in_1 = zeros(n,1); ind_stg_in_2 = zeros(n,m2); pop_stg_in_2 = zeros(n,1); K = 100; % Start with initial random strategies rng ('default'); for i = 0 : n-1 % Repeat with different initials for j = 0 : n-1 k = rem(i+j,n)+1; ind_stg_in_1(k,1:m1) = (j+1)*rand(1,m1); ind_stg_in_2(k,1:m2) = (j+1)*rand(1,m2); end for l = 1 : m1 ind_stg_in_1(1:n,l) = h1.*ind_stg_in_1(1:n,l); end for l = 1 : m1 ind_stg_in_1(1:n,l) = ind_stg_in_1(1:n,l) / sum(ind_stg_in_1(1:n,l)); end for l = 1 : m2 ind_stg_in_2(1:n,l) = h2.*ind_stg_in_2(1:n,l); end for l = 1 : m2 ind_stg_in_2(1:n,l) = ind_stg_in_2(1:n,l) / sum(ind_stg_in_2(1:n,l)); end for l = 1 : n pop_stg_in_1(l) = sum(ind_stg_in_1(l,1:m1)) / m1; end for l = 1 : n pop_stg_in_2(l) = sum(ind_stg_in_2(l,1:m2)) / m2; end % Start simulation, to reach equilibrium strategies function [ind_stg_eq_1,pop_stg_eq_1,ind_stg_eq_2,pop_stg_eq_2] = soc_dis_sim (ind_stg_in_1,pop_stg_in_1,act_set_1,ind_stg_in_2,pop_stg_in_2,act_set_2,A,w) % % Simulation of collective behavior of social distancing % % A --connectivity matrix of social activities, n x n, n = 10 % w --contact weights assigned to social activities, n x 1 % % ind_stg_in_1 --initial strategies of individuals in subpop 1, n x m1 % pop_stg_in_1 --initial strategy of subpop 1, n x 1 % % ind_stg_in_2 --initial strategies of individuals in subpop 2, n x m2 % pop_stg_in_2 --initial strategy of subpop 2, n x 1 % % ind_stg_eq_1 --equilibrium strategies of individuals in subpop 1, n x m1 % pop_stg_eq_1 --equilibrium strategy of subpop 1, n x 1 % % ind_stg_eq_2 --equilibrium strategies of individuals in subpop 2, n x m2 % pop_stg_eq_2 --equilibrium strategy of subpop 2, n x 1 % % act_set_1 --activity set for subpop 1 % act_set_2 --activity set for subpop 2 % % Zhijun Wu, 12/20/2020, Math Dept, Iowa State University % W = diag(w); A = (A*W + W*A) / 2; n = size(ind_stg_in_1,1); m1 = size(ind_stg_in_1,2); m2 = size(ind_stg_in_2,2); ind_stg_eq_1 = ind_stg_in_1; pop_stg_eq_1 = pop_stg_in_1; while (max_dev > 1.0e-8 && k < K) for j = 1 : m1 x1 = ind_stg_eq_1(1:n,j); y1 = pop_stg_eq_1; ind_stg_eq_1(1:n,j) = soc_dis_upd (x1,y1,h1,y2,h2,A); for l = 1 : n pop_stg_eq_1(l) = y1(l) + (ind_stg_eq_1(l,j) -x1(l)) / m1; end end for j = 1 : m2 x2 = ind_stg_eq_2(1:n,j); y2 = pop_stg_eq_2; [formula] [ind_stg_eq_1,pop_stg_eq_1,ind_stg_eq_2,pop_stg_eq_2] = soc_dis_sim (ind_stg_in_1,pop_stg_in_1,h1,ind_stg_in_2,pop_stg_in_2,h2,A,w); dlmwrite(['pop_stg_eq_1_',num2str(k),'.dat'],pop_stg_eq_1,'precision','%8.6f'); dlmwrite(['pop_stg_eq_2_', [/formula] ind_stg_eq_2(1:n,j) = soc_dis_upd (x2,y2,h2,y1,h1,A); for l = 1 : n pop_stg_eq_2(l) = y2(l) + (ind_stg_eq_2(l,j) -x2(l)) / m2; end end k = k + 1; e = ones(n,1); e1 = ones(m1,1); c1 = sqrt(sum(((ind_stg_eq_1 -pop_stg_eq_1*e1').*(h1*e1')).^2)); d1 = sum(c1) / m1; e2 = ones(m2,1); c2 = sqrt(sum(((ind_stg_eq_2 -pop_stg_eq_2*e2').*(h2*e2')).^2)); d2 = sum(c2) / m2; close all; end function ydata = myfun(x,xdata) ydata = -x(1)*atan(x(2)*(xdata -x(3))) + x(4); end end end %strategy i has higher contact, reduce its frequency: if (rel_con(i) < 0) if (x1(i) > y1(i)) x1(i) = x1(i) -1.0 * (x1(i) -y1(i)); else x1(i) = x1(i) -0.5 * min(y1(i)-x1(i),x1(i)-0.0); end end end end ind_stg_out_1 = x1 / sum(x1); end

Repeat sequences limit the effectiveness of lateral gene transfer and favored the evolution of meiotic sex in early eukaryotes ## Supporting information text supplementary materials and methods At the beginning of each simulation, every individual possesses a genome composed of unique protein-coding genes, each of which can exist in either a wildtype or deleterious mutant state. The genome is assumed to be circular (locus is contiguous with locus 1). The genome is interspersed at random intervals with a generic repeat sequence, at an initial density per protein-coding gene. The initial positions of the repeats are randomly sampled from a uniform distribution (i.e., all loci regions have the same probability of harbouring a repeat sequence at the beginning of the simulation). For simplicity, protein-coding genes and repeats are treated as unitary entities, and we neglect sequence variability within these genes. The new generation is obtained by sampling individuals, with replacement, from the old population [fig_ref] Figure S1: Illustration of the model dynamics [/fig_ref]. The probability of reproduction is proportional to the individual fitness. Following previous theoretical studies (1-4), we assume no epistatic interactions and measure fitness as a multiplicative function [formula] ! ( ) = (1 − ) "#$ ! (&) ,(1) [/formula] where ! ( ) is the number of different functional protein-coding genes possessed by an individual at time ( − ! ( ) is the number of genes that have been lost, either because of mutations or deletions). To avoid unnecessary complexity, we assume that gene duplication has a negligible effect on fitness, and only consider whether there is at least one functional copy of each protein-coding gene. Once a new generation is formed, the old generation dies, and its DNA forms the genetic pool from which the new generation acquires environmental DNA (eDNA) for recombination. We assume that eDNA strands are only stable for one generation before decaying irreversibly. Individuals of the new generation undergo LGT with a probability , which is taken to be a constant throughout each simulation (i.e. is independent of genome size and content or the amount of available eDNA, which may change during a simulation run). For each individual that undergoes LGT, a sequence of eDNA of length is randomly sampled from the eDNA pool. The requisite for successful recombination is homology between the terminal loci of the eDNA sequence and the host genome. Homology can be either to a protein-coding gene or a repeat sequence. These dynamics follow experimental evidence that recombination of nonhomologous DNA can take place fairly easily in the presence of homologous flanking sequences, but not in their absence (5, 6); integration of homologous DNA is estimated to be very considerably more likely than strongly divergent "foreign" DNA (5, 6). After a sequence is sampled from the eDNA pool, one of its terminal loci is randomly selected and matched with a homologous locus * in the host genome. If multiple homologous loci are present, one site is selected at random. The other terminal locus of the eDNA is then matched to a homologous locus ) in the host genome. If there is a single homologous locus ) in the recipient genome, the recombination probability is generated according to a Gaussian distribution, [formula] ( ) ) = * +√-. # " # / $ % &' ( 0 # (2) [/formula] where ) is the distance between * and ) , and the standard deviation is = √ (where is the length of the eDNA). Note that no recombination occurs with probability 1 − ( ) ). This function penalizes the probability of exchange over large distances (e.g., it is more likely that an eDNA sequence spanning 2 loci would recombine over a length spanning 2 loci than 20 loci). This is in agreement with experimental data on recombination length in bacteria, showing the rarity of large recombination events and deletions [bib_ref] Extensive cotransformation of natural variation into chromosomes of naturally competent Haemophilus influenzae, Mell [/bib_ref]. It also reflects the assumption that longer eDNA sequences have greater variance in the distribution of recombination probabilities. If there are multiple homologous loci ) in the recipient genome, one of the ) loci is selected to be the other terminal region of recombination given weights proportional to ( ) ). This favors loci with a small mismatch between ) and . We then as before apply the recombination probability ( ) ) at this chosen site. If recombination occurs, all the elements between * and ) (included) are substituted by the recombining eDNA sequence. If there is no match to either * or ) , recombination with the eDNA sequence is not possible and no genetic exchange takes place. ## 5 To model fully homologous recombination, we select two homologous loci * and ) as above. If the eDNA and genomic sequences contain exactly the same genes in the same order (either as wild-type or mutant alleles) recombination successfully takes place (we assume that point mutations do not significantly affect the probability of homologous recombination). The sequence between * and ) is excised and replaced by the eDNA sequence. Otherwise, no genetic exchange takes place. After LGT, each individual acquires new deleterious mutations, where is a random integer drawn from a Poisson distribution with mean . The genome wide mutation rate is given by = ′, where ′ is the number of wildtype protein-coding genes, as we assume that mutated genes cannot mutate again. The position of the particular locus or loci in the genome that mutates is then randomly determined. For simplicity, the possibility of back mutation in protein-coding genes is neglected. [fig] Figure S1: Illustration of the model dynamics. (A) Each generation, an individual has a probability of acquiring a fragment of eDNA of length from the environment and recombining it. Following LGT, mutations are randomly introduced at a rate per locus. The new generation is then generated by 8 sampling with replacement from the old generation in proportion to individual reproductive fitness 1 . The old generation dies and its DNA is released into the environment and constitutes the eDNA pool for the new generation. (B) The wildtype genome is represented as a circular series of genes indicated by different colours. (C) The wildtype genome is subject to mutation pressure, resulting in the accumulation of deleterious alleles through Muller's ratchet. (D) In a repeat-free population ( = 0), LGT ( = 0.1) allows homologous recombination, increasing genetic variation and favouring the elimination of deleterious mutations. (E) In the presence of repeats ( = 0.1), the possibility of ectopic recombination limits the benefits of LGT, causing gene deletions and duplications. Other simulation parameters: [/fig] [fig] Figure S2: Genome size and recombination length. At low initial repeat density ( = 0.01), (A) increases in recombination length ( ) limit mutation accumulation as genome size increase, (B) without increasing the rate of deletion. But in repeat-rich genomes ( = 0.3), (C) higher provides virtually no benefits, (D) while introducing a large number of new deletions. Error bars show the standard deviation over 100 independent simulations. Gene loss rate was calculated over 123 = 5,000 generations. Other parameters: = 2,500, = 10 #4 and = 0.1. [/fig] [fig] Figure S3: Time series of key model variables. Illustration of the change in key variables during the course of a standard simulation ( 123 = 5,000 generations) when there is a high repeat density ( = 0.3 ). Change in (A) mean genome size and (B) mean repeat numbers are slight. (C) The presence of repeats allows ectopic recombination, resulting in the mean duplication content (green line) and total gene loss (the number of wildtype genes that have been lost; yellow line) rising through time. In addition, the mean mutation load (calculated as the sum of all mutated genes, including duplicated ones; purple line) increases through time. All these processes occur with considerable variation and some reversals. Other parameters: = 2,500, = 300, = 10 #5 and = 0.1. [/fig] [fig] Figure S4: Distribution of key model variables. Illustration of the final distribution at the end of a standard simulation ( 123 = 5,000 generations) using the same parameter values in Figure S3. Population frequency distribution of (A) genome size, (B) repeat content, (C) total gene loss, (D) mutation load and (E) duplication content. Other parameters: = 2,500, = 300, = 10 #5 , = 0.3, and = 0.1. [/fig]

Risk factors and mortality of pulmonary embolism in COVID-19 patients: Evidence based on fifty observational studies Background: At present, many studies have described acute pulmonary embolism (PE) as a frequent and prognostically relevant complication of coronavirus disease 2019 infection. Thus we performed the present analysis of 50 studies to evaluate the risk factors and mortality of PE in COVID-19 patients.Method: Databases including PubMed, Embase, Cochrane Library and Web of Science were searched to October, 2021. Odds ratio (OR), mean difference (MD) or standard MD was used to evaluate the outcomes. The primary outcomes were the difference of mortality between PE and non-PE COVID-19 patients as well as relevant risk factors of PE in COVID-19 patients. All statistical analyses were performed using the standard statistical procedures provided in Review Manager 5.2.Result: A total of 50 studies including 10053 patients were included in this meta-analysis. Our results indicated that COVID-19 patients with PE experienced significantly higher mortality than non-PE patients (21.9% vs. 10.7%), with a pooled OR of 2.21 (95% CI 1.30 -3.76; P = .003). In addition, COVID-19 patients with PE also experienced more mechanical ventilation (MV) (OR 2.21; 95% CI 1.30 -3.75; P = .003) and invasive mechanical ventilation (IMV) (OR 3.58; 95% CI 2.47 -5.20; P < .0001) respectively. Univariate analysis (UVA) results indicated the Sequential Organ Failure Assessment (SOFA) score, time to deep venous thrombosis (DVT), nonintensive care unit (non-ICU) patients and no anticoagulation as risk factors of PE for COVID-19 patients. In addition, multivariate analysis also found that SOFA score, D-dimer, BMI > 30 kg/m 2 and history of PE were risk factors of PE for COVID-19 patients.Conclusion: The present analysis indicated that PE increased the mortality of COVID-19 patients. Mechanical ventilation, especially invasive mechanical ventilation, is correlated with an increased incidence of PE in patients with COVID-19. The incidence of PE for COVID-19 patients may be multifactorial and further researches focused on risk factors were needed in the future.Abbreviations: AMSTAR = assessing the methodological quality of systematic reviews, COVID-19 = coronavirus disease 2019, DVT = deep venous thrombosis, HFNC = high-flow nasal cannula, ICU = intensive care unit, IMV = invasive mechanical ventilation, MD = mean difference, MVA = multivariate analysis, NMV = non-invasive mechanical ventilation, NOS = Newcastle-Ottawa Scale, OR = odds ratio, PE = pulmonary embolism, PRISMA = preferred reporting items for systematic reviews and metaanalyses, SOFA = sequential organ failure assessment, UVA = univariate analysis. # Introduction The outbreak of coronavirus disease 2019 (COVID-19) remains a severe public health emergency of international concern. Over the past months, several investigations have suggested an association between the COVID-19 pathogenesis and a pro-coagulant pattern that seems to be implicated in a higher risk of both arterial and venous thrombotic events. [bib_ref] Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic..., Lodigiani [/bib_ref] [bib_ref] Pulmonary embolism and COVID-19: a comparative analysis of different diagnostic models performance, Silva [/bib_ref] [bib_ref] Pulmonary embolism in COVID-19: the actual prevalence remains unclear, Van Twist [/bib_ref] [bib_ref] Pulmonary thromboembolism in COVID-19, Woodard [/bib_ref] In this regard, acute pulmonary embolism (PE) has emerged as a potential severe complication of the infection and both American and European consensus statement have suggested general recommendations to deal with these clinical events. [bib_ref] Confirmation of the high cumulative incidence of thrombotic complications in critically ill..., Klok [/bib_ref] [bib_ref] Acute pulmonary embolism in patients with COVID-19 at CT angiography and relationship..., Léonard-Lorant [/bib_ref] Many studies have reported a high incidence of PE in patients with COVID-19, ranging from 10.5% to 14.7% in patients who were admitted to general wards and from 23.4% to 24.7% in patients who were admitted to the intensive care unit (ICU). [bib_ref] Incidence and mortality of pulmonary embolism in COVID-19: a systematic review and..., Liao [/bib_ref] [bib_ref] Incidence of acute pulmonary embolism in COVID-19 patients: systematic review and meta-analysis, Roncon [/bib_ref] [bib_ref] Pulmonary embolism and deep vein thrombosis in COVID-19: a systematic review and..., Suh [/bib_ref] It was believed that complications of PE, such as pulmonary infection, pulmonary consolidation, pulmonary arterial hypertension and increasing of right heart load, increased or resulted in in-hospital death of COVID-19 patients. [bib_ref] Incidence and mortality of pulmonary embolism in COVID-19: a systematic review and..., Liao [/bib_ref] However, the influence of PE to mortality of COVID-19 patients was still unclear. In addition, the risk factors of PE are not evaluated at present in patients with COVID- [bib_ref] COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection, Al-Samkari [/bib_ref]. Thus we performed the present analysis to evaluate the risk factors and mortality of PE in COVID-19 patients. We aim to explore the risk factors of PE in COVID-19 patients and hope that this will be helpful to prophylaxis or diagnosis of PE in COVID-19 patients. # Methods ## Search strategy and study selection A systematic search of PubMed, Embase, Cochrane Library and Web of Science up to October, 2021 was conducted for relevant studies using a search strategy developed by a medical information specialist that involved controlled vocabulary and keywords related to our research question (e.g., "coronavirus disease", "COVID-19", "pulmonary embolism", "PE"; "prognosis", "outcome", "survival", "death", "mortality", "prevalence", "risk factors"). The search strategy was limited to English language articles. Two assessors independently screened the titles and abstracts of each study. When a relevant study was identified, its full text was obtained for further evaluation. The full text of related references was also obtained for review. ## Criteria for considering studies We included studies if they met the following criteria: studies that: (1) compared the death or other outcomes between PE and non-PE patients with COVID-19; (2) explored the risk factors of PE in patients with Studies were excluded if they met the following criteria: (1) experimental trial on animals or a nonhuman studies; [bib_ref] Pulmonary embolism and COVID-19: a comparative analysis of different diagnostic models performance, Silva [/bib_ref] study population included non-COVID-19 patients; (3) study reported in the form of an abstract, letter, editorial, expert opinion, review, or case report; or (4) lack of sufficient data or failure to meet the inclusion criteria. ## Quality assessment and data extraction Two reviewers assessed the quality of each study using the 9-star Newcastle-Ottawa Scale (NOS).The scores were judged according to the three aspects of NOS of evaluation: selection, comparability, and outcome between the case group and control group. In addition, the risk of bias for each studies and the risk of bias across all studies were evaluated and shown with figures generated by RevMan 5.2 software.Baseline characteristics and outcomes from the included studies were extracted using a standardized extraction form. Key study characteristics including country, sample size, mean age, location, setting, end points and main outcomes were extracted. Data were extracted by 1 reviewer and then examined for accuracy and completeness by a second reviewer. ## Data synthesis and statistical methods The data of comparable outcomes between PE and non-PE patients with COVID-19 were combined-analyzed, using the standard statistical procedures provided in RevMan 5.2.Dichotomous data were measured with odds ratio (OR) and continuous variable data were measured with mean difference (MD). The heterogeneity between studies was evaluated by the chi-square-based Q statistical test, [bib_ref] Quantitative synthesis in systematic reviews, Lau [/bib_ref] with P h value and I 2 statistic, ranging from 0% to 100 %, to quantify the effect of heterogeneity. P h ≤ 0.10 was deemed to represent significant heterogeneity,and pooled estimates were estimated using a random-effect model (the DerSimonian and Laird method [bib_ref] Meta-analysis in clinical trials revisited, Dersimonian [/bib_ref]. On the contrary, if statistical study heterogeneity was not observed (P h > 0.10), a fixed effects model (the Mantel-Haenszel method [bib_ref] Statistical aspects of the analysis of data from retrospective studies of disease, Mantel [/bib_ref] was used. The effects of outcome measures were considered to be statistically significant if pooled ORs with 95% www.md-journal.com CI did not overlap with 1 or pooled MDs with 95% CI did not overlap with 0. This work has been reported in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] and Assessing the methodological quality of systematic reviews (AMSTAR) Guidelines. [bib_ref] AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised..., Shea [/bib_ref] Because this work in fact was a second analysis of previous study, the ethical approval for this study was not applicable. # Results ## Included studies, study characteristics, and quality assessment At the beginning of the search, a total of 114 records of citations were obtained; 105 of records were reviewed further after duplicates were removed. By screening titles and abstracts, 41 studies were preliminarily excluded and the remaining 64 studies The characteristics of included studies in this meta-analysis. were retrieved in full text for further evaluation. After reading the full texts, 14 studies were excluded further. Eventually, 50 studies [bib_ref] Venous and arterial thromboembolic complications in COVID-19 patients admitted to an academic..., Lodigiani [/bib_ref] [bib_ref] Confirmation of the high cumulative incidence of thrombotic complications in critically ill..., Klok [/bib_ref] [bib_ref] Acute pulmonary embolism in patients with COVID-19 at CT angiography and relationship..., Léonard-Lorant [/bib_ref] (10053 patients) were included in this systematic review and meta-analysis. Six studies were from USA, 14 from France, 8 from China, 3 from Switzerland, 5 from Spain. 15 studies were conducted by multicenter and the others by single center. The detailed search process and summary of studies are shown in the study flow diagram [fig_ref] Figure 1: Flow diagram following the PRISMA template of the search strategy for the... [/fig_ref]. The other characteristics of each study are shown in . ## Author/year Risk-of-bias graphs were generated to further identify the risk of bias of the including studies. The risk of bias for each study was presented as percentages across all included studies, and the risk-of-bias item for each included study was displayed [fig_ref] Figure 2: Risk of bias graph [/fig_ref]. The risk-of-bias graphs indicated generally low risk of selection and comparability. In addition, all studies experienced low risk of bias in "assessment of outcomes" item. A high risk of bias was mainly observed in "ascertainment of exposure" and "adequacy of follow-up of cohorts". Unclear risk of bias was mainly observed in "ascertainment of exposure" and "other bias". ## The clinical outcomes of covid-19 patients with pe We compared the mortality of COVID-19 patients between PE and non-PE. As [fig_ref] Figure 4: Forest plot of the mortality between PE and non-PE patients with COVID-19 [/fig_ref] shows, our pooled results indicated that COVID-19 patients with PE experienced significantly higher mortality than non-PE patients (21.9% vs. 10.7%), with a pooled OR of 2.21 (95% CI 1.30 -3.76; P = .003). As significant heterogeneity between studies was observed (P = .0003 and I 2 = 65%), the randomized effect model was used. In addition, it was observed that COVID-19 patients with PE also experienced more ICU admission than non-PE, with a pooled OR of 2.79 (95% CI 1.88 -4.13; P < .0001). However, no significant difference was found between PE and non-PE patients in the incidence of acute respiratory failure (OR 2.25; 95% CI 0.52 -9.70; P = .28) and arrhythmia (OR 5.74; 95% CI 0.25 -130.37; P = .27) [fig_ref] Table 2: The comparison of clinical outcomes in hospital between PE and non-PE patients... [/fig_ref]. ## Anthropometric and clinical characteristics between pe and non-pe patients with covid-19 In order to explore the difference of characteristics of COVID-19 patients with PE, we compared the characteristics between PE and non-PE patients with COVID-19. As [fig_ref] Table 3: The comparison of characteristics between PE and non-PE patients with COVID-19 [/fig_ref] shows, compared with non-PE, COVID-19 patients with PE experienced longer time from illness onset to admission, with a pooled MD of 1.50 days (95% CI 0.45 -2.55; P = .005). No significance was found in age (MD 3.99 years; 95% CI -0.77 -8.76; P = .10), gender (OR 1.81; 95% CI 0.96 -3.41; P = .07), BMI (MD -1.30 Kg/m 2 ; 95% CI -3.42 -0.82; P = .23), time to CTPA (MD 1.23 days; 95% CI -0.33 -2.79; P = .12), hospitalization (MD 4.15 days; 95% CI -0.48 -8.77; P = .08) respectively. We found no difference between PE and non-PE COVID-19 patients in symptoms such as cough (OR 0.86; 95% CI 0.51 -1.45; P = .57), fever (OR 1.71; 95% CI 0.88 -3.33; P = .11), dyspnea (OR 1.27; 95% CI 0.73 -2.22; P = .40), chest pain (OR 1.12; 95% CI 0.26 -4.78; P = .88) [fig_ref] Table 4: The comparison of symptoms between PE and non-PE patients with COVID-19 [/fig_ref]. The comparison of physical examination between PE and non-PE patients with COVID-19 similarly found no significant difference in respiratory rate (MD 2.00 breaths/min; 95% CI -4.69 -8.69; P = .56), heart rate (MD -2.00 beats/min; 95% CI -17.54 -13.54; P = .80), systolic BP (MD -2.00 mm Hg; 95% CI -17.56 -13.56; P = .80), diastolic BP (MD 4.00 mm Hg; 95% CI -10.88 -18.88; P = .60), temperature (MD 0.00 ˚C; 95% CI -1.57 -1.57; P = 1.0), and lower limb edema (OR 0.56; 95% CI 0.02 -17.92; P = .74) . We compared relevant risk factors between PE and non-PE patients with COVID-19. As [fig_ref] Table 6: The comparison of relevant PE risk factors between PE and non-PE patients... [/fig_ref] shows, no significant difference between PE and non-PE patients with COVID-19 was found in diabetes mellitus (OR 0.98; 95% CI 0. [bib_ref] Proximal deep vein thrombosis and pulmonary embolism in COVID-19 patients: a systematic..., Longchamp [/bib_ref] [fig_ref] Table 7: The comparison of treatment in hospital between PE and non-PE patients with... [/fig_ref]. ## Comparison of oxygen therapy in hospital between pe and non-pe patients with covid-19 In order to explore the risk factors of PE in COVID-19 patients, we also compared the oxygen therapy in hospital between PE and non-PE patients with COVID-19. Our pooled results indicated that COVID-19 patients with PE experienced more mechanical ventilation (MV) (OR 2.21; 95% CI 1.30 -3.75; P = .003) and invasive mechanical ventilation (IMV) (OR 3.58; 95% CI 2.47 -5.20; P < .0001) respectively [fig_ref] Figure 5: Forest plot of the mechanical ventilation use between PE and non-PE patients... [/fig_ref]. However, no significant difference was observed in maximum FiO2 (MD -0.27; 95% CI -0.89 [fig_ref] Table 8: The comparison of oxygen therapy in hospital between PE and non-PE patients... [/fig_ref]. ## Laboratory findings between pe and non-pe patients with covid-19 We compared the laboratory indicators between PE and non-PE COVID-19 patients. Our pooled analysis indicated that compared to non-PE, COVID-19 patients with PE had higher baseline and peak serum D-dimer, with pooled MDs of 5.98 μg/mL (95% CI 4.15 -7.81; P < .0001) and 1.10 μg/mL (95% CI 0.13 -2.07; P = .03) respectively. In addition, COVID-19 patients with PE had higher NT-pro BNP (MD 94.24 pg/mL; 95% CI 45.21 -143.27; P = .0002), hs Troponin I (MD 5.00 ng/L; 95% CI 1.01 -8.99; P = .01), but lower albumin (MD -3.58 g/L; 95% CI -5.18 to -1.98; P < .0001) . However, no significant difference was found in ferritin, platelets, lymphocytes, NLR, IL-6 (MD -2.23 pg/mL; 95% CI -33.02 -28.56; P = .89), fibrinogen (MD 1.96 mg/dL; 95% CI -1.95 -5.87; P = .33) and SOFA score (MD -1.00; 95% CI -4.03 -2.03; P = .52) . ## Risk factors associated with pe for patients with covid-19 Univariate analysis (UVA) results indicated SOFA score (OR 1.87; 95% CI 1.39 -2.52; P < .0001), time to DVT (OR 1.04; 95% CI 1.01 -1.07; P = .009), non-ICU patients (OR 6.50; 95% CI 2.10 -20.12; P = .001), no anticoagulation (OR 3.00; 95% CI 1.10 -8.18; P = .03) and dyslipidemias (OR 9.06; 95% CI 1.88 -43.67; P = .006) as risk factors of PE for COVID-19 patients. In addition, multivariate analysis (MVA) also found that SOFA score (OR 2.07; 95% CI 1.38 -3.11; P = .0004), D-dimer (OR 2.82; 95% CI 1.05 -7.58; P = .04), BMI > 30 kg/m 2 (OR 2.70; 95% CI 1.30 -5.61; P = .008) and history of PE (OR 3.50; 95% CI 1.20 -10.21; P = .02) were risk factors of PE for COVID-19 patients. Inversely, MVA indicated that COVID-19 patients receiving statin therapy had negative correlation to PE, with a pooled OR of 0.40 (95% CI 0.20 -0.80; P = .01). However, age, gender, PaO2/FiO2 ratio, and hypertension were not indicated as risk factors of PE for patients with COVID-19 [fig_ref] Table 10: The pooled results of univariate and multivariate analysis of risk factors associated... [/fig_ref]. ## Publication bias Funnel plots were conducted for assessing the publication bias of included literatures and we could roughly assess the publication bias by seeing whether their shapes were of any obvious asymmetry. The funnel plots showed no clear evidence of publication bias for mortality between PE and non-PE patients with COVID-19 (see supplemental digital content, http://links.lww. com/MD/G909). # Discussion PE is a life-threatening complication in patients with COVID-19, and given the data presented by previous studies, patients with COVID-19 always experienced a high incidence of PE and mortality. [bib_ref] Incidence and mortality of pulmonary embolism in COVID-19: a systematic review and..., Liao [/bib_ref] [bib_ref] Incidence of acute pulmonary embolism in COVID-19 patients: systematic review and meta-analysis, Roncon [/bib_ref] However, the risk factors of PE for patients with COVID-19 are still unclear. Thus, we conducted the present analysis with 50 observational studies including 10053 patients in order to explore the relevant risk factors of PE for patients with COVID-19. Our results indicated that COVID-19 patients with PE always experienced more ICU admission, longer time from illness onset to admission, more mechanical ventilation and IMV, higher baseline and peak serum D-dimer, higher NT-pro BNP and hs Troponin I, but lower albumin. In addition, SOFA score, time to DVT, non-ICU patients, no anticoagulation and dyslipidemias was indicated as risk factors of PE for COVID-19 patients. Multivariate analysis also found that SOFA score, D-dimer, BMI > 30 kg/m 2 and history of PE may be independent risk factors of To the best of our knowledge, the present analysis is the first systematic review and meta-analysis designed to focus on the clinical relevant risk factors instead of the prevalence of PE in patients with COVID-19. [bib_ref] Incidence and mortality of pulmonary embolism in COVID-19: a systematic review and..., Liao [/bib_ref] [bib_ref] Incidence of acute pulmonary embolism in COVID-19 patients: systematic review and meta-analysis, Roncon [/bib_ref] [bib_ref] Pulmonary embolism and deep vein thrombosis in COVID-19: a systematic review and..., Suh [/bib_ref] [bib_ref] Venous thromboembolism in COVID-19: a systematic review and meta-analysis, Kollias [/bib_ref] [bib_ref] Proximal deep vein thrombosis and pulmonary embolism in COVID-19 patients: a systematic..., Longchamp [/bib_ref] However, prior to our analysis, 1 meta-analysis was performed to summarize evidence on the incidence of clinically relevant VTE-defined as VTE excluding isolated subsegmental PE and distal deep vein thrombosisin adult critically ill patients with COVID-19. [bib_ref] Risk of clinically relevant venous thromboembolism in critically ill patients with COVID-19:..., Gratz [/bib_ref] The author reported longer mean ICU stay, advanced age and overweight, critical illness, immobility were associated with increased VTE risk. [bib_ref] Risk of clinically relevant venous thromboembolism in critically ill patients with COVID-19:..., Gratz [/bib_ref] This was in line with our results to a large extent. Severe COVID-19 disease is accompanied by excessive cytokine release, which in turn activates the coagulation cascade, resulting in typical laboratory alterations such as elevated fibrinogen and D-dimer levels. [bib_ref] Clinical features of patients infected with 2019 novel coronavirus in Wuhan, Huang [/bib_ref] Thus we compared the laboratory indicators between PE and non-PE patients aiming to found the difference and possible risk factors of PE in COVID-19 patients. As a results, serum D-dimer, higher NT-pro BNP and hs Troponin I as well as albumin level were found significant difference and may be associated with the incidence of PE. Several relevant limitations of our work need to be recognized. First, as our results indicated that statin therapy may beneficial to PE and no anticoagulation may increase the incidence of PE for COVID-19 patients, some patients included in our included studies may receive this treatment. However, these studies did not report this part of patients, which resulted in our failure to perform subgroup analysis further. To date, only 1 prospective, randomized, controlled trial has compared different anticoagulation regimens in critically ill patients (n = 20) with COVID-19.Therefore, it seems unlikely that a meta-analysis could shed light on this important question at this point. In line with this, a recently published Cochrane review concluded that there is currently insufficient evidence to determine the risks and benefits of anticoagulation in patients with COVID-19. [bib_ref] Prophylactic anticoagulants for people hospitalised with COVID-19, Flumignan [/bib_ref] Second, we observed substantial heterogeneity among studies that-apart from distinct outcome definitions-may have been caused by differences in study designs and settings. In particular, the absence of uniform diagnostic procedures to detect The comparison of physical examination between PE and non-PE patients with COVID-19. ## Examination Sample size Pooled results PE needs to be borne in mind when interpreting the results of our study. Furthermore, we cannot exclude that the different included patient cohorts and different treatment strategies used in studies might have resulted in distinct PE risks. Third, the inherent limitations of retrospective data reporting applied to the majority of the included studies. This is a likely explanation for our finding that all of the included studies had a moderate to high risk of bias. # Conclusion In conclusion, the present study summarizes the globally available risk factors of PE in patients with COVID-19. We calculated and compared the mortality of COVID-19 patients and found more than twofold mortality in PE than non-PE patients. Though it is multifactorial, we found several relevant risk factors of PE in patients with COVID-19 which may be helpful to www.md-journal.com The comparison of laboratory findings between PE and non-PE patients with COVID-19. ## Laboratory indicators sample size Pooled results clinical precaution and patients with these risk factors should be vigilant for PE. [fig] Figure 1: Flow diagram following the PRISMA template of the search strategy for the association between ALBI grade and the prognosis of patients with HCC. [/fig] [fig] Figure 2: Risk of bias graph: review authors' judgments about each risk of bias item presented as percentages across all included studies. [/fig] [fig] Figure 3: Risk of bias summary: review authors' judgments about each risk of bias item for each included study. Medicine [/fig] [fig] Figure 4: Forest plot of the mortality between PE and non-PE patients with COVID-19. [/fig] [fig] Figure 5: Forest plot of the mechanical ventilation use between PE and non-PE patients with COVID-19. [/fig] [table] Table 2: The comparison of clinical outcomes in hospital between PE and non-PE patients with COVID-19.COVID-19 = coronavirus disease 2019, ICU = intensive care unit, OR = odds ratio, PE = pulmonary embolism. [/table] [table] Table 3: The comparison of characteristics between PE and non-PE patients with COVID-19. [/table] [table] Table 4: The comparison of symptoms between PE and non-PE patients with COVID-19. [/table] [table] Table 6: The comparison of relevant PE risk factors between PE and non-PE patients with COVID-19.COVID-19 = coronavirus disease 2019, OR = odds ratio, PE = pulmonary embolism, VTE = venous thrombus embolism. [/table] [table] Table 7: The comparison of treatment in hospital between PE and non-PE patients with COVID-19.COVID-19 = coronavirus disease 2019, OR = odds ratio, PE = pulmonary embolism. [/table] [table] Table 8: The comparison of oxygen therapy in hospital between PE and non-PE patients with COVID-19.COVID-19 = coronavirus disease 2019, HFNC = high-flow nasal cannula, IMV = invasive mechanical ventilation, MD = mean difference, MV = Mechanical ventilation, NMV = noninvasive mechanical ventilation, OR = odds ratio, PE = pulmonary embolism. [/table] [table] Table 10: The pooled results of univariate and multivariate analysis of risk factors associated with PE for patients with COVID-19.BMI = body mass index, COVID-19 = coronavirus disease 2019, DVT = deep vein thrombosis, MVA = multivariate analysis, OR = odds ratio, PE = pulmonary embolism, SOFA = sequential organ failure assessment, UVA = univariate analysis. [/table]

Recent advances in energy-saving chemiresistive gas sensors: A review A B S T R A C TWith the tremendous advances in technology, gas-sensing devices are being popularly used in many distinct areas, including indoor environments, industries, aviation, and detectors for various toxic domestic gases and vapors. Even though the most popular type of gas sensor, namely, resistive-based gas sensors, have many advantages over other types of gas sensors, their high working temperatures lead to high energy consumption, thereby limiting their practical applications, especially in mobile and portable devices. As possible ways to deal with the high-power consumption of resistance-based sensors, different strategies such as self-heating, MEMS technology, and room-temperature operation using especial morphologies, have been introduced in recent years. In this review, we discuss different types of energy-saving chemisresitive gas sensors including self-heated gas sensors, MEMS based gas sensors, room temperature operated flexible/wearable sensor and their application in the fields of environmental monitoring. At the end, the review will be concluded by providing a summary, challenges, recent trends, and future perspectives. # Introduction Due to rapid industrialization and urbanization, the world is facing the problem of severe air pollution, as witnessed by the increasingly large amounts of deleterious and pollutant gases being introduced into the environment. To name a few, these pollutants consist of several toxic gases such as CO, SO 2 , NO 2 and H 2 S and/or chemicals and volatile organic compounds (VOCs) such as benzene, toluene, ethanol, acetaldehyde and formaldehyde. The presence of toxic gases and VOCs can be detrimental to humans and the surrounding atmosphere [bib_ref] Metal oxide semi-conductor gas sensors in environmental monitoring, Fine [/bib_ref] [bib_ref] Gas sensing using porous materials for automotive applications, Wales [/bib_ref] [bib_ref] Oxidative stress and the cardiovascular effects of air pollution, Free Radic, Miller [/bib_ref]. For instance, an estimated 3.8 million people per year face severe illnesses that may prove fatal and can be attributed to air pollution. Moreover, near 20% of cardiovascular deaths and about 20% of deaths due to stroke can be attributed to household pollution [bib_ref] Air pollution and noncommunicable diseases: a review by the forum of international..., Schraufnagel [/bib_ref]. Moreover, toxic gases can significantly affect the respiratory tract and may even be harmful to the nervous and the immune systems [bib_ref] Air pollution and its effects on the immune system, Free Radic, Glencross [/bib_ref] [bib_ref] Metal-oxide semiconductors for carbon monoxide (CO) gas sensing: a review, Mahajan [/bib_ref]. In particular, severe air pollution can cause abnormalities in lung surfactant composition and damage to the lungs, making humans more vulnerable to diseases such as COVID-19 [bib_ref] Effects of meteorological conditions and air pollution on COVID-19 transmission: evidence from..., Zhang [/bib_ref]. Accordingly, air pollution has become a pressing global problem, prompting several health agencies to recommend short-time exposure limits (STELs) for various toxic and VOC gases [bib_ref] Coordination polymers: opportunities and challenges for monitoring volatile organic compounds, Kumar [/bib_ref] [bib_ref] Detection of hazardous volatile organic compounds (VOCs) by metal oxide nanostructures-based gas..., Mirzaei [/bib_ref]. Moreover, if the potential risk to human health is to be reduced, pollutant gases in the air must be continuously monitored using sensitive and reliable electronic devices. Nowadays, the detection of dangerous gases is becoming increasingly important in different industries, indoor and outdoor air quality monitoring, public safety, mines, and so on [bib_ref] Coordination polymers: opportunities and challenges for monitoring volatile organic compounds, Kumar [/bib_ref] [bib_ref] Detection of hazardous volatile organic compounds (VOCs) by metal oxide nanostructures-based gas..., Mirzaei [/bib_ref] [bib_ref] Review-resistivetype hydrogen sensors on zinc oxide nanostructures, Ren [/bib_ref] [bib_ref] Performance analysis of gas sensing device and corresponding IoT framework in mines, Nath [/bib_ref] , As a result, numerous sensing techniques, such as the electrochemical [bib_ref] Electrochemical sensors and biosensors, Kimmel [/bib_ref] , optical fiber [bib_ref] Metal-organic framework thin film coated optical fiber sensors: a novel waveguide based..., Kim [/bib_ref] , quartz crystal microbalance (QCM) [bib_ref] The quest for highly sensitive QCM humidity sensors: the coating of CNT/MOF..., Chappanda [/bib_ref] and capacitive [bib_ref] A silver nanoparticle-anchored UiO-66 (Zr) metal-organic framework (MOF)-based capacitive gas sensor, Surya [/bib_ref] techniques, have been applied for this purpose. However, most of these techniques have disadvantages such as relatively high price, low sensitivity and selectivity, sophisticated design, and a need for additional equipment; some even lack portability [bib_ref] Realization of an ultrasensitive and highly selective OFET NO 2 Sensor: the..., Yuvraja [/bib_ref]. Among them, metal oxide semiconductor (MOS) gas sensors, working on the basis of changes in resistance in the presence of a target gases, have attracted great attention and have become a hot research topic [bib_ref] New perspectives of gas sensor technology, Yamazoe [/bib_ref] because of their numerous and unique characteristics, such as high sensitivity, short response/recovery time, ease of fabrication, high stability, simple operation and low price [bib_ref] Metal oxides for solid-state gas sensors: what determines our choice?, Korotcenkov [/bib_ref]. The modulation of the electrical resistance or conductance in a target gas environment is known to be the basic gas sensing mechanism of a resistive-based sensor [bib_ref] Nanoscale metal oxide-based heterojunctions for gas sensing: a review, Miller [/bib_ref] [bib_ref] Gas sensors based on one dimensional nanostructured metal-oxides: a review, Arafat [/bib_ref]. Initially in air, upon adsorption of oxygen molecules, an electron depletion layer (EDL) for n-type materials and a hole accumulation layer (HAL) for p-type materials will be formed on the sensor's surface. When an n-type MOS is exposed to an oxidizing gas, its resistance increases while for reducing gases the resistance decreases [bib_ref] Room temperature chemiresistive gas sensors: challenges and strategies-a mini review, Srinivasan [/bib_ref]. [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] -(b) schematically show the principle underlying the gas sensing mechanism of n-and p-type MOS-based resistive gas sensors [bib_ref] Electrospun metal oxide composite nanofibers gas sensors: a review, Abideen [/bib_ref]. The typical sensing mechanisms can be described by using the band bending theory. MOS materials are known to have many nanograins of different sizes and electrons are believed to flow in the grain boundaries. After oxygen gas molecules have been adsorbed on the sensor's surface, an electron will be extracted from the conduction band (CB), and an EDL will be formed in n-type MOS gas sensors [bib_ref] The morphologies of the semiconductor oxides and their gas-sensing properties, Lin [/bib_ref]. This process results in energy-band bending as shown in. Due to presence of grains, Schottky barriers with a height of V 1 will be initially formed in air, as shown in [bib_ref] Metal oxide gas sensors: sensitivity and influencing factors, Wang [/bib_ref]. The reaction of oxygen ionic species with reacted gases can change the height of the Schottky barrier to (V 2 ), which changes the conductivity, and thus the resistance, of the gas sensor [bib_ref] Vapor trace recognition using a single nonspecific chemiresistors, Dobrokhotov [/bib_ref] [bib_ref] Recent progress and perspectives of gas sensors based on vertically oriented ZnO..., Ahmad [/bib_ref] [bib_ref] Resistive gas sensors based on metal-oxide nanowires, Mirzaei [/bib_ref]. Traditionally, chemiresistive-based sensors consist of three components: namely, the insulating substrate (typically alumina or silicon oxide), interdigitated electrodes, and a heater under the substrate to heat the sensor [bib_ref] Oxygen-plasma-assisted enhanced acetone-sensing properties of ZnO nanofibers by electrospinning, Du [/bib_ref]. Generally, MOS-based gas sensors work at high temperatures (100-450 - C) [bib_ref] Engineering approaches to improvement of conductometric gas sensor parameters. Part 2: Decrease..., Korotcenkov [/bib_ref] ; however, this heating process results in some issues, such as degradation of the long-term stability due to the aggregation and coarsening of nanograins, an increase in fabrication cost due to the addition of the heater and high power consumption (100 mW up to 1 W) [bib_ref] Self-heated Ag-decorated SnO 2 nanowires with low power consumption used as a..., Ngoc [/bib_ref] [bib_ref] Low power consumption gas sensor created from silicon nanowires/TiO 2 core-shell heterojunctions, Liu [/bib_ref] [bib_ref] MEMS-based NO 2 gas sensor using ZnO nano-rods for low-power IoT application, Lee [/bib_ref] [bib_ref] Function and application of gas sensors, Kohl [/bib_ref]. In this regard, power consumption is a challenging issue that limits the integration of MOS-based gas sensors into some important portable devices. Due to the advances in smart phone technology, various sensors are now being integrated into cell phones for various applications, such as healthcare, public safety, and environmental and food monitoring [bib_ref] Surface free-carrier screening effect on the output of a ZnO nanowire nanogenerator..., Xue [/bib_ref]. The current chemiresistive sensors are not suitable for battery or portable operation due to the high temperatures needed for operation. Thus, the design of portable, low-cost and low power consuming or energy-saving gas sensors is essential for their use in wireless and portable devices [bib_ref] Ultralow power consumption gas sensor based on a self-heated nanojunction of SnO..., Ngoc [/bib_ref] [bib_ref] A low-cost approach to low-power gas sensors based on self-heating effects in..., Monereo [/bib_ref]. Thus, many attempts, such as the uses of low-power light-emitting diodes (LEDs) [bib_ref] Monolithic micro light-emitting diode/metal oxide nanowire sensor with microwatt-level power consumption, Cho [/bib_ref] , noble-metal functionalization [bib_ref] Metal-core@metal oxide-shell nanomaterials for gas-sensing applications: a review, Mirzaei [/bib_ref] and hybrid materials [bib_ref] Synergistic effects of codecoration of oxide nanoparticles on the gas sensing performance..., Park [/bib_ref] , have been made to lower the energy consumption of gas sensors by decreasing the optimum sensing temperature. Moreover, two other promising techniques to reduce [bib_ref] Highly sensitive and selective gas sensors using p-type oxide semiconductors: Overview, Kim [/bib_ref] with permission from Elsevier (Copyright 2020). power consumption significantly are operation of gas sensor in a self-heating mode [bib_ref] Novel self-heated gas sensors using on-chip networked nanowires with ultralow power consumption, Tan [/bib_ref] and integration of gas sensors into MEMS platforms [bib_ref] MEMS based microheaters integrated gas sensors, Singh [/bib_ref]. In this review article, we have discussed the current advancements of energy-saving gas sensors based on chemiresistive principle [fig_ref] Figure 3: An overview of the main techniques and applications of the energy-saving gas... [/fig_ref]. This review article discusses several topics: self-heated gas sensors, lowpower MEMS-based gas sensors, and gas sensors operating at room temperature with their applications including flexible/wearable gas sensors, and their associated gas sensing mechanisms. At the end, the review is concluded with brief summary and future perspectives on the possible opportunities for further developing energy-saving gas sensors. ## Self-powered gas sensors Piezoelectric nanogenerators (PENGs) and triboelectric nanogenerators (TENGs) enabling the monitoring of gases at RT in a selfpowered way have been introduced by Zhong Lin Wang's group [bib_ref] Piezoelectric nanogenerators based on zinc oxide nanowire arrays, Wang [/bib_ref] [bib_ref] Flexible triboelectric generator, Fan [/bib_ref] [bib_ref] Capsule triboelectric nanogenerators: toward optional 3D integration for high output and efficient..., Wu [/bib_ref] In regard to PENG-based gas sensors, pure ZnO has been investigated, with the piezoelectric output being generated by ZnO acting as both a gas sensing signal and power supply [bib_ref] Tuning oxygen vacancies and improving UV sensing of ZnO nanowire by microplasma..., Yang [/bib_ref] [bib_ref] Self-powered room temperature NO 2 detection driven by triboelectric nanogenerator under UV..., Su [/bib_ref]. Also, several metals, such as Cu [bib_ref] High response and selectivity of a Cu-ZnO nanowire nanogenerator as a self-powered/active..., Fu [/bib_ref] , Pd [bib_ref] Room-temperature selfpowered ethanol sensing of a Pd/ZnO nanoarray nanogenerator driven by human..., Lin [/bib_ref] , Au [bib_ref] Realizing roomtemperature self-powered ethanol sensing of Au/ZnO nanowire arrays by coupling the..., Xing [/bib_ref] , and Pt [bib_ref] Pt/ZnO nanoarray nanogenerator as self-powered active gas sensor with linear ethanol sensing..., Zhao [/bib_ref] , have been incorporated in order to enhance the catalytic reactions and, thus, the sensing performances. In addition, due to the many benefits, including increased modulation of the resistance, of ZnO-comprising heterostructures, including ZnO/SnO 2 [bib_ref] Portable room-temperature self-powered/active H 2 sensor driven by human motion through piezoelectric..., Fu [/bib_ref] , ZnO/NiO [bib_ref] High and fast H 2 S response of NiO/ ZnO nanowire nanogenerator..., Qu [/bib_ref] , and ZnO/In 2 O 3 [bib_ref] Core-shell In 2 O 3 /ZnO nanoarray nanogenerator as a self-powered active..., Zang [/bib_ref] , they have been employed to enhance the sensing capabilities, providing excellent performances, owing to their stronger output signal [bib_ref] Capsule triboelectric nanogenerators: toward optional 3D integration for high output and efficient..., Wu [/bib_ref]. TENGs can be used to monitor gases because the surface adsorbed species strongly affect the effectiveness of triboelectric charging [bib_ref] Triboelectric nanogenerator as self-powered active sensors for detecting liquid/gaseous water/ethanol, Zhang [/bib_ref]. The first TENG-based gas sensor was comprised of polymer films such as polyamide (PA) and polytetrafluoroethylene (PTFE) [bib_ref] Triboelectric nanogenerator as self-powered active sensors for detecting liquid/gaseous water/ethanol, Zhang [/bib_ref]. Subsequently, a variety of materials, such as ZnO [bib_ref] Self-powered, roomtemperature electronic nose based on triboelectrification and heterogeneous catalytic reaction, Kim [/bib_ref] , polyaniline (PANI) [bib_ref] Outputting olfactory bionic electric impulse by PANI/PTFE/PANI sandwich nanostructures and their application..., Xue [/bib_ref] , and poly :poly(styrenesulfonate) (PEDOT:PSS) [bib_ref] Improving the working efficiency of a triboelectric nanogenerator by the semimetallic PEDOT:..., Uddin [/bib_ref] , has been adopted. Because we are dealing with chemiresistive-based gas sensors and the number of pages is limited, in this article, we will focus mainly on a special category of self-powered gas sensors, namely, self-heated gas sensors. ## Self-heated gas sensors As discussed, the energy consumption must be lowered if batteryoperated gas-sensing devices are to be designed. In practice, for the gas sensors used in mobile or wireless network devices power consumption is in the range of several microwatts [bib_ref] Recent progress and perspectives of gas sensors based on vertically oriented ZnO..., Ahmad [/bib_ref]. Typically, a conventional thick or thin film gas sensor equipped with a heater consumes about 1-5 W during its operation [fig_ref] Figure 4: Schematics of [/fig_ref]. On the other hand, MEMS-based gas sensors consume less than 30-50 mW to reach temperatures up to 500 - C. Also, for a self-heated gas sensor without an external heater, much less power is consumed [bib_ref] Self-heated hydrogen gas sensors based on Pt-coated W 18 O 49 nanowire..., Zhu [/bib_ref]. The main factors underlying self-heating gas sensors are the application of an appropriate voltage, with Joule heating generating heat inside the sensor, thereby increasing its temperature. As shown schematically in [fig_ref] Figure 4: Schematics of [/fig_ref] , in this mode of operation, an external voltage is directly applied to the sensor electrodes. Nevertheless, for self-heated gas sensors, realization of high sensitivity at an appropriate bias voltage still remains a challenge [bib_ref] A review on efficient self-heating in nanowire sensors: prospects for very-low power..., Fàbrega [/bib_ref]. The efficient self-heating (ESH) coefficient for a self-heated gas sensor can be expressed as [formula] ESH = ΔT/Q(1) [/formula] where ΔT is the temperature increase due to the applied voltage, and Q is the electrical power dissipation. Typically, an ESH value greater than 1 (ESH > 1) is beneficial for self-heating, which means an increase of a few Kelvin/microwatt consumed. Conventional self-heating sensors (ΔT ~ 500 - C and P ~ 10 mW) have ESH values of ~ 0.05 [bib_ref] Effective design and fabrication of low-power-consumption self-heated SnO 2 nanowire sensors for..., Ngoc [/bib_ref]. In general, MOS materials with nanowire (NW) morphology are preferred for self-heating studies [bib_ref] Effective design and fabrication of low-power-consumption self-heated SnO 2 nanowire sensors for..., Ngoc [/bib_ref]. As the NWs serve as pathways for electron transfer, the lengths of the NWs directly affect the heat generated inside the gas sensor, thereby allowing Joule heating to warm effectively the NWs and ensuring a higher response to the target gas. Furthermore, power is consumed at microwatt levels owing to the minimal thermal capacitance of the NWs [bib_ref] Engineering approaches to improvement of conductometric gas sensor parameters. Part 2: Decrease..., Korotcenkov [/bib_ref] [bib_ref] A self-heating gas sensor with integrated NiO thin-film for formaldehyde detection, Lee [/bib_ref] [bib_ref] Ultralow power consumption gas sensors based on self-heated individual nanowires, Prades [/bib_ref]. Moreover, the diameter of the NW is important because the temperature increase has an indirect relation with the radius of the NW; thinner NWs produce larger increases in the temperature [bib_ref] Effective design and fabrication of low-power-consumption self-heated SnO 2 nanowire sensors for..., Ngoc [/bib_ref]. In 2003 and 2006, Salehi et al. and Lee at al. introduced self-heated gas sensors based on SnO 2 and NiO thin films for the detection of CO and formaldehyde, respectively [bib_ref] A highly sensitive self-heated SnO 2 carbon monoxide sensor, Salehi [/bib_ref] [bib_ref] A self-heating gas sensor with integrated NiO thin-film for formaldehyde detection, Lee [/bib_ref]. These systems showed lower power consumption as compared to conventional gas sensors. For further decreases in the energy consumption of the MOS gas sensors, NW morphologies are preferred due to the aforementioned advantages. The first NW-based self-heated sensor was introduced by Strelcov et al. [bib_ref] Evidence of the self-heating effect on surface reactivity and gas sensing of..., Strelcov [/bib_ref] , and so far, the self-heating properties of single NW [bib_ref] Ultralow power consumption gas sensors based on self-heated individual nanowires, Prades [/bib_ref] [bib_ref] Comparative NO 2 gas-sensing performance of the self-heated individual, multiple and networked..., Chinh [/bib_ref] [bib_ref] Ultralow-power hydrogen sensing with single palladium nanowires, Offermans [/bib_ref] [bib_ref] Quantitative analysis of CO-humidity gas mixtures with self-heated nanowires operated in pulsed..., Prades [/bib_ref] , multiple NWs [bib_ref] Evidence of the self-heating effect on surface reactivity and gas sensing of..., Strelcov [/bib_ref] [bib_ref] Comparative NO 2 gas-sensing performance of the self-heated individual, multiple and networked..., Chinh [/bib_ref] [bib_ref] A self-heated silicon nanowire array: selective surface modification with catalytic nanoparticles by..., Jin [/bib_ref] and networked NWs [bib_ref] Self-heated hydrogen gas sensors based on Pt-coated W 18 O 49 nanowire..., Zhu [/bib_ref] [bib_ref] Comparative NO 2 gas-sensing performance of the self-heated individual, multiple and networked..., Chinh [/bib_ref] have been reported. As compared to the single-NW and the network-type NW sensors synthesized by using off-chip methods, on-chip NW sensors have many advantages [bib_ref] Novel self-heated gas sensors using on-chip networked nanowires with ultralow power consumption, Tan [/bib_ref] [bib_ref] Effective design and fabrication of low-power-consumption self-heated SnO 2 nanowire sensors for..., Ngoc [/bib_ref] [bib_ref] Nanoscale Thermal management of single SnO 2 nanowire: pico-Joule energy consumed molecule..., Meng [/bib_ref] [bib_ref] Novel fabrication of and SnO 2 nanowire gas sensor with high sensitivity, Choi [/bib_ref]. Therefore, on-chip networked NW gas sensors are being developed for self-heating purposes; in this context, the on-chip methods are used to obtain power consumption at μW level [bib_ref] Self-heated hydrogen gas sensors based on Pt-coated W 18 O 49 nanowire..., Zhu [/bib_ref] [bib_ref] Evidence of the self-heating effect on surface reactivity and gas sensing of..., Strelcov [/bib_ref]. Hieu and co-workers reported the synthesis of on-chip grown networked SnO 2 NWs [bib_ref] Novel self-heated gas sensors using on-chip networked nanowires with ultralow power consumption, Tan [/bib_ref]. Prior to the on-chip growth, the electrode was designed using a photolithography method. After that, SnO 2 NWs having average diameters around 95 nm were grown using a well-known vapor-liquid-solid (VLS) method. The sensor showed a response of 2.1-2.5 ppm NO 2 gas with a power consumption of only 25 mW, along with good selectivity. The authors further addressed reducing the size of the electrodes with a gap width of 10 μmand growing SnO 2 NWs on the electrode. The sensor exhibited an enhanced response (R g /R a = 4.6) towards NO 2 at 20 ppm. The sensing mechanism was explained based on modifications of the potential barriers at the SnO 2 NW-NW junctions and establishment of depletion layers at the SnO 2 NWs [bib_ref] A gradient microarray electronic nose based on percolating SnO 2 nanowire sensing..., Sysoev [/bib_ref]. When the sensors were exposed to NO 2 gas, NO 2 molecules were adsorbed on the surface of the SnO 2 NW sensor, and electrons were extracted from the conduction band of SnO 2 NWs to form NO 2 − ionic species [bib_ref] Selective improvement of NO 2 gas sensing behavior in SnO 2 nanowires..., Kwon [/bib_ref] , which resulted in an increased number of depletion layers and potential barriers with increased heights. The reduction of power consumption in the self-heated sensors depends on the sensing area of the electrode, and the design of head-tohead electrodes is an important aspect that is often neglected. Ngoc et al. introduced a self-heated NW gas sensor based on SnO 2 NWs grown using a chemical vapor deposition (CVD) technique, in which SnO 2 NW networks bridged the electrode gaps [bib_ref] Effective design and fabrication of low-power-consumption self-heated SnO 2 nanowire sensors for..., Ngoc [/bib_ref]. They investigated the effect of the electrode gap size on the sensing performance. For that purpose, the head-to-head electrodes gaps were maintained at 2, 5, 10 and 20 μm, which were labelled as G2, G5, G10 and G20, respectively. The mean diameter of the fabricated SnO 2 NWs was 80 nm. The electrode with a small gap was seen to yield a dense SnO 2 NW network (G2 with densest nanojunction) whereas the electrode with a large gap filled with a sparse network (G20) had the sparsest nanojunction. The sensor with a 20-μm electrode gap (G20) displayed a good response (3.75) toward C 2 H 5 OH (250 ppm) at a 10-mW power supplied. One of the merits in designing head-to-head electrodes is reduced power consumption because the self-heated sensor is dependent on the electrode area [bib_ref] Novel self-heated gas sensors using on-chip networked nanowires with ultralow power consumption, Tan [/bib_ref]. The nanojunctions between the SnO 2 NWs established the Schottky barriers, of S.M. which increase the resistance at the nanojunction (R j ) as compared to the resistance at the NWs. Thus, one can conclude that for a self-heated NW sensor, warm-up at the junction in a nanoscale range is more important than network heating [bib_ref] Evidence of the self-heating effect on surface reactivity and gas sensing of..., Strelcov [/bib_ref]. Noble metal decoration is a good strategy to enhance the gas sensing characteristics of MOS-based gas sensors, which is due to the sensitization effects of noble metals [bib_ref] Ag nanoparticles sensitized In 2 O 3 nanograin for the ultrasensitive HCHO..., Zhou [/bib_ref] [bib_ref] Seiyama Effects of additives on semiconductor gas sensors, Yamazoe [/bib_ref]. In this regards, the effect of self-heating and the density of the catalytic material were investigated in Ag-decorated SnO 2 NW networks by Ngoc et al. [bib_ref] Self-heated Ag-decorated SnO 2 nanowires with low power consumption used as a..., Ngoc [/bib_ref]. SnO 2 NWs were synthesized using a thermal evaporation method, and Ag nanoparticles (NPs) were sputter deposited on the surfaces of SnO 2 NWs for different durations of 10, 20, 40 and 80 s to control the density of Ag NPs; accordingly, the sensors were named ST10, ST20, ST40, and ST80, respectively. They used as H 2 S gas sensor in the self-heating mode under different powers of 2-20 mW [bib_ref] Self-heated Ag-decorated SnO 2 nanowires with low power consumption used as a..., Ngoc [/bib_ref]. Among the four sensors, the ST80 sensor showed the highest response of 21.2 to 0.5 ppm to H 2 S gas at a low heating power of 2 mW with an acceptable response/recovery time of 18/980 s. Ag NPs are known for their ability to selectively react with H 2 S at low concentrations at temperatures below 200 - C [bib_ref] Synthesis of Ag-doped SnO 2 thin films for the evaluation of H..., Kolhe [/bib_ref]. However, when the power reached 20 mW, the authors reported serious thermal damage of the sensing material. When the sensor is exposed to H 2 S gas, Ag 2 O NPs react with H 2 S gas molecules as follows: [formula] Ag 2 O + H 2 S → Ag 2 S + H 2 O(2) [/formula] which is an exothermic chemical reaction that can occur at low temperatures. During the recovery period, the following reaction is expected: [formula] 2Ag 2 S + 3O 2 → 2Ag 2 O + 2SO 2(3) [/formula] which is an endothermic chemical reaction occurring at high temperatures. Under low heating power, the reaction in Eq. (2) is dominant and converts Ag 2 O into Ag 2 S with a metallic nature, leading to a loss of the pn junctions between Ag 2 O and SnO 2 and decreased sensor resistance. Under high heating powers, the reaction in Eq. (3) increases, but never dominates the reaction in Eq. [bib_ref] Gas sensing using porous materials for automotive applications, Wales [/bib_ref]. Therefore, under lower heating powers, a higher gas response is observed. Also, with increasing Ag catalytic density, the response time decreases due to the high rate of the reaction between Ag 2 O and H 2 S while the recovery time increases because a longer time is required to change a large amount of Ag 2 S back to Ag 2 O [bib_ref] Synthesis of Ag-doped SnO 2 thin films for the evaluation of H..., Kolhe [/bib_ref] [bib_ref] Micromachined nanocrystalline silver doped SnO 2 H 2 S sensor, Gong [/bib_ref] [bib_ref] Nano Ag-doped In 2 O 3 thick film: a low-temperature H 2..., Chavan [/bib_ref]. In addition to Ag catalysts, Pt, Pd, and Au NPs have also been used in studies of self-heating gas sensors [bib_ref] Tolueneand benzene-selective gas sensors based on Pt-and Pd-functionalized ZnO nanowires in self-heating..., Kim [/bib_ref] [bib_ref] Low power-consumption CO gas sensors based on Au-functionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref] [bib_ref] Self-heating effects on the toluene sensing of Ptfunctionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref] [bib_ref] Pd-functionalized core-shell composite nanowires for self-heating, sensitive, and benzene-selective gas sensors, Kim [/bib_ref] [bib_ref] Realization of H 2 S sensing by Pd-functionalized networked CuO nanowires in..., Kim [/bib_ref] [bib_ref] Realization of Au-decorated WS 2 nanosheets as low power-consumption and selective gas..., Kim [/bib_ref]. These noble metal NPs are generally functionalized on the surface of the gas sensor by using various approaches, such as UV-irradiation [bib_ref] One-pot synthesis of Au-loaded SnO 2 nanofibers and their gas sensing properties, Katoch [/bib_ref] , γ-ray irradiation [bib_ref] Sputtered PdO decorated TiO 2 sensing layer for a hydrogen gas sensor, Lee [/bib_ref] , and sputtering [bib_ref] Extremely sensitive and selective sub ppm CO detection by the synergistic effect..., Kim [/bib_ref]. In a recent study of gas sensors operating in the self-heating mode, Kim et al. used UV-irradiation and sputtering techniques to deposit both Pd and Pt NPs on the on-chip grown ZnO NWs for a selective study of benzene and toluene gases [bib_ref] Seiyama Effects of additives on semiconductor gas sensors, Yamazoe [/bib_ref]. Initially, the ZnO NWs were synthesized through a VLS method followed by ultraviolet (UV) irradiation technique to deposit Pd NPs while the Pt NPs were deposited by using a magnetron sputtering method, followed by an annealing treatment. Morphologies of the synthesized Pd/ZnO NWs and Pt/ZnO NWs are shown inand (C), respectively. For self-heating mode, both 5 nm-Pt-ZnO NWs and 5s-Pd-ZnO NWS sensors were studied with different applied voltages from 1 to 20 V. The Pt/Pd-ZnO NWs sensors showed high responses to toluene and benzene, respectively, among other reducing gases. The highest response of the 5 nm-Pt/ZnO NW sensor operating in the self-heating mode to 50-ppm toluene was 2.74 at RT under a 20-V applied voltage whereas the highest response obtained for the Pd/ZnO NW sensor under the same conditions towards benzene (50 ppm) was 2.20. Also, the power consumptions for the Pt-and Pd-ZnO NW gas sensors were found to be 208 and 139 μW at 5 V, respectively. The good selectivity toward benzene and toluene can be ascribed to the combination of catalytic effects of both Pt and Pd NPs, respectively. For benzene, the following reaction has been reported: C 6 H 6 (g)→C 6 H 6 (ads) (4) [formula] C 6 H 6 (ads) + 15O − → 6CO 2 + 3H 2 O + 15e −(5) [/formula] and for toluene, the following reaction was reported: Accordingly, the higher response of the Pt/ZnO NW sensor to toluene relative to the response of the Pd/ZnO NW sensor to benzene was related to the higher electron donating capacity of toluene. Schematic diagrams of the sensing mechanisms underlying pristine and Pt/Pd-ZnO NW sensors are provided in [formula] C 7 H 8 (g)→C 7 H 8 (ads)(6)C 7 H 8 (ads) + 18O − → 7CO 2 + 4H 2 O + 18e − .(7)(F). [/formula] When a voltage is applied, heat is generated because the electrons lose kinetic energy when they collide with other electrons or other obstacles in their paths. The relation between Joule heating per volume (W/m 3 ) and the applied voltage can be expressed as follows [bib_ref] An analytical model of Joule heating in piezoresistive microcantilevers, Ansari [/bib_ref] : [formula] J=V 2 /ρL 2 ,(8) [/formula] where ρ is the resistivity of the sensing layer, and L is its length. The temperature increase due the self-heating effect was found to be 0, 1.6, 12.9, 25.8, and 80 - C, respectively, for applied voltages of 0, 1, 5, 10, and 20 V. Two possible locations of Joule heating were the ZnO grains and the networked ZnO-ZnO homojunctions [bib_ref] Self-heating effects in large arrangements of randomly oriented carbon nanofibers: application to..., Monereo [/bib_ref]. Other than the single components, binary/ternary structures or heterostructures with different noble metal catalysts have been used for sensing studies due to the synergistic effect resulting from the combination of the MOS structure and a noble metal, which results in enhanced sensing reactions, surface activities and selectivities towards specific gases [bib_ref] Synergistic effects in gas sensing semiconducting oxide nano heterostructures: a review, Walker [/bib_ref] [bib_ref] Nanoscale metal oxide-based heterojunctions for gas sensing: a review, Miller [/bib_ref] Kim et al. reported Au-decorated SnO 2 -ZnO core-shell NW-based sensors for the investigation of the CO gas-sensing properties under self-heating [bib_ref] Low power-consumption CO gas sensors based on Au-functionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref]. The SnO 2 NWs were synthesized vertically on chip by using the typical VLS method [bib_ref] Tolueneand benzene-selective gas sensors based on Pt-and Pd-functionalized ZnO nanowires in self-heating..., Kim [/bib_ref]. Then, ultrafine Au NPs were deposited on the sensor's surface by using the γ-ray radiolysis technique. The ZnO shell thickness was varied from 10 to 80 nm by performing 50 to 500 cycles of atomic layer deposition (ALD). The relationship between the applied voltage (V > 1) and the increase in the temperature of the sensor due to the self-heating effect can be formulated as follows: [formula] ΔT = − 1.3575 + 0.120V + 0.1645V 2 For V > 1,(9) [/formula] where ΔT is the increase in the temperature due to self-heating, and V is the applied voltage.The self-heating effects with different applied voltages from 0 to 20 V were checked, and the temperatures obtained 0-20 V were 23-103 - C. The Joule heating process in the SnO 2 -ZnO C-S NWs can contribute to three sources of resistances: (i) in the ZnO grains, (ii) in the grain ZnO boundaries, and (iii) through ZnO-ZnO homojunctions(f)). Accordingly, as the number of NW contacts in the sample increased with increasing thickest of the ZnO shell, the corresponding increase in the number of Joule heating sources led to a higher temperature and, thus, a higher gas response. Also, the good selectivity of the sensor to CO gas was related to a combination of the catalytic effect of Au, the special architecture of the sensor, and the sensing temperature (due to selfheating). However, the most important factor was the catalytic effect of Au to CO gas, where a low energy was required for oxidation of CO over Au NPs [bib_ref] Tolueneand benzene-selective gas sensors based on Pt-and Pd-functionalized ZnO nanowires in self-heating..., Kim [/bib_ref]. The lowest power for the sensor was found to be 0.81 nW at its corresponding voltage of 1 V whereas the highest power consumption of 8.3 μW was obtained at 20 V. The previous reports that we reviewed here are all showed energy consumption values from mW to μW. However, the obtained low power consumption in this study clearly indicates that Au/SnO 2 -ZnO C-S NW sensors are very promising in the design of portable sensors with extremely low power consumption. In addition to Au NPs, Pt and Pd NPs have been deposited on SnO 2 -ZnO core-shell NWs to study their gas-sensing properties under selfheating conditions [bib_ref] Pd-functionalized core-shell composite nanowires for self-heating, sensitive, and benzene-selective gas sensors, Kim [/bib_ref] [bib_ref] Realization of H 2 S sensing by Pd-functionalized networked CuO nanowires in..., Kim [/bib_ref]. For example, Kim et al. reported SnO 2 -ZnO C-S NWs-(c)) and studied the selective/sensitive detection of toluene gas without using external heating [bib_ref] Self-heating effects on the toluene sensing of Ptfunctionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref]. The SnO 2 NWs were synthesized by using a VLS method, whereas the ALD technique was applied to coat a ZnO shell, followed by γ-ray radiolysis to deposit Pt NPs on the sensor surface. The thicknesses of the ZnO shells was set to 10, 30, and 85 nm for 50, 200 and 500 ALD cycles, respectively. Subsequently, the self-heating effect of Pt-decorated SnO 2 -ZnO core-shell NW sensors having an 85-nm shell thickness was measured at different voltages from 0 to 20 V.shows thermograph images obtained at 0, 1, 3, 5, 10 and 20 V. The 10-nm-thick shell shows a negligible self-heating effect whereas the 85-nm-thick ZnO shell sensor exhibits a quite good self-heating effect. The response of the Pt-decorated SnO 2 -ZnO core-shell NW sensor with an 85-nm thickness to toluene was found to be 3.14. Moreover, the response of the sensor was explored at applied voltages from 1 to -20 V. The highest response of 3.14 was obtained at 20 V, and the lowest response of 1.00 was obtained at 1 V. The amount of power consumption of the as-prepared sensor was found to be 31 μW at 5 V, which provides promise for applications such as mobile and portable gas sensors. The mechanism of Joule heating can be explained based on mainly three possible sources. Firstly, the Joule heating occurs inside the ZnO grains as current flows through them. Secondly, the Joule heating occurs in the ZnO grain boundaries. Thirdly, due to the entangled NW structure of the NWs, it occurs in the ZnO-ZnO homojunctions as electrical currents flow through those homojunctions [bib_ref] Tolueneand benzene-selective gas sensors based on Pt-and Pd-functionalized ZnO nanowires in self-heating..., Kim [/bib_ref]. The sensing mechanism inshows the formation of heterojunctions between ZnO/Pt and ZnO/SnO 2 , which greatly affect the sensing response. To further continue their self-heating study, Kim et al. reported onchip grown SnO 2 NWs Si/SiO 2 substrate by using a VLS technique for ultra-low power consumption sensors in the nW range when operating in the self-heating mode [bib_ref] Pd-functionalized core-shell composite nanowires for self-heating, sensitive, and benzene-selective gas sensors, Kim [/bib_ref]. The ZnO shell, which had an 80-nm thickness, coated on SnO 2 NWs by using the ALD method, after which the Pd NPs were coated on the SnO 2 -ZnO core-shell NWs by using γ-rays radiolysis. The heat produced because of Joule heating is known to be directly proportional to the voltage and indirectly to the resistance according to following formula: [formula] Q ∝ V 2 /R(10) [/formula] Thus, Joule heating can be increased by increasing the applied voltage. The responses of a Pd-SnO 2 -ZnO core-shell NW sensor toward So far, only n-type MOS materials were discussed for sensing studies. This is due to the fact that in general, p-type MOS-based gas sensors show lower responses relative to those of their n-type counterparts [bib_ref] Development of nanocrystalline CrNbO 4 based p-type semiconducting gas sensor for LPG,..., Balamurugan [/bib_ref]. However, a few studies of p-type gas sensors operating in the self-heating mode have been published. Among many p-type MOS materials, CuO has been investigated for H 2 S sensing due to its being highly sensitive to H 2 S [bib_ref] Synthesis and H 2 S sensing properties of CuO-SnO 2 core/shell PN-junction..., Xue [/bib_ref] [bib_ref] Importance of the nanograin size on the H 2 S-sensing properties of..., Choi [/bib_ref]. Kim et al. prepared pristineand Pd-functionalized CuO NWsfor H 2 S gas sensing in self-heating mode (1-5 V) as shown in in [bib_ref] Realization of H 2 S sensing by Pd-functionalized networked CuO nanowires in..., Kim [/bib_ref]. The Pd functionalization on the CuO NWs (Pd/CuO NWs) was carried out using ultraviolet irradiation. The Pd-functionalized CuO NW sensor was seen to exhibit its best response of 1.894-100 ppm H 2 S gas at 5 V which was higher than that of pristine sensor. The high sensitivity of the current sensor was ascribed to the transformation of CuO to CuS with metallic conductivity during exposure to H 2 S(f)). The change in conductivity from CuO (E g =1.2 eV) to CuS and the catalytic effect of Pd NPs resulted in a high response to H 2 S gas, in accordance with literature [bib_ref] Importance of the nanograin size on the H 2 S-sensing properties of..., Choi [/bib_ref]. The self-heating effects were attributed to two phenomena), (i) the passing of electrons through the CuO NWs and (ii) the CuO-CuO homojunctions network, in which the homojunctions were in direct contact with one another. Thus, the Joule heating was generated in the CuO-CuO homojunctions network [bib_ref] Low power-consumption CO gas sensors based on Au-functionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref] [bib_ref] Localized self-heating in large arrays of 1D nanostructures, Monereo [/bib_ref] , leading to temperature increase inside of the sensor and appearance of a sensing signal. In an another experiment, a p-type MOS material was synthesized by oxidizing metal NPs such as Pt NPs. Prajapati et al. reported on the use gas sensors with a single NW of PtO x and a NW array of PtO x /Pt for H 2 sensing at RT under a self-heating condition [bib_ref] Self-heating oxidized suspended Pt nanowire for high performance hydrogen sensor, Prajapati [/bib_ref]. The diameter of the Pt NW arrays was varied from 10 to 80 nm. For the formation of a single PtO x NW, a 60-nm Pt NW was oxidized in an oxygen ambient under a constant voltage. Upon exposure to hydrogen gas, the single NW PtO x sensor showed an excellent response (ΔR/R a × 100) of about 818% to H 2 (1000 ppm) at RT under an ultra-low 0.25-V applied voltage. However, the NW array of PtO x /Pt showed a higher response (~936%) with a quicker response/recovery time of ~25 s/~108 s under the same conditions. The higher sensitivity and the faster response of the oxidized NW array were related to the increased sensing area and the different oxidation lengths. The oxidation (PtO x ) length of the PtO x /Pt NW array was 50 nm to ~100 nm, which facilitated a reduction in the response time with the electrode having a nanoscale gap while the higher oxidation lengths (500 nm-1 μm) offered higher surface areas for gas sensing. Other than NWs, two dimensional (2D) nanosheets (NSs), especially 2D layered transition metal dichalcogenides (TMDs) have been also used for self-heating studies due to their several unique properties [bib_ref] Two dimensional transition metal dichalcogenides in Biosystems, Kalantar-Zadeh [/bib_ref] [bib_ref] The ultrahigh NO 2 response of ultra-thin WS 2 nanosheets synthesized by..., Xu [/bib_ref]. In this context, Kim et al. prepared Au NP decorated WS 2 NSs [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] (a)-(b)) for CO sensing in the self-heating mode [bib_ref] Realization of Au-decorated WS 2 nanosheets as low power-consumption and selective gas..., Kim [/bib_ref]. Microscopically analyses which are shown in [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , demonstrate the formation of desired morphology. The actual sensor temperatures were obtained using an IR imaging camera [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , and the sensor showed its highest response to CO gas at only 2 V [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] with a power consumption of only 28.6 μW. The very low power consumption was related to the intrinsic resistivity of WS 2 NSs, which facilitated heat generation within the WS 2 NSs. In addition, many WS 2 -WS 2 homojunctions acted as power sources of Joule heating upon application of a voltage. The good response was related to the catalytic role of Au towards CO gas at low temperatures. Also, MOS materials with nanorod morphology have been used for self-heating studies. For example, Seo et al. and Choo et al. designed selfheated gas sensors with high-sensitivity to H 2 [bib_ref] Self-heating hydrogen gas sensor based on an array of single suspended carbon..., Seo [/bib_ref] [bib_ref] A novel self-heating zinc oxide/indium tin oxide-based hydrogen gas sensor: dual sensing..., Choo [/bib_ref]. A cost-effective, self-heating (2-8 V), ZnO-based H 2 gas sensor was made from ZnO nanorods grown using a hydrothermal process on the surface of indium tin oxide (ITO) [bib_ref] A novel self-heating zinc oxide/indium tin oxide-based hydrogen gas sensor: dual sensing..., Choo [/bib_ref]. Joule heating was mostly generated inside the ITO whereas ZnO acted as a resistor to modulate the electrical resistance of the sensor. At 4 V, evidence of Joule heating in the sensor was obvious, and with further increases in the operating voltage, higher temperatures were obtained for the sensor. At a maximum 8 V, the working temperature was 75 - C, and the highest gas response to H 2 was the result. However, no selectivity study was performed. The above section can be summarized as follows: the reasons for operating the gas sensors in the self-heating mode are to overcome the issue of integrating a heater element and to lower the power consumption. Mostly MOS materials with NWs morphology, including single NW, multiple NWs and networked NWs, have been used for self-heated gas sensors. However, the design of the individual NW sensors involves complicated process steps and laboratory fabrication methods with low yield, which are hardly transferable for large-scale production [bib_ref] Comparative NO 2 gas-sensing performance of the self-heated individual, multiple and networked..., Chinh [/bib_ref]. Also, core-shell NWs are very promising for use in the self-heating mode due to maximization of the interfaces between the core and the shell. Sensing materials decorated with noble metals can operate with low power consumptions due to their high sensitivity resulting from the presence of the noble metals. [fig_ref] Table 1: Summary of different self-heating gas sensors showing their sensing properties and power... [/fig_ref] shows the properties and the power consumptions of some self-heated gas sensors reported in the literature. ## Low-power mems-based gas sensors During the fabrication of sensing devices, MOS sensors are connected to a heater, which is necessary to activate the sensing reaction between the target gas and the sensing material. Therefore, many MOS-based commercial gas sensors use heater resistors to warm the sensing surface to the desired temperature [bib_ref] Gas-kinetic interactions of nitrous oxides with SnO 2 surfaces, Ruhland [/bib_ref]. However, the power consumption of such gas sens0rs is high [bib_ref] A transparent ZnO nanowire MEMS gas sensor prepared by an ITO micro-heater, Hsueh [/bib_ref] , which limits their application to smartphones and portable electronic devices. Therefore, a micro heater with low power consumption can play a vital role in overcoming the above issue [bib_ref] MEMS-based NO 2 gas sensor using ZnO nano-rods for low-power IoT application, Lee [/bib_ref]. The recent advances in different fabrication technologies have lead to the downsizing of sensing chips and heater resistors, resulting in tiny, reduced-power gas sensors [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. Especially, the micro-electromechanical system (MEMS) has enabled very compact, low power consuming (a few tens of mW), MOS gas sensors to be fabricated [bib_ref] Low power operation of temperature-modulated metal oxide semiconductor gas sensors, Burgués [/bib_ref]. Gas sensors based on MOS materials can be integrated into MEMS platforms to achieve miniaturized and portable sensing devices with very low power consumptions. MEMSs are electro-mechanical systems with a 3D geometry. They are constructed on silicon-wafer platforms by using microelectronic fabrication of a photolithography method and post-process techniques. These processes allow designers to accumulate different sensor arrays on a single sensor platform that can be utilized for mass-production at a low cost [bib_ref] Development of a micromachined hazardous gas sensor array, Mitzner [/bib_ref]. Nanoscale MOS materials integrated with MEMS technology are widely employed in gas sensing applications due to the drastically reduced size, low cost and significantly lower power consumption of such devices [bib_ref] Facile synthesis and ultrahigh ethanol response of hierarchically porous ZnO nanosheets, Zhang [/bib_ref] [bib_ref] Development of high sensitivity ethanol gas sensors based on Pt-doped SnO 2..., Ivanov [/bib_ref] [bib_ref] Design and simulation of MEMS gas sensor topologies for detection of inert..., Umesh [/bib_ref]. In comparison with traditional MOS-based gas sensors [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , MEMS-based gas sensors that use MOS materials typically consist of three basic components: (i) a micro-heater, (ii) a sensing material in the form of a thin film (receptor layer), and (iii) interdigited electrodes [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. The micro-heater embedded in the substrate layer (suspension), a dielectric layer or an insulation layer, a passivation layer, and a microheater element, as shown in [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , are components of a conventional MOS gas sensor [bib_ref] MEMS based integrated gas sensor for NO 2 and NH 3, Prajesh [/bib_ref] [bib_ref] Microhotplates for metal oxide semiconductor gas sensor applications-towards the CMOS-MEMS monolithic approach, Liu [/bib_ref]. Different materials, such as platinum (Pt), nichrome (Ni) and tungsten (W), can be used for the fabrication of the heater part [bib_ref] Investigation of high-temperature degradation of platinum thin films with an in-situ resistance..., Firebaugh [/bib_ref] [bib_ref] Design and electrothermal analysis of MEMS based microheater array for gas sensor..., Modal [/bib_ref] [bib_ref] On-chip deposition of carbon nanotubes using CMOS microhotplates, Haque [/bib_ref]. Five categories of microheater geometries are typically used: rectangular, square, circular, irregular shapes (like honeycombs, drive wheels or others), and 3D structures. The most widely reported structure is the meander shape with either a square or a rectangular pattern [bib_ref] Technological journey towards reliable microheater development for MEMS gas sensors: a review, Bhattacharyya [/bib_ref]. Microheaters can be categorized into different configurations, such as a closed membrane, a suspended membrane or a bridge [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. In all of them, the substrate just below the sensing region is etched away. Among them, the suspended membrane substrate [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] is the most popular because of its much reduced power consumption [bib_ref] Integration of ZnO nanostructures with MEMS for ethanol sensor, Pandya [/bib_ref]. The suspended membrane may either be released from the front side [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] or the back side [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. The main aim in making bridge/diaphragm architecture is to reduce the heat loss through air, which has a low thermal conductivity: 0.026 WK − 1 m − 1 at 20 - C, by using thermal isolation, thereby inducing low power consumption. The back-side etching of a substrate is good due its simplicity compared to front side etching because the front side etching for creating the cavity needs extra protection of the structure layer or the sacrificial layer. MOS-based MEMS gas sensors were first realized in the early 1990s and were derived from the so-called micro-hotplate devices [bib_ref] Growth of SnO 2 films on micromachined hotplates, Cavicchi [/bib_ref]. Many reports on MOS-based MEMS gas sensors have been published [bib_ref] MEMS-based NO 2 gas sensor using ZnO nano-rods for low-power IoT application, Lee [/bib_ref] [bib_ref] A low power MEMS gas sensor based on nanocrystalline ZnO thin films..., Bhattacharyya [/bib_ref] [bib_ref] Low power consumption micro C 2 H 5 OH gas sensor based..., Moon [/bib_ref] [bib_ref] A transparent ZnO nanowire MEMS gas sensor prepared by an ITO micro-heater, Hsueh [/bib_ref]. For example, Hsueh et al. fabricated a transparent MEMS-type gas sensor [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] based on ZnO NWs (length: 2.5 μm, diameter: ~75 nm) synthesized using a hydrothermal method as the sensing material, ITO as a transparent electrode, and a micro heater [bib_ref] A transparent ZnO nanowire MEMS gas sensor prepared by an ITO micro-heater, Hsueh [/bib_ref]. The highest power consumption at an applied voltage of 6.73 V was only 124 mW whereas the maximum power consumption obtained at an applied voltage of 11.55 V was 386 mW. The temperature obtained at 6.73 V was 150 - C, and that obtained at 11.55 V was 350 - C. The sensing results showed that the response to NO gas was higher than the responses to other gases due to the fact that the bond energy of NO (N-O = 200 kJ/mol) is lower than those of other gases. Also, a blue-light-emitting diode (LED) was integrated into the MEMS sensor, and its lighting effects on the gas-sensing characteristics at RT were investigated. When the LED was switched on, the sensor current increased while it decreased when the LED was switched off, which was related to the presence of defects in the ZnO NWs. A large number of electrons were generated by LED-emitted light, and the presence of defects and photo-generated electrons led to an enhanced response to NO gas. MOS composites have also been used in MEMS configurations to enhance the sensitivity of MEMS-based gas sensors. Behera et al. synthesized p-n ZnO-CuO nanoflake heterojunction nanostructures based on a MEMS platform [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] [bib_ref] An innovative gas sensor incorporating ZnO-CuO nanoflakes in planar MEMS technology, Behera [/bib_ref]. The simulated temperature distribution profile of the microheater indicated that the heat was uniform throughout the substrate and confined within the sensor platform [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. At an increased sensing temperature of 259 - C, the sensor consumed only 100 mW of power. At 300 - C, the sensor exhibited a high sensing response [(ΔR/R a ) × 100] of ~275% to 50 ppm of acetone. The enhanced gas-sensing performance was attributed to the charge carrier modulation at the surfaces of ZnO and CuO, the barrier height modulation at the ZnO-CuO interface, the presence of defects in the interfaces between ZnO and CuO, the catalytic effects of CuO, and the higher dipole moment of acetone relative to those of other gases. MOS-decorated gas sensors also have been used in MEMS configurations. For instance, Yuan et al. reported a MEMS-based acetone sensor made from CeO 2 nanodots decorated on WO 3 NWs, prepared through a hydrothermal and subsequent thermal decomposition [bib_ref] Fabrication of a micro-electromechanical system-based acetone gas sensor Using CeO 2 nanodot-decorated..., Yuan [/bib_ref]. The sensor exhibited a response (R a /R g ) of 1.30-500 ppb acetone with good stability, which indicates that this sensor can be used to diagnose diabetes via breath analyses. The sensing mechanism was related to the high surface area of the gas sensor, the fast carrier transport of the WO 3 NWs, the formation of WO 3 -CeO 2 heterojunctions, and the existence of large numbers of oxygen vacancies in CeO 2 . In summary, even though gas sensors based on MEMS technology can operate with reduced power consumption, the slurry-based drop coating of sensing materials on the small sensing areas of MEMS micro hot plates is an extremely difficult task. Furthermore, fabrication of MEMS-based sensing devices on a large-scale has difficulties such as low yield and large sample-to-sample deviation, which ultimately limit their practical use. One good strategy to deal with this is to deposit the sensing material by using advanced physical techniques such as sputtering, which is a simple technique, to integrate the sensing materials on micro-hot-plate platform [bib_ref] Sputtered SnO 2 : NiO thin films on self-assembled Au nanoparticle arrays..., Wang [/bib_ref]. ## Low or room-temperature operated mos gas sensors So far we reviewed the MOS-based gas sensors in self-heating mode without external heater. Mostly, various MOS materials with NW morphology have been reported for operation in self-heating mode. However, a very few reports on other kinds of morphologies based on self-heating method for gas sensing application have been also published [bib_ref] Realization of Au-decorated WS 2 nanosheets as low power-consumption and selective gas..., Kim [/bib_ref] [bib_ref] Self-heating hydrogen gas sensor based on an array of single suspended carbon..., Seo [/bib_ref] [bib_ref] Selfactivated ultra high chemosensitivity of oxide thin film nanostructures for transparent sensors, Moon [/bib_ref]. Conventional MOS-based gas sensors usually operate at high temperatures ranging from 100 to 450 - C in order to achieve enhanced sensing performance because of their larger band gap [bib_ref] Ag nanoparticles sensitized In 2 O 3 nanograin for the ultrasensitive HCHO..., Zhou [/bib_ref] [bib_ref] Facile fabrication of semiconducting oxide nanostructures by direct ink writing of readily..., Siebert [/bib_ref] , which leads to serious problems such as high-power consumption and increase of overall price of the gas sensor due to need for the heater. Thus, RT operation of gas sensors is highly desirable to minimize the risk of gas explosion, reduction of energy consumption, increases sensor life and possibility of integration into smart phone devices [bib_ref] Advances in designs and mechanisms of semiconducting metal oxide nanostructures for high-precision..., Li [/bib_ref] [bib_ref] Semiconductor metal oxides as chemoresistive sensors for detecting volatile organic compounds, Lin [/bib_ref]. Recently, many strategies have been carried out to enhance the sensing properties at MOS gas sensors RT such as morphology engineering of pristine MOSs [bib_ref] Room temperature H 2 S gas sensor based on rather aligned ZnO..., Hosseini [/bib_ref] [bib_ref] Room temperature high-performance H 2 S sensor based on porous CuO nanosheets..., Li [/bib_ref] [bib_ref] A room temperature sub-ppm NO 2 gas sensor based on WO 3..., Zhao [/bib_ref] , noble metal NPs-decorated MOSs [bib_ref] Improved NO 2 sensing performance of electrospun WO 3 nanofibers with silver..., Jaroenapibal [/bib_ref] [bib_ref] Ultra-sensitive and selective hydrogen nanosensor with fast response at room temperature based..., Lupan [/bib_ref] , doped MOSs [bib_ref] Semi shield driven p-n heterostructures and their role in enhancing the room..., Jayababua [/bib_ref] [bib_ref] High and fast H 2 S response of NiO/ ZnO nanowire nanogenerator..., Qu [/bib_ref] , construction of heterojunction structures [bib_ref] Highly selective detection of NH 3 and H 2 S using the..., Zhou [/bib_ref] [bib_ref] Gas sensors using carbon nanomaterials: a review, Llobet [/bib_ref] , hybrid composites with conducting polymers [bib_ref] Conducting polymers as chemiresistive gas sensing materials: a review, Wong [/bib_ref] , carbon materials [bib_ref] Reduced graphene oxide decorated with CuO-ZnO hetero-junctions: towards high selective gas-sensing property..., Wang [/bib_ref] [bib_ref] Two-dimensional nanostructured materials for gas sensing, Liu [/bib_ref] [bib_ref] Graphene-metal oxide nanohybrids for toxic gas sensor: a review, Gupta Chatterjee [/bib_ref] and 2D materials such transition metal chalcogenides, metal carbides [bib_ref] Chemical sensing of 2D graphene/ MoS 2 heterostructures device, Cho [/bib_ref] [bib_ref] Enhanced ammonia response of Ti 3 C 2 T x nanosheets supported..., Tai [/bib_ref]. Also UV-irradiation is a good technique to reduce the sensing temperature [bib_ref] UV-LED photo-activated chemical gas sensors: a review, Espid [/bib_ref] [bib_ref] A comparative study on UV light activated porous TiO 2 and ZnO..., Chen [/bib_ref]. In following section we discuss about low temperature or RT gas sensors based on MOS materials. ## Pristine mos gas sensors It is known that morphology greatly affects the gas sensing properties, and therefore various morphologies have been used for sensing studies [bib_ref] Approaches to enhancing gas sensing properties: a review, Yuan [/bib_ref]. For example, Wang et al. prepared WO 3 hierarchical hollow spheres by a facile hydrothermal method and explored its NO 2 sensing properties at RT [bib_ref] A room temperature sub-ppm NO 2 gas sensor based on WO 3..., Zhao [/bib_ref]. The sensor exhibited a high response (R g /R a ) of 15.1 to 300 ppb NO 2 gas with excellent selectivity and moderate response/recovery times (670 s/2940 s) and good repeatability. The high NO 2 sensing performance of the sensor was related to the large surface area, presence of high oxygen vacancies and electronic interactions which occurred between NO 2 gases with the oxygen ions on the surface of the sensor and electronic interactions occur between target gases with the oxygen ions of the sensor [bib_ref] Fabrication of highly sensitive and selective room-temperature nitrogen dioxide sensors based on..., Song [/bib_ref] [bib_ref] Ultrasensitive ppb-level NO 2 gas sensor based on WO 3 hollow nanospheres..., Zhang [/bib_ref] [bib_ref] La 2 O 3 -sensitized SnO 2 nanocrystalline porous film gas sensors..., Zhang [/bib_ref]. In addition to n-type MOSs, p-type MOSs has also been studied for RT gas sensing. For instance, Li et al. reported a highly sensitive and selective H 2 S gas sensor using porous CuO nanosheets, synthesized using a hydrothermal method [bib_ref] Room temperature high-performance H 2 S sensor based on porous CuO nanosheets..., Li [/bib_ref]. The sensor exhibited a response of 1.25 towards 10 ppb H 2 S with response/recovery time of 234 s and 76 s and excellent selectivity to H 2 S. According to the literature p-type CuO is the dominant material for H 2 S sensing [bib_ref] Hydrothermal synthesis of hierarchically flower-like CuO nanostructures with porous nanosheets for excellent..., Li [/bib_ref]. The sensing mechanism was mainly related to the transformation of semiconducting CuO to metallic CuS. In fact, H 2 S molecules can react with CuO to form CuS, based on the following reaction [bib_ref] Hydrothermal synthesis of hierarchically flower-like CuO nanostructures with porous nanosheets for excellent..., Li [/bib_ref]. [formula] H 2 S (g) + CuO (g) → CuS (s) + H 2 O (g)(11) [/formula] The transformation to CuS was confirmed by using XPS analysis, where after exposure to H 2 S gas a new peak related to the Cu 2p 3/2 state at 930.8 eV appears which can be attributed to CuS. Similar transition to metal sulfide also has been reported for In 2 O 3 gas sensor [bib_ref] Advances in In 2 O 3 -based materials for the development of..., Kumar [/bib_ref]. ## Mos modified with noble metal nps Noble metal NPs (Au, Ag, Pd, and Pt) not only can remarkably enhance the sensitivity, shorten the response/recovery times and enhance the selectivity, but also can decrease the operation temperature of gas sensor. Because at RT, the detection of a few gases like CO and H 2 by pristine MOS sensor is difficult. But, this issue can be solved by modifying the surface of the sensor using these noble metal NPs. Hence, the noble metal functionalization is regarded as one of the best approaches for enhancing gas sensing properties [bib_ref] Seiyama Effects of additives on semiconductor gas sensors, Yamazoe [/bib_ref] [bib_ref] Noble metal@metal oxide semiconductor core@shell nano-architectures as a new platform for gas..., Rai [/bib_ref]. For example, Arunkumar et al. [bib_ref] Au Decorated ZnO hierarchical architectures: facile synthesis, tunable morphology and enhanced CO..., Arunkumar [/bib_ref] prepared ZnO nanostars using a hydrothermal followed by the decoration of different wt% of Au NPS (as shown in [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] The ZnO nanostars with 3 wt% Au NPs showed highest surface area among other morphologies such as ZnO nanoflowers, marigold and nanorods. The Au-ZnO nanostars also exhibited excellent sensitivity (R s = 35) towards 500 ppm CO with a very fast response/recovery times (41 s/40 s) at RT and excellent selectivity to CO among other interfering gases. Due to the spillover effect [bib_ref] Facile approach to synthesize Au@ZnO core-shell nanoparticles, and their application for highly..., Majhi [/bib_ref] of Au NPs, the thickness of EDL formed at the interface between Au and ZnO increases [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] and upon exposure to CO gas, significant reduction in the thickness of EDL, contributing to the sensing signal. Moreover, the hierarchical structure with numerous channels provided plenty of adsorption sites for CO gas molecules [bib_ref] Gold-nanoparticle-functionalized In 2 O 3 nanowires as CO gas sensors with a..., Singh [/bib_ref]. ## P-n heterojunction nanostructures Composite structures comprising of n-p heterojunctions are highly demanded in gas sensing application due to their synergistic effects [bib_ref] Self-heating effects on the toluene sensing of Ptfunctionalized SnO 2 -ZnO core-shell..., Kim [/bib_ref] [bib_ref] Nanoscale metal oxide-based heterojunctions for gas sensing: a review, Miller [/bib_ref]. The sensing performance can be greatly improved due to the electronic sensitization at p-n heterojunctions and broadening of EDL on the interfaces between the sensing materials [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. An ethanol sensor based on p-n NiO/SnO 2 nanocomposite reported recently [bib_ref] Semi shield driven p-n heterostructures and their role in enhancing the room..., Jayababua [/bib_ref]. The sensor exhibited a high sensitivity of (R a /R g =140) to 100 ppm ethanol at RT with fast response/recovery times of 23 and 13 s, respectively. The enhanced ethanol gas sensing was attributed to the formation of depletion layer at the p-n junction and catalytic activity of the NiO [bib_ref] High and fast H 2 S response of NiO/ ZnO nanowire nanogenerator..., Qu [/bib_ref]. Another p-n heterojunction sensor was prepared by an electrospinning method using mesoporous In 2 O 3 -CuO composite nanofibers [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , with a high surface area of 48.7 m 2 /g. Due to a mesoporous morphology and high surface area, as well as formation of p-n heterojunction [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , the sensor showed a response 1.5-100 ppm NH 3 at RT with good selectivity and fast dynamics [bib_ref] Highly selective detection of NH 3 and H 2 S using the..., Zhou [/bib_ref]. ## Mos structure modified with 2d materials Recently, various 2D nanomaterials, such as graphene and its derivatives, transition-metal dichalcogenides (TMDs), black phosphorous and transition-metal carbides/nitrides (MXene), have been used for the fabrication of RT gas sensors [bib_ref] Two-dimensional nanostructured materials for gas sensing, Liu [/bib_ref]. However, their RT sensing properties need to be enhanced in terms of sensitivity, selectivity and response/recovery [bib_ref] Synergistic effects in gas sensing semiconducting oxide nano heterostructures: a review, Walker [/bib_ref] [bib_ref] Graphene-metal oxide nanohybrids for toxic gas sensor: a review, Gupta Chatterjee [/bib_ref] [bib_ref] Preparation and applications of mechanically exfoliated single-layer and multilayer MoS 2 and..., Li [/bib_ref] [bib_ref] Sensing behavior of atomically thin-layered MoS 2 transistors, Late [/bib_ref]. The combination of these 2D materials with a MOS structure has led to significant improvements in the sensing properties [bib_ref] Realization of Au-decorated WS 2 nanosheets as low power-consumption and selective gas..., Kim [/bib_ref]. Due to its excellent conductivity, high surface area, and unique 2D structure, reduced graphene (rGO) has been recognized as a promising sensing material [bib_ref] mimicking a dog's nose: scrolling graphene nanosheets, Chen [/bib_ref] [bib_ref] Highly selective and ultra-low power consumption metal oxide based hydrogen gas sensor..., Rascha [/bib_ref]. Especially, it is used in composite form to enhance its sensing properties. For example, a nanocomposite with a rGO-ZnO-SnO 2 heterostructure was synthesized by using the hydrothermal method [bib_ref] Construction of ZnO SnO 2 heterostructure on reduced graphene oxide for enhanced..., Wang [/bib_ref]. The as-prepared ternary composite sensor exhibited a response (ΔR/R a × 100) of 141% towards 5 ppm of NO 2 at RT. The presence of rGO led to rapid electron transfer between ZnO and SnO 2 . Furthermore, the formation of p-n and n-n heterojunctions contributed to the enhanced NO 2 sensing characteristics. In another study, Ma et al. reported RT gas-sensing three-dimensional (3D) Fe 3 O 4 -SiO 2 -rGO core-shell spheres synthesized using electrostatic self-assembly, followed by thermal reduction. The as-prepared 3D Fe 3 O 4 -SiO 2 -rGO nanosphere suspensions (0.5 mg/mL) were dropped onto the sensor chip under a magnetic field (0.28 T) applied perpendicular to the electrodes, and after evaporation, the materials were aligned in the sensor electrodes to form uniform multichannel sensing devices. The sensor exhibited an ultrahigh response of 34.41 toward 5 ppm of NO 2 at RT. The high sensing performance was related to the large sensing area resulting from the presence of rGO, to the 3D nature of the sensing layer, and to the high electron transport between rGO and Fe 3 O 4 -SiO 2 . Transition-metal dichalcogenides (TMDs) such as molybdenum disulfide (MoS 2 ) have grabbed tremendous attention due to their having high stability, numerous surface active sites for functionalization and gas adsorption, and good conductivity at RT [bib_ref] Chemical sensing of 2D graphene/ MoS 2 heterostructures device, Cho [/bib_ref] [bib_ref] Room-temperature SO 2 gas sensing properties based on a metal-doped MoS 2..., Zhang [/bib_ref]. However, the pristine MoS 2 gas sensors exhibit low sensitivity and slow dynamics at RT [bib_ref] High-performance sensors based on molybdenum disulfide thin films, Lee [/bib_ref]. Accordingly, it is mostly used in a composite form. For example, Han et al. reported the fabrication of MoS 2 /ZnO heterostructures via a wet chemical route [bib_ref] Design of hetero-nanostructures on MoS 2 nanosheets to boost NO 2 roomtemperature..., Han [/bib_ref]. The sensor showed a response (ΔI/I a ) of 30.50 towards 5 ppm of NO 2 with an ultrafast response time (40 s). The sensing mechanism was explained on the basis of the synergistic effects of the unique 2D/0D morphology and the formation of p-n junctions between ZnO and MoS 2 [bib_ref] Interface bonds determined gas-sensing of SnO 2 -SnS 2 hybrids to ammonia..., Xu [/bib_ref]. In another study related to MoS 2 , Chen et al. reported the surface functionalization of MoS 2 by Au NPs for selective detection of acetone at RT [bib_ref] Surface functionalization of layered molybdenum disulfide for the selective detection of volatile..., Chen [/bib_ref]. The sensor exhibited high a response to acetone gas, which was related to the strong adsorption of acetone molecules on the Au-MoS 2 sensor and to the high catalytic activity of Au NPs towards acetone. Another category of 2D materials with sheet-like or layered morphology is known as MXenes, which have a general formula of Mn +1 AX n , (MAX phase), where M stands for a transition metal, A is a IIIA or IVA element, and X is N and/or C with n = 1, 2, or 3 [bib_ref] Recent advance in MXenes: a promising 2D material for catalysis, sensor and..., Zhu [/bib_ref] [bib_ref] Two-dimensional transition metal carbides, Naguib [/bib_ref] [bib_ref] Guidelines for synthesis and processing of two-dimensional titanium carbide (Ti 3 C..., Alhabeb [/bib_ref]. The advantages of these materials are high conductivity, high surface area due to the unique 2D morphology, and excellent flexibility, which make them attractive materials for many applications, including gas sensing [bib_ref] Guidelines for synthesis and processing of two-dimensional titanium carbide (Ti 3 C..., Alhabeb [/bib_ref] [bib_ref] 2D metal carbides and nitrides (MXenes) for energy storage, Anasori [/bib_ref] [bib_ref] Room temperature gas-sensing of Two dimensional titanium carbide (MXene), Lee [/bib_ref]. Some studies related to the gas-sensing properties of a pristine Mxene and its composites with a MOS structure have been published [bib_ref] Room temperature gas-sensing of Two dimensional titanium carbide (MXene), Lee [/bib_ref] [bib_ref] Metallic Ti 3 C 2 T x MXene gas sensors with ultrahigh..., Kim [/bib_ref] [bib_ref] A flexible VOCs sensor based on a 3D MXene framework with a..., Yuan [/bib_ref] [bib_ref] Two-dimensional vanadium carbide MXene for gas sensors with ultrahigh sensitivity toward nonpolar..., Lee [/bib_ref] [bib_ref] Room-Temperature, Highly Durable Ti 3 C 2 T x MXene/Graphene hybrid fibers..., Lee [/bib_ref]. For example, Tai et al. designed an ammonia gas sensor made from Ti 3 C 2 T x and TiO 2 composite nanostructures [bib_ref] Enhanced ammonia response of Ti 3 C 2 T x nanosheets supported..., Tai [/bib_ref]. TiO 2 NPs were deposited on the synthesized Ti 3 C 2 T x nanosheets to fabricate TiO 2 /Ti 3 C 2 T x nanosheet composites. The fabricated gas sensor exhibited a higher response to ammonia relative to other gases at RT. The enhanced NH 3 sensing response of the sensor was explained using the model of self-built electric-field (space-charge layer) regulation. Upon intimate contact between Ti 3 C 2 T x and TiO 2 , electrons in the conduction band of TiO 2 migrated to Ti 3 C 2 T x due to its excellent metallic properties and to the higher work function of Ti 3 C 2 T x , leading to a self-built electric field (Schottky barrier) at their interface. In air, the density of holes on the surface of Ti 3 C 2 T x increases whereas the number of electrons on the surface of TiO 2 decreases because the electrons are consumed by the generated ionized adsorbed oxygen, resulting in a weakening of the self-built electric field due to the increased number of holes in Ti 3 C 2 T x and decreased resistance of TiO 2 /Ti 3 C 2 T x . On the contrary, in NH 3 , the self-built electric field was strengthened due to a decrease in the number of holes on the surface of Ti 3 C 2 T x and an increase in the number of electrons on the surface of TiO 2 (4NH 3 + 5O 2 -→ 4NO + 6H 2 O + 5e -), leading to increased resistance. ## Mos structure modified with conducting polymers In addition to the MOS/rGO nanocomposites used for sensing studies [bib_ref] Recent development in nanocarbon materials for gas sensor applications, Xiao [/bib_ref] [bib_ref] Fabrication of flexible roomtemperature NO 2 sensors by direct laser writing of..., You [/bib_ref] , many MOS/conducting polymer (CP) composites have been used for such purposes. CPs such as polyaniline (PANI), polythiophene (PTh) and polypyrrole (PPy) are the most common polymers for RT gas-sensing studies due to their unique electrical and optical characteristics, low cost, ease of fabrication, and flexibility [bib_ref] Gas sensors based on conducting polymers, Bai [/bib_ref] [bib_ref] Highly sensitive, room temperature gas sensor based on polyaniline-multiwalled carbon nanotubes (PANI/MWCNTs)..., Abdulla [/bib_ref] [bib_ref] Low power consumption and fast response H 2 S gas sensor based..., Alia [/bib_ref]. Most CPs have good sensitivity to ammonia gas, but in pristine form, their recovery times are long; thus, they are mostly used in composite form with MOS structures. For example, Beniwal et al. demonstrated NH 3 SnO 2 /PPy electrospun composite sensors [bib_ref] Electrospun SnO 2 /PPy nanocomposite for ultra-low ammonia concentration detection at room..., Beniwal [/bib_ref]. The sensor showed an excellent response (ΔR/R a ) of 57% with a short response/recovery time of 18 s/30 s for 100 ppb of NH 3 at RT. The intrinsically good sensitivity of SnO 2 to NH 3 , along with formation of p-n heterojunctions, led to the enhanced response to NH 3 at RT. Furthermore, PPy has a -NH-group, which can easily interact with NH 3 gas, thereby increasing the electrical resistance and, hence, the gas response. PPy/MnO 2 and PANI/Fe 2 O 3 are other examples of composites used for RT gas-sensing studies [bib_ref] Highly selective and sensitive response of polypyrrole-MnO 2 based composites towards ammonia..., Malook [/bib_ref] [bib_ref] One step synthesis of PANI/Fe 2 O 3 nanocomposites and flexible film..., Zhu [/bib_ref]. Recently, a novel CuO-Chitosan nanocomposite H 2 S gas sensor was introduced by Fajr et al. [bib_ref] Low power consumption and fast response H 2 S gas sensor based..., Alia [/bib_ref]. The sensor revealed a fast response of 14 s and a high response (ΔR/R a ) of 217.89% for 100 ppm of H 2 S gas at 40 - C. They also measured the power consumption of the sensor and found that it showed a power consumption of 0.45 W at 40 - C. The formation of heterojunctions and the catalytic effect of CuO contributed to the sensing signal. ## Mos treated with uv-irradiation A good strategy for decreasing the operation temperature of gas sensors is UV illumination. When the energy of UV light is equal or higher than the band gap energy of the sensing material, photogenerated electrons and holes can be produced by the absorption of UV light. In fact, the energy required to excite these electrons and holes is supplied by the light, instead of by heat. Furthermore, illumination facilitates the chemisorption of oxygen molecules on the surface to generate more O 2 by improving the chemical activity of the surface [bib_ref] Design and simulation of MEMS gas sensor topologies for detection of inert..., Umesh [/bib_ref] at RT as follows [bib_ref] A comparative study on UV light activated porous TiO 2 and ZnO..., Chen [/bib_ref] [bib_ref] Facile approach to synthesize Au@ZnO core-shell nanoparticles, and their application for highly..., Majhi [/bib_ref] [bib_ref] Gold-nanoparticle-functionalized In 2 O 3 nanowires as CO gas sensors with a..., Singh [/bib_ref] [bib_ref] Pt-functionalized PdO nanowires for room temperature hydrogen gas sensors, Cho [/bib_ref] : [formula] hν → h + + e -(12)O 2 + e− (hν) → O 2 -(hν) → O 2 - (hν) ,(13) [/formula] where ν is the frequency of the illuminated light and h is Planck's constant. Many reports about RT operation of gas sensors under UV illumination [bib_ref] MEMS based integrated gas sensor for NO 2 and NH 3, Prajesh [/bib_ref] [bib_ref] UV-activated room-temperature gas sensing mechanism of polycrystalline ZnO, Fan [/bib_ref] [bib_ref] Metal oxide semiconductors with highly concentrated oxygen vacancies for gas sensing materials:..., Zhang [/bib_ref] [bib_ref] Room temperature responses of visible-light illuminated WO 3 sensors to NO 2..., Zhang [/bib_ref] have been published. For example, Cui et al. reported ZnO nanostructures with different morphologies (nanofibers, nanoflowers, nanoplates) for HCHO detection under illumination with UV light at RT [bib_ref] UV-light illumination room temperature HCHO gas-sensing mechanism of ZnO with different nanostructures, Cui [/bib_ref]. The ZnO morphology played a significant role in the sensing capacity, and the ZnO nanofiber sensor was found to display a high response of 12.61 (I g /I a ) to 100 ppm of HCHO under illumination with 365-nm UV light at RT. Initially, under exposure to UV light, chemisorbed oxygen ions are formed due to the removal of electrons from the conduction band of ZnO, resulting in the formation of a depletion layer. The photo-induced oxygen O 2 − ions (hν) are weakly bound to ZnO and can be removed just by turning-off the UV light. When a ZnO sensor is exposed to HCHO, the following reaction occurs: [formula] O 2 − (hν) + HCHO (g) → CO 2 + H 2 O + e − .(14) [/formula] The release of electrons and the desorption of oxygen species lead to a reduction in the thickness of the depletion layer and a decrease in the electrical resistance [bib_ref] UV-light illumination room temperature HCHO gas-sensing mechanism of ZnO with different nanostructures, Cui [/bib_ref]. The sensing enhancement in ZnO nanofibers was related to the surface area (9.61 m 2 /g) of this sensor with a fibrous structure being larger relative to those of other morphologies. In this section, we discussed various strategies to enhance the RT sensing properties of MOS-based gas sensors. In general, different strategies such as composite made using n-n MOSs or p-n MOSs, decoration with noble-metal NPs, and morphology engineering, as well as hybrid composites with carbon materials or CPs, have been used to realize RT gas sensors. Gas sensor operating at low temperatures with little or no power consumption can be used for different applications. In the next section, we will discuss the most important applications of gas sensors , namely, flexible and wearable gas sensors. ## Applications of low or rt gas sensors ## Flexible and wearable sensors Considering the types of substrates used for gas sensor fabrication, gas sensors can be divided into flexible and non-flexible (rigid) gas sensors. The non-flexible sensors, which are currently the most common types, are fabricated on rigid substrates. However, they are rigid and cannot be used in applications that require high flexibility. For example, in some applications such as personal use, a gas sensor must be flexible under mechanical deformations and must have properties making it suitable for wearing. Flexible gas sensors are realized on flexible substrates, and upon stretching, titling or bending, their properties should not change significantly [bib_ref] Wearable flexible sensors: a review, Nag [/bib_ref]. Flexible gas sensors with low weight, low price, high flexibility, high stretchability, and high conformability can act as good platforms for wearable gas sensors and has significance in monitoring in ambient atmosphere at RT [bib_ref] Wearable and flexible electronics for continuous molecular monitoring, Yang [/bib_ref] [bib_ref] Review of wearable device technology and its applications to the mining industry, Mardonova [/bib_ref] [bib_ref] Wearable, wireless gas sensors using highly stretchable and transparent structures of nanowires..., Park [/bib_ref]. Wearable sensors with integrated wireless technology can be integrated to an end-user with the Internet of Things (IoT) and offer real-time information for personal use [bib_ref] A survey on wearable sensor-based systems for health monitoring and prognosis, Pantelopoulos [/bib_ref] , which has opened a new path for smart wearable devices. Different flexible substrates, such as polyethylene-terephthalate (PET) [bib_ref] Light-controlling, flexible and transparent ethanol gas sensor based on ZnO nanoparticles for..., Zheng [/bib_ref] , polyimide (PI) [bib_ref] Microheater-integrated single gas sensor array chip fabricated on flexible polyimide substrate, Kim [/bib_ref] [bib_ref] A flexible, transparent and high-performance gas sensor based on layer-materials for wearable..., Zheng [/bib_ref] , Kapton ® [bib_ref] Low temperature trimethylamine flexible gas sensor based on TiO 2 membrane nanotubes, Perillo [/bib_ref] , have been used for the realization of flexible and wearable sensors. Graphene and its derivatives have been shown to be ideal candidates for flexible gas sensors due to their high mechanical strength, good stability, high carrier mobility, and good flexibility [bib_ref] Flexible graphene-based wearable gas and chemical sensors, Singh [/bib_ref] [bib_ref] Graphene-based gas sensor: metal decoration effect and application to a flexible device, Cho [/bib_ref]. Moreover, gas sensors based on fibers or textiles have been investigated due to their potential applications in wearable devices. For example, Yun et al. reported a bendable and washable electronic-textile (e-textile) gas sensor based on a composite of RGO-decorated cotton yarn, the so-called RGOCY [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] [bib_ref] Ultrasensitive and highly selective graphene-based single yarn for use in wearable gas..., Yun [/bib_ref]. The sensor was able to sense NO 2 gas at RT. [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] [formula] (b)-(e) [/formula] show the performance of the fabricated gas sensor under different conditions, such as bending (bend radius of 1.0 mm) and straightening; even after washing with a detergent, the sensor showed stable sensing properties, demonstrating its excellent performance under different conditions. Moreover, after 1000 bending-straightening cycles, the sensor exhibited stable electrical and sensing properties. The stability under washing was attributed to the chemical against water and detergent and to the mechanical resistance to shearing resulting from friction between the yarn and the magnetic stirrer during the washing of the gas sensor. Lee et al. first introduced a flexible hybrid sensor based on graphene fibers and Ti 3 C 2 T x Mxene fabricated by using a wet spinning process [bib_ref] A comparative study on UV light activated porous TiO 2 and ZnO..., Chen [/bib_ref]. Initially, Ti 3 C 2 T x MXene was synthesized, after which it was dispersed with a GO suspension to be drop casted onto the Si/SiO 2 substrate to realize a composite sensor. After thermal activation at 200 - C, the composite Ti 3 C 2 T x /GO was reduced to Ti 3 C 2 T x /rGO, and its gas-sensing properties at RT were measured. The sensor displayed an enhanced response to NH 3 (ΔR/R 0 × 100 = 6.77%) with excellent flexibility and stability, enabling its use in portable and wearable sensing devices. At RT, the band gap of the Ti 3 C 2 T x /rGO composite was widened from 1.05 eV to 1.57 eV, which had a positive effect on its sensing properties. Also, the optimum content of Ti 3 C 2 T x in the Ti 3 C 2 T x /rGO composite was 40 wt%. For higher amounts of Ti 3 C 2 T x , the band gap decreased, leading to a weak gas response due to the simultaneous increase in the density of charge carriers. Many reports on rGO composite gas sensors with good flexibility have been published [bib_ref] Room-Temperature, Highly Durable Ti 3 C 2 T x MXene/Graphene hybrid fibers..., Lee [/bib_ref] [bib_ref] Recent development in nanocarbon materials for gas sensor applications, Xiao [/bib_ref]. You et al. reported a flexible sensing device made of a composite film of graphene and In 2 O 3 for NO 2 sensing at RT [bib_ref] Fabrication of flexible roomtemperature NO 2 sensors by direct laser writing of..., You [/bib_ref]. They designed a direct laser writing (DLW) process to integrate the In 2 O 3 -RGO sensor. The coated In 2 O 3 -GO film on a flexible substrate was converted to In 2 O 3 -RGO by using a CO 2 laser and a programmable scanning procedure [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. One of the benefits of laser treatment is that it makes the sensor conducting due to the photo reduction of GO. Even though the initial In 2 O 3 -GO film was an insulator with high resistance, after laser treatment it was conductive. Although the In 2 O 3 -RGO composite sensor showed a good sensitivity to NO 2 gas, its response and recovery times were ~ 4.2 min/~ 13.3 min to 500 ppb of NO 2 gas at RT, which limit its applications in real situations. In addition to the composites made from rGO/MOS [bib_ref] Fabrication of flexible roomtemperature NO 2 sensors by direct laser writing of..., You [/bib_ref] , rGO/Mxene [bib_ref] Room-Temperature, Highly Durable Ti 3 C 2 T x MXene/Graphene hybrid fibers..., Lee [/bib_ref] and rGO-cotton Yarn [bib_ref] Ultrasensitive and highly selective graphene-based single yarn for use in wearable gas..., Yun [/bib_ref] , CP-based composites [bib_ref] Highly sensitive, room temperature gas sensor based on polyaniline-multiwalled carbon nanotubes (PANI/MWCNTs)..., Abdulla [/bib_ref] [bib_ref] Low-cost and flexible printed graphene-PEDOT: PSS gas sensor for ammonia detection, Seekaew [/bib_ref] have also been studied for flexible, wearable sensors at RT. PEDOT:PSS is a conjugated polymer that is widely used for flexible and printed electronics due to its good electrical conductivity, high transparency, and good processability. Seekaew et al. demonstrated a flexible gas sensor based on a PEDOT-PSS/graphene nanocomposite [bib_ref] Low-cost and flexible printed graphene-PEDOT: PSS gas sensor for ammonia detection, Seekaew [/bib_ref]. Yamazoe et al. demonstrated a flexible gas sensor based on a PEDOT-PSS/graphene nanocomposite [bib_ref] Seiyama Effects of additives on semiconductor gas sensors, Yamazoe [/bib_ref]. The sensing film was deposited on a flexible substrate by using ink-jet printing. The ink for ink-jet printing was prepared using a PEDOT:PSS and graphene solution. The sensor showed high selectivity to NH 3 at RT and exhibited a p-type conducting behavior. The increased NH 3 sensing response for increased bending was related to the swelling effect of the sensor, where NH 3 gas molecules diffused into the graphene-PEDOT-PSS layer [bib_ref] Conductivity, morphology, interfacial chemistry, and stability of poly(3,4-ethylene dioxythiophene)-poly(styrenesulfonate): a photoelectron spectroscopy..., Crispin [/bib_ref]. The enhanced sensing properties of the sensor were related to the direct charge-transfer process between the graphene-PEDOT-PSS surface and the NH 3 molecules due to the improved interactions via π electrons, the high specific surface area due to the presence of graphene with a sheet-like morphology, and the swelling due to the diffusion of NH 3 molecules into the graphene/polymer chain matrix [bib_ref] Enhanced sensitivity of ammonia sensor using graphene/polyaniline nanocomposite, Wu [/bib_ref]. Other than the rGO, another carbon material, namely, a single wall carbon nanotube (SWCNT), has been utilized to realize flexible gas sensors due to its having a high surface area, porous structure, high conductivity, and strong affinity for the adsorbed gas molecules. For example, Hua et al. prepared SWNT-Fe 2 O 3 composite films by combination of CVD and subsequent heat treatment in air [bib_ref] A flexible gas sensor based on single-walled carbon nanotube-Fe 2 O 3..., Hua [/bib_ref]. As presented in [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref] , the gas sensor was deposited on a flexible plastic substrate, and it was able to be bent to large angles (90 - and 180 - ) and then recover to its initial flat shape. The gas sensor's responses to 20 ppm of H 2 S under different bending angles at RT are presented in [fig_ref] Figure 1: Schematic illustration of the basic sensing principle for [/fig_ref]. The sensor was deformed from a straight to a bent shape 16 times. Under all conditions, the sensor showed almost similar responses to H 2 S gas. RT gas sensors are in high demand for different applications because no external energy is required for their operation. [fig_ref] Table 2: Gas-sensing properties of RT-based gas sensors [/fig_ref] summarizes the RT gas-sensing properties of resistive-based gas sensors. The table clearly demonstrates that different sensing materials, whether in pristine, composite or decorated form, with various morphologies have been successfully used for RT gas sensors. ## Conclusions, challenges and future perspectives In this comprehensive review paper, we discussed the recent progress in the field of energy-saving gas sensors in terms of various sensing materials, sensing mechanisms, and strategies used to save energy. Due to the fast development of 5G technology and IoT and to further improvements of life service, in the near future, energy-saving gas sensors will be in high demand and will be extensively integrated into common electronic devices, such as smartphones, tablets, smart watches and so on. For these applications, realization of flexible, highly sensitive, selective, stable and cheap gas sensors with fast response and recovery times are in great demand. Self-heating is a promising strategy for MOS-based gas sensors, especially for MOS materials with NW morphology. Operation of gas sensors in the self-heating mode can greatly decrease the power consumption from several μW to nW level. Such reductions in the power consumed can remarkably increase the sensor life and save a great amount of energy. Single, ordered, and networked NWs can be used for this purpose. However, synthesis of networked NWs is easier than that of single or ordered NWs, and as a result, currently, it is the most widely used morphology for self-heating gas sensors. However, mostly MOS materials with NW morphology have been reported for operation in selfheating mode. Thus, there is much room available for the future development of other morphology based self-heated gas sensors. Although, the self-heated gas sensors could able to show the power consumption in the range of nW level, however, the response value of most the sensors in this category are very less, which needed to be studied further. Utilization of UV light is another technique used to decrease the operating temperature of gas sensors. UV light with energies higher than the band-gap energy of the sensing layer can excite electrons to the conduction band, and reactions of the electrons with oxygen and target gases can enhance the response of the sensor at RT. However, positioning a source of UV light just above the sensing layer is not always possible due to size limitations and economic considerations. Sensors that can work at RT without the need for UV light or operate in the selfheating mode do not need any external heater and, therefore, hold promise for energy-saving purposes. In addition to materials, morphology greatly affects the sensing properties of sensors that work at RT. Among different strategies to improve the gas-sensing properties of RT based gas sensors, the noble-metal functionalization and construction of heterojunction nanostructures are widely used. However, the operation of a sensor at RT has some challenges. The catalytic and sensitization effects are the main reasons for enhancement of sensitivity and selectivity of RT gas sensors. On the other hand, the synergistic effects of heterojunctions are the primary reason of the high sensing properties of p-n heterojunction based RT gas sensors. However, the humidity presence in the RT sensing ambient can significantly reduce the sensitivity of a gas sensor. Furthermore, most often the reactions cannot be completed at RT; accordingly, the response at RT is lower relative to those at higher temperatures. MOS-based MEMS gas sensors are another choice as they have simple architectures, are easy to fabricate, and are efficient in term of energy use. In fact, the use of MEMS gas sensors not only can significantly decrease the final dimensions of the gas sensors but can also lead to reductions in power consumption in gas sensors and electronic devices. However, considering the power consumption of the above studied gas sensors, MEMS based gas sensor still behind the self-heated gas sensors (Scheme 1). One of the problems of MEMS gas sensors is they still use the external heater and gas sensing measurement still requires the elevated temperatures for the operation. Hence, it is still challenging to overcome the issue. However, in case of RT based gas sensors, there are a few reports have been reported on the power consumption study. Despite of significant advances made so far in the developing low power consumption based gas sensors, several challenges and issues should be focused towards achieving high sensitivity, selectivity long-term stability with quick response/recovery time. In the future, with further developments of synthesis techniques, novel materials and creative technologies, high-performance gas sensors with high flexibility that can work at RT will be more readily available. ## Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. [fig] Figure 1: Schematic illustration of the basic sensing principle for (a) n-and (b) p-type MOS gas sensors. Reprinted from Ref. [/fig] [fig] Figure 2: (a) Schematic of the resistance change of an n-type MOS gas sensor during reducing gas (H 2 ) detection, (b) surface charge layer model, and (c) grain boundary barrier model. Reproduced from the Ref. [30] under the Creative Commons attribution 4.0 License (CC BY 4.0/). Copyright 2020, IOP Science. [/fig] [fig] Figure 3: An overview of the main techniques and applications of the energy-saving gas sensors discussed in this review paper. [/fig] [fig] Figure 4: Schematics of (a) a conventional MOS-based gas sensor and (b) a MOS-based gas sensor in the self-heating mode. Adapted from Ref. 62 with permission from Elsevier (Copyright 2020). S.M. Majhi et al. [/fig] [fig] Figure 5: (a) Schematic illustration of the fabrication of networked SnO 2 NWs. (b-e) FE-SEM images of networked SnO 2 NWs grown on the sensor substrate after reducing the electrode size. Reproduced from Ref. 74 with permission from the American Chemical Society (Copyright 2020). [/fig] [fig] Figure 6: (A) Scheme illustrating the procedures for the fabrication of the sensor chip (Step-1) followed by the on-chip growth of ZnO NWs (Step-II), and Pd and Pt functionalization on ZnO NWs (Step-III); (B, C) morphology of Pt and Pd-ZnO NWs; (D, E) response values obtained for Pt and Pd-ZnO NWs sensors with respect to applied voltages; (F) toluene gas-sensing mechanism of (a) pure ZnO NWs and (c) Pt/Pd-ZnO NWs. (b) Energy band diagram of Pt, Pd NPs and ZnO NWs. Reprinted from Ref. 82 with permission from Elsevier (Copyright 2020). [/fig] [fig] Figure 7: (a) Schematic presentation of the synthesis procedures for Au-decorated SnO 2 -ZnO core-shell NWs, (b-c) FE-SEM images of bare SnO 2 NWs and Au functionalized SnO 2 -ZnO core-shell NWs, and (d) variation of Au/SnO 2 -ZnO core-shell NW sensor's response with the thickness of the ZnO shell. (e) Temperature versus applied voltage and (f) schematic of the self-heating mechanism in SnO 2 -ZnO C-S NWs and the corresponding process of resistance generation. Reprinted from Ref. 83 with permission from Elsevier (Copyright 2020). benzene at different concentrations were measured under different applied voltages from 1 to 20 V. With increasing concentration and applied voltages, the response also increased, and the highest response of 2.62 was obtained for 50-ppm benzene at 20 V. The high selectivity of the Pd-SnO 2 -ZnO core-shell NW sensor toward benzene can be attributed to the interaction between the π electrons of benzene and the dband electrons of Pd NPs [76]. A response of 43.13 was obtained for 50-ppm benzene at a temperature of 200 • C. The response of the sensor was mainly attributed to the ZnO shell because the heterojunction barrier hinders the electron flow from SnO 2 to ZnO. Therefore, the majority of electrons flow through the ZnO shells. The Joule heating in SnO 2 -ZnO C-S NWs typically occurs in different ways viz. inside the ZnO grains and grain boundaries as well as the ZnO-ZnO homojunctions. The highest value of power consumptions of 22.6 μW was obtained at 20 V whereas the lowest value of 0.99 nW was obtained at 1 V. [/fig] [fig] Figure 8: (a) Synthesis procedure for realizing Pt-SnO 2 -ZnO C-S NWs, (b, c) corresponding FE-SEM images, (d) thermographic images of Pt-SnO 2 -ZnO C-S NW sensors for different shell thicknesses (10-85 nm) with varying operating voltage (0-20 V), and (e) self-heating and sensing mechanisms. Reproduced from Refs. [84] with permission from Elsevier (Copyright 2020). [/fig] [fig] Figure 9: (a) Schematic for the preparation of Pd-decorated CuO NWs. TEM micrographs of the as-synthesized (c) CuO NWs and (d) Pd/CuO NWs. (c) Heat generated under the self-heating condition and the corresponding thermographic image of the CuO NW sensor at 5 V. (d) Self-heating mechanism of the CuO NW sensor showing the self-heating effects in CuO NWs and CuO-CuO junctions. Reproduced from Ref. [86] with permission from Elsevier (Copyright 2020). [/fig] [fig] Scheme 1: An overview of resistive-based gas sensors based on their energy consumption point of view. S.M. Majhi et al. [/fig] [table] Table 1: Summary of different self-heating gas sensors showing their sensing properties and power consumptions. [/table] [table] Table 2: Gas-sensing properties of RT-based gas sensors. [/table]

Gene expression and translation have been extensively studied in human post-mortem brain tissue from subjects with psychiatric disease. Post-translational modifications (PTMs) have received less attention despite their implication by unbiased genetic studies and importance in regulating neuronal and circuit function. Here we review the rationale for studying PTMs in psychiatric disease, recent findings in human post-mortem tissue, the required controls for these types of studies, and highlight the emerging mass spectrometry approaches transforming this research direction. # Introduction Psychiatric disease imparts a substantial burden on the global population. For example, depression, schizophrenia, bipolar disorder , and autism spectrum disorderare estimated to collectively impair the lives of over 350 million individuals across the globe, with limited treatment options and a relatively small number of compounds in FDA trials. Thus, psychiatric disease is deservedly the focus of intense scientific study. Psychiatric disorders have varying levels of heritabilityand genome wide association studies (GWAS) have identified risk loci for some, such as schizophrenia (Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014) and autism spectrum disorder. Studies in live patients [e.g., TMS, EEG, and fMRI] and of post-mortem brain tissue have found distinct impairments in discreet brain areas, circuits, and cellular structuresthat could plausibly underlie disease symptoms. For example, individuals with schizophrenia display impairments in working memory tasks, which are associated with altered activation of the dorsolateral prefrontal cortexas well as impairments in the processing of auditory sensory informationwhich are associated with altered event-related potentials localized to the primary auditory cortex. Alterations in dendritic spine density have been reproducibly observed in layer 3 of both the dorsolateral prefrontal cortexand primary auditory cortexand are believed to contribute to the observed impairments in working memory and auditory sensory processing. It is important to note that the limited studies that have investigated layer 5 in a cortical region did not observe decreased spine density. Additionally, studies a have also not found a concurrent decrease in presynaptic boutons in layer 3 of cortical regions. Thus, synaptic alterations in cortical regions in schizophrenia are limited to specific layers and structures. Interesting, subcortical regions appear to have distinct pathologies. For example, while the hippocampus displays similar decreases in dendritic spine density, it displays different activity alterations and GABA cell pathology. Thus, it is essential to investigate the molecular pathology of individual brain areas, layers, cell types, and cellular structures. Alterations in dendritic spines have also been observed in bipolar disorderand autism spectrum disorder. While many areas of research benefit greatly from the use of animal models, polygenetic psychiatric disorders are difficult, if not impossible to model in animals, complicating the investigation of disease etiology. In an effort to elucidate the molecular mechanisms driving these structural and functional impairments the field has turned to transcriptomic and proteomic analyses of human post-mortem brain tissue to quantify disease associated differences in transcriptsand proteins, providing many valuable insights into disease pathology. More recently, multi-omics analyses, grounded in GWAS, have identified quantitative trait loci (QTLs) for common risk variants associated with gene expression (eQTLs)and protein levels (pQTLs). eQTL studies have started to provide insight into the biological effects of common risk loci. To date at least one human brain pQTL study has been published, finding that only a subset of pQTLs were also eQTLs, highlighting the disconnect between the transcriptome and the proteome. Several groups are currently pursuing well powered proteomic investigations of the human brain, suggesting that additional proteogenomic studies will further map associations between common variants and protein expression in psychiatric disease. While these studies are powerful, they fail to capture the more dynamic aspects of the proteome, such as post-translational modifications (PTMs), that are among the final mediators of cell and circuit activity, and are difficult, if not impossible, to infer from the transcript or protein levels. Post-translational modifications have been studied in neurological disorders, most notably Tau hyperphosphorylation in Tauopathies such as Alzheimer's disease, but they have received less attention in psychiatric disease and their study in human post-mortem brain tissue is often viewed with skepticism. This skepticism is not entirely unwarranted, as many PTMs are highly dynamic. However, many are stable post-mortem and, as PTMs regulate protein activity, this information is likely more valuable than protein levels alone. Here we review the rationale for studying PTMs in psychiatric disease, recent findings in human post-mortem brain tissue, the common pitfalls and required controls for these types of studies, and highlight the emerging mass spectrometry approaches transforming this research direction. Investigations of transcript levels in human post-mortem brain tissue from autism spectrum disorder and schizophrenia subjects further implicate PTMs. The most recent meta-analysis of RNAseq studies from PsychENCODEfound that 52 protein kinases, 14 protein phosphatases, 9 proteasome subunits, and 22 ubiquitin ligases were differentially expressed in autism spectrum disorder; while 123 protein kinases, 41 protein phosphatases, 7 proteasome subunits, and 62 ubiquitin ligases were differently expressed in schizophrenia. ## Ptms are implicated in psychiatric disease These findings are not surprising as schizophrenia and autism spectrum disorder are both widely viewed as developmental synaptopathies and a multitude of studies have demonstrated the essential roles of protein phosphorylation and the ubiquitin-proteasome system in synaptic plasticity, long term potentiation, and learning. As stated above, while these genetic and transcriptomic investigations can, and have, implicated specific classes of PTMs and enzymes, they cannot capture their effects on the broader synaptic proteome. Furthermore, while altered levels of a given kinase or ubiquitin ligase can be modeled in cell culture or animal models, the complex genetic risk factors and environmental circumstances that give rise to psychiatric disease, as well as the unique circuitry and neuronal populations of the human brain, cannot. It is important to note here that neurons and organoids derived from patient pluripotent stem cells can mimic the genetic risk profiles of psychiatric disease and provide a powerful window into pathological neurodevelopmental processes. However, these models still lack the longevity of human adolescence and adulthood as well as interactions with systemic features (e.g., circulating hormones or the microbiome) and environmental risk factors. Thus, post-mortem brain studies are essential to investigating the molecular changes associated with psychiatric disease. In next section we will seek to answer the following questions: Can disease associated PTM differences be observed in human post-mortem brain tissue and do these PTMs have biological validity. The history of Tau gives us some hope that PTMs observed in human post-mortem brain tissue can yield insights into disease etiology. ## A brief history of modern ptm studies in schizophrenia and other psychosis related disorders By the early 2000s dendritic spinesand NMDA receptorshad been implicated in schizophrenia pathology. As the decade progressed, genetic, transcriptomic, and early mass spectrometry studies continued to implicate postsynaptic ligands, receptors, and scaffolding proteins, such as ERBB4, AKT, NRG1, and PSD95in schizophrenia. Several groups then began utilizing traditional antibody-based approaches and eventually mass spectrometry for targeted phospho-analyses in patient tissue.found that phosphorylation of NR1 S897 was decreased (while total NR1 levels were unaltered) in frontal cortex tissue from individuals with schizophrenia. This site was of particular interest as antipsychotic drugs were known to increase NR1 S897 phosphorylation in primary neuronal culturesand S897 is essential for antipsychotic drug-mediated gene expression. A later study further demonstrated S897's importance in NMDAR synaptic incorporation, NMDAR-mediated synaptic transmission, AMPA receptor mediated synaptic transmission, and long-term potentiation.found that S770 phosphorylation on the transcription factor SP4 was positively correlated with negative symptoms in schizophrenia subjects. Importantly, they also found that SP4 phosphorylation was inversely correlated with SP4 levels. SP4 regulates dendritic arborizationand phosphorylation at SP4 S770 regulates stability of the protein. More recently,found that phosphorylation of PI3K, AKT, and MTOR was decreased in cortical tissue from a subset of subjects with bipolar disorder. Subsequent studies in mice found that overexpression of dominant negative AKT impaired dendritic spine maintenance and performance in cognitive tasks. Finally,used mass spectrometry-based proteomics to investigate 18 phosphorylation sites on MAP2 in cortical tissue from schizophrenia subjects, finding differential phosphorylation at 9 while total levels of MAP2 were unaltered. Generating a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) they found reductions in basilar dendritic length and complexity along with reduced spine density. The studies above measured static phosphorylations, presumably preserved at death. But a few adventurous groups have pushed these studies further, attempting to capture dynamic phosphorylation activities in human post-mortem brain tissue. In two publications, Hahn and Wang combined targeted phosphorylation studies with a post-mortem tissuestimulation paradigm to identify phosphorylation differences in schizophrenia after receptor stimulation. In the first study they found that NRG1 stimulation of ERBB4 decreased glutamate/glycine induced phosphorylation of NMDAR2A and PLCγ, likely driven by increased association between ERBB4 and PSD95, but independent of the levels of any of the assayed proteins. In the second study, they observed increased phosphorylation of mGluR5 after stimulation, which was accompanied by decreased coupling with Gq/11, indicating decreased mGluR5 activity, again independent of the levels of any of the assayed proteins. Taking a different approach,utilized kinase arrays to interrogate signaling cascades in cortical tissue from schizophrenia subjects, finding significant alterations in kinome activity that further implicate cellular and ion homeostasis as well as cytoskeletal organization in schizophrenia. A key driver underlying the incomplete correlation between mRNA and protein abundance is the fact that protein turnover is dynamic. The principal mechanism of turnover is the ubiquitinproteasome system in which polyubiquitinated proteins are targeted to the proteasome for degradation. Additionally, the ubiquitin-proteasome system regulates synaptic protein stability and is essential for LTP and learning. Thus, while it has received less attention than phosphorylation, the ubiquitin-proteasome system is beginning to be investigated in schizophrenia.first observed differences in both free and protein ubiquitination. More recently,found that increased protein ubiquitination was correlated with increased protein insolubility in cortical tissue from schizophrenia subjects. Finally, paralleling the kinome arrays used to assess kinase activity in schizophrenia, Scott and Meador-Woodruff (2020) utilized proteasome activity assays, finding altered trypsin and chymotrypsin like activity in schizophrenia tissue. While this review has focused on ubiquitination and phosphorylation a growing body of work implicates additional PTMs such as glycosylation and myristylation in schizophrenia, reviewed in detail in. Briefly, alterations in N-Glycosylation on GABA, NMDA, and AMPAreceptors have been observed in schizophrenia. The studies reviewed above suggest several points: (1) That differences in PTMs can be observed in brain tissue from subjects with psychiatric disease; (2) that levels of multiple classes of PTMs are altered across multiple protein families; and (3) when tested in forward genetic models, individual PTMs can significantly impact disease relevant biology such as glutamatergic signaling and dendritic spine plasticity, that could not be predicted by genetics, transcriptomics, or even protein quantification. For example, MAP2 is not found at any schizophrenia risk loci and its protein levels are unaltered in schizophrenia tissue, yet it is hyperphosphorylated at multiple sites in schizophrenia and modeling just one of these sites induces a loss of dendritic spines. The breadth of the PTM alterations observed in schizophrenia via mostly targeted approaches highlights the need for broad and systematic investigations of PTMs in psychiatric disease. Specifically, next generation studies should be rigorously designed to catalog post-mortem effects on individual PTM sites, be performed in well powered and well-balanced cohorts, utilize state-of-the-art mass spectrometry approaches, target selected brain areas, cortical layers, cell types, and microdomains, and take advantage of new informatic and statistical approaches to multiomic integration to map associations between genes, multiple PTMs, and phenotypes. ## Experimental considerations for investigating ptms in post-mortem brain tissue The impact of post-mortem interval (PMI; the time between when a subject becomes deceased and the brain tissue is fixed and/or frozen) on molecular integrity has long been appreciated and three main strategies have been employed to account and control for this confound. (1) The effect of PMI on individual PTM sites can be modeled, using either mouseor human (Gallegotissue. Several groups have used this approach to either correct for mRNA degradationin human studies or to identify proteins that degrade nonlinearly (MacDonald et al., 2019b) across PMI for removal from case-control statistical comparisons. The same approach should be employed in PTM studies in human post-mortem brain tissue, identifying which specific modifications at which sites degrade non-linearly over time. (2) PMI is often included as a co-factor in statistical analysis. (3) When possible, diagnostic groups or subject pairs should be matched as closely as possible for PMI [as well as other factors that are known to impact proteins and PTMs such as sexand age]. Given the dynamic nature of many PTMs, all of these approaches should be utilized, and it is especially important to identify PTMs that are rapidly degraded early in PMI and to remove them from downstream statistical analyses. In the past, generating a well-balanced and powered cohort was a significant challenge, but with the recent unification of multiple brain tissue repositories under the aegis of the NIH NeuroBioBank, researchers now have access to quality tissue from thousands of well cataloged cases and appropriate controls. ## Emerging mass spectrometry approaches for ptm quantification in post-mortem brain tissue Advances in mass spectrometry instrumentation and sample preparation techniques continue to increase the throughput, breadth and depth of PTM coverage, and some of these approaches have begun to see use in human post-mortem brain tissue. Next generation proteomics instruments such as the timsTOF (Bruker) and Orbitrap instruments (ThermoFisher) with increased scan speeds facilitate deep coverage of modified peptides. For example,utilized a tandem mass tag (TMT) based approach to quantify over 48,000 phosphopeptides (representing over 33,000 unique phosphorylation sites) in human post-mortem brain tissue. In addition to increased instrument speed and sensitivity, modern mass spectrometers now offer an array of dissociation methods (e.g., CID, HCD, and ETD) enhancing the identification, and subsequent quantification, of high energy and complex PTMs, such as phosphorylationand glycosylation. Protocols to enrich phosphopeptides and glycopeptides from tryptic digests are now well developed and can be accomplished with high efficiency using commercially available kits and specialized liquid handling robots with pre-programmed proteomic applications, such as the AssayMAP BRAVO (Agilent), enabling both throughput and quantitative depth. Quantification of protein ubiquitination initially proved more challenging in standard proteomic work flows as trypsin digests off the larger ubiquitin side chain leaving only a lysine bound gly-gly which was difficult to capture. However, the recent availability of commercial antibodies and refined sample preparation now allow for deep profiling of protein ubiquitination in human samples as well; for example, over 14,000 ubiquitination sites were recently quantified in human tumor cells. ## The spatial resolution limits of ptm quantification in the human brain As reviewed above, the activity, structure, and molecular pathologies associated with psychiatric illness are highly spatially localized, displaying brain area, layer, cellular, and microdomain specificity. Multiple groups have used mass spectrometry to quantify protein levels in cortical layer capturesand synaptic microdomains obtained by either biochemical fractionationor Fluorescence-activated Cell Sortingfrom human post-mortem brain tissue. Synaptic microdomain enrichments generated by biochemical fractionation can provide sufficient material for phosphopeptide enrichment and quantificationwhich has been accomplished in fresh human brain tissue. While it has not yet been demonstrated in human brain tissue, laser capture microdissection and Fluorescenceactivated Cell Sorting both likely generate sufficient material for investigation of the phosphoproteome with the aid of isobaric labeling such as TMT and iTRAQ. Glycopeptide enrichment is sufficiently robust that it could likely be applied in the same scenarios as phosphopeptides. Conversely, ubiquitin-motif peptide enrichment still requires significant amounts of starting material, likely limiting its application to brain areas for the present. Finally, while recent advances in single cell proteomics such as SCoPE-MSnow allow for the quantification of hundreds of proteins in single cells, deep mass spectrometry based quantification of PTMs is still in the future. Given the pace of instrument development, single cell PTM quantification is likely not too far off. ## Informatics Keeping a pace with the advancements in instrumentation, the last few years has seen the release of multiple software packages for exploring PTMs in the context of known kinase-substrate relationships, kinase motifs, protein-protein interactions, and protein networks. Packages such as iGPSand KEA2identify known or presumptive kinase-substrate interaction from phosphoproteomics data, potentially identifying upstream kinases driving observed changes in protein phosphorylation. Other tools such as ProteoVizintegrate the identification of sequence motifs and kinases with gene set enrichment pathway analysis while CausalPath "identifies potentially causal dependencies between measured protein features (such as phosphorylations or global protein levels) using literature-curated biological pathways". Thus, researchers in the PTM space have a rapidly expanding number of informatics resources to draw upon in exploring their datasets. ## Closing thoughts In closing, PTMs are implicated in the etiology of neuropsychiatric diseases by unbiased genetic and transcriptomic studies, most notably autism spectrum disorder and schizophrenia. When investigated in human post-mortem brain tissue using targeted approaches, robust alterations in phosphorylation, glycosylation, and ubiquitination are observed in psychiatric disease, suggesting a much broader set of changes, with likely associations between different PTMs as well as the genome, transcriptome, and proteome. Advances in mass spectrometry instrumentation and proteomic sample preparation methods now allow for sufficiently powered studies to map these interactions, which, when combined with emerging informatics tools, will surely provide insight into the etiology of many psychiatric diseases as PTMs are the ultimate mediators of so many neuronal and circuit activities. # Author contributions MM wrote the first draft of the mini review. MG, RS, and MM revised and reviewed the second draft of the mini review. All authors contributed to the article and approved the submitted version. # Funding This work was funded by NIH grants R01MH118497, R01MH125235, and K08 MH118513.

Don’t let up: implementing and sustaining change in a new post-licensure education model for developing extended role practitioners involved in arthritis care Journal of MultidisciplinaryHealthcare Dovepress submit your manuscript | www.dovepress.com Dovepress 389 P e r s P e c t i v e s open access to scientific and medical research Open Access Full text Article # Introduction After the first set of short-term wins, a change effort will have direction and momentum. In successful situations, people build on this momentum to make a vision a reality by keeping urgency up and a feeling of false pride down; by eliminating unnecessary, exhausting, and demoralizing work; and by not declaring victory prematurely. -Cohen DS. # Background The Advanced Clinician Practitioner in Arthritis Care (ACPAC) program is a unique, interprofessional, clinical, and academic training program currently offered for physical therapists, occupational therapists, and nurses experienced in the musculoskeletal (MSK) field. It is a post-licensure program offered through the Department of Continuing Professional Development, Faculty of Medicine, at The University of Toronto, Canada.The ACPAC program is offered principally at two main academic health care centers -St Michael's Hospital (adult) and the Hospital for Sick Children (pediatric), but additionally relies upon a broad network of health care (community and academic) institutions and involves over 90 faculty. The vision for the ACPAC program, [fig_ref] 1: The efforts of the Arthritis Alliance of Canada and the Ontario Rheumatology... [/fig_ref] AcPAc program-trained extended role Practitioner (ERP) profile. AcPAc program-trained erPs have been prepared to work in settings for ongoing management and triage, two identified requirements to establish new interprofessional models of care. 2. these practitioners, who are physical therapists, occupational therapists, or nurses with expertise in the musculoskeletal field, have expanded and advanced knowledge, clinical skills, and attitudes related to arthritis care obtained through additional training and formal evaluation with measured competencies. 3. erP roles can be autonomous or require additional performance expectations of authorized activities which are currently achieved through delegation or medical directives * Notes: *Medical directives are defined as "indirect physician orders, used to expedite patient care by competent health professionals". 23 Abbreviation: AcPAc, Advanced clinician Practitioner in Arthritis care. ## 390 Lundon et al including competency development, was formulated under the leadership of two academic rheumatologists (the adult and pediatric medical directors) and a physical therapist with a PhD in bone pathophysiology (program director) all of whom have worked collaboratively to direct and coordinate the program since its inception. [bib_ref] Development and early evaluation of an inter-professional post-licensure education programme for extended..., Lundon [/bib_ref] The focus of the ACPAC program is on the assessment, diagnosis, triage, and independent but collaborative management of select MSK and arthritis-related disorders with the rationale for and processes involved in the inception, development, implementation, [bib_ref] Development and early evaluation of an inter-professional post-licensure education programme for extended..., Lundon [/bib_ref] and leadership 4 of the ACPAC program described fully elsewhere. In a brief review, experienced physical therapists, occupational therapists, and nurses are prepared, through advanced academic and clinical training, for extended practice roles [fig_ref] 1: The efforts of the Arthritis Alliance of Canada and the Ontario Rheumatology... [/fig_ref] within the context of a variety of arthritis care settings. The ACPAC program consists of five intensive on-site weeks interspersed with advanced clinical training and self-directed learning opportunities offered between September and June of each academic year. The foundation of the curriculum is based on the integration of material from the rheumatology and orthopedic fields and consists of four core components: Component I -Advanced Rheumatology Theory (online); Component II -Advanced Assessment and Integrative Sessions; Component III -Advanced Clinical Training in Rheumatology (general adult and pediatric rheumatology, as well as surgical/specialty clinics); and Component IV -Health Promotion, Advocacy, and Leadership in Arthritis Care. Both program and candidate evaluation involve extensive theoretic and practical skill examinations, clinical competency appraisals based on CanMEDS, clinical case reports, portfolios, and structured learning projects as well as online advanced rheumatology learning assessments. Each ACPAC trainee must achieve at least 70% and/or "meets expectations" for each evaluation component (http://www.acpacprogram.ca). The expectations for increased clinical responsibilities in the extended practice role are fully described by Lundon et al. [bib_ref] Development and early evaluation of an inter-professional post-licensure education programme for extended..., Lundon [/bib_ref] The goal is to engage this new human health care resource as integral participants in the design and delivery of innovative models of arthritis care. There are currently 47 graduates working across a spectrum of urban-academic (n=30), community (n=8), and remote (n=9) clinical settings. These graduates are filling the gap in arthritis care to contend with 1) the need to improve an interprofessional approach to care that is regarded to optimize care of a growing population of patients living with arthritis 5,6 and 2) a relatively insufficient number of traditional arthritis care specialists resulting in increasingly long wait times and poor access to care. 7,8 Finally, and not insignificantly, the maximizing of scopes of practice of existing and experienced human health resource (ie, OT, PT, RN), which entails formal training processes with measured competencies to support extended practice roles, has the intuitive benefit of improving access to and efficiency of arthritis care in supportive collaborative care environments. ## The challenge Despite the recent changes in health care service provisions for numerous health professionals to broaden scope of practice in Ontario (MOHLTC 2012), 9 related academic, regulatory, and professional bodies have been slow to accommodate for the training logistics required to support such change. To date, it has been left up to the individual and/or institution to drive new initiatives. Introduction of regulatory structure to broaden diversity of workforce roles in health care has lagged significantly behind the realities of practice in Ontario, a phenomenon also observed in other health care systems. [bib_ref] Policy on new workforce roles: a discussion paper, Bridges [/bib_ref] It is under these conditions that the ACPAC program leadership has substantially increased their effort across the past few years to sustain this educational initiative. In anticipation of rapid changes in health care policy, these ## 391 implementing and sustaining change in education model efforts include overseeing both continued generation of competent Extended Role Practitioners (ERPs) and the evaluation of these roles at the health services level. We feel strongly that this is not a time to let up or let go. ## Early wins Across a 9-year period, the ACPAC program has achieved a set of short-term "wins" giving direction and momentum to the development of new models of arthritis care in Ontario. ## Community reach The longer term viability of the ACPAC program required an early and sustained effort to develop strong financial and in-kind linkages with key stakeholders including: the government (The Ministry of Health and Long-Term Care -MOHLTC); industry; academia (Faculty of Medicine, University of Toronto) and related academic health care institutions (in particular, the St Michael's Hospital and The Hospital for Sick Children); and The Arthritis Society (advocacy group). ## The ministry of health and long-term care In terms of being recognized by and relevant to the Ministry of Health in Ontario, in 2009 the ACPAC program was selected as one of three top innovations in human health resource initiatives in Ontario. ## Academic health care institutions The success of the ACPAC program has relied upon the firm commitment of St Michael's Hospital and The Hospital for Sick Children which as large academic health care institutions have provided the necessary infrastructure including physical environment, administration support, and expert faculty resources. Administrators within these institutions recognized the urgent nature of impending demographic changes and simultaneous human health resource shortages in the area of arthritis care and as such were receptive and willing to espouse this novel program. The supportive role of core academic teaching hospitals has been critical to lead and foster the development of new human health resource which is an integral part of the design of a larger chronic disease (arthritis) management strategy. The synergistic relationship between commitment as host site and the training program itself is evident as the ACPAC program has in turn drawn local, national, and international attention to these institutions for their role in supporting innovation in post-licensure, interprofessional training opportunities. ## The arthritis society Graduates of the ACPAC program employed at The Arthritis Society have become integral to the advanced clinical delivery of primary health care for individuals with arthritis in diverse clinical settings throughout the province. The specialized skills acquired by these extended role practitioners have been instrumental to The Arthritis Society's increased capacity to provide advanced therapy and consultation services to Ontarians, particularly those in rural, remote, and underserviced communities. The proven effectiveness of the training program has served as an important foundation upon which The Arthritis Society can build its efforts to identify and deliver new innovative models of care that respect current health care priorities, build on existing community resources, and supports improved health care capacity, patient outcomes, and access to coordinated, interprofessional care. # Strengthened interprofessional relationships The Canadian Rheumatology Association, a physician-based group, and the Arthritis Health Professions Association have endorsed the ACPAC program. research activity evaluation of program outcome 1. Competency: pre-post theory and practical skills evaluation An extensive pre-post data set -generated from results of theoretical and practical (OSCE) examinations -measured significant change (growth) in advanced skills and knowledge in arthritis and supports competency in these new practitioners. [bib_ref] Development and early evaluation of an inter-professional post-licensure education programme for extended..., Lundon [/bib_ref] These changes in knowledge and skills are considerable as they were demonstrated in health care professionals already highly trained in arthritis care, underscoring the importance of this educational initiative. ## Health services evaluation (2009-2011) The early impact of ACPAC program-trained ERPs on the Ontario health care system was evaluated using the Balanced Scorecard approach of Kaplan and Norton.The evaluation framework was a further modified version of the Province of Ontario Hospital Report: Rehabilitation series,with a four quadrant approach representing four perspectives supporting translation of strategic objectives into measurable performance indicators. The four perspectives included that of patients and stakeholders, clinical utilization and outcomes, system integration and change, and financial performance and condition. 14 There is evidence that, in general, ACPAC program-trained ERPs have roles which are responsive to a dynamically evolving health care environment. [bib_ref] Arthritis extended-role practitioners: impact on community practice (An exploratory study), Lineker [/bib_ref] [bib_ref] Evaluation of perceived interprofessional care behaviour amongst stakeholders and emergent advanced clinician..., Lundon [/bib_ref] [bib_ref] System integration and clinical utilization of the Advanced Clinician Practitioner in Arthritis..., Passalent [/bib_ref] ## Key implications of system-level evaluation findings for health care provision in ontario Detailed results and implications based on the extensive health services evaluationwere presented at the government-level to the Ministry of Health (Human Resources Division). Key implications of health care provision by ACPAC programtrained ERPs supported positive changes identified by improved access (especially in rural and remote regions), strengthened human health resource capacity, perceived impact on patient outcomes, and an abundance of potential opportunities for broadened role promotion and utilization. ## Relevance to key stakeholders: the ministry of health and long-term care The optimizing of human health resource utilization and maximizing scopes of practice through development of extended role, scope, or advanced practice practitioners, particularly in the MSK field, [bib_ref] Advanced practice physiotherapy in patients with musculoskeletal disorders: a systematic review, Desmeules [/bib_ref] is of global interest. The ACPAC program initiative is also aligned with a current mandate in Ontario to expand roles of existing human health care providers to meet health care service needs.Furthermore, it represents a robust approach to collectively train and generate competent practitioners from multiple professions with expertise in arthritis care. While changes in regulation are broadly welcomed to accommodate and support new and expanded roles, the professionals involved in extended role practice must both assure and be assured that new human health resource positions are competent, transportable, secure, and not vulnerable to politically driven changes in priority. The ACPAC program has been a leading interprofessional educational initiative offered at the post-licensure level that has effectively generated a new type of health care worker. At this juncture, a critical opportunity exists for our academic institutions to embrace post-licensure, clinical, and academic education training programs (such as ACPAC). This will ensure standardized processes are in place to train competent health care workers essential to develop new models of care, as well as optimize the use of existing human health resources. It is clear that an opportunity exists for relevant health professionals willing to undergo additional formal training to achieve the necessary competencies to fill a system-level gap in arthritis/MSK care at a broader (national and international) level. Recent effort to broaden our reach at the national level has been made to secure the best candidates and ensure a standardized training process is accessible across the country so that care is distributed in regions of most need. In Canada, as we contend with the impending demographic statistics outlined in [fig_ref] Table 2: Arthritis burden million canadians live with OA [/fig_ref] , 20 the ongoing generation of this new human health resource will be critical to the success of new models of care. There is a renewed sense of urgency as we plan the next steps for the ACPAC program as it transitions from a human health resource initiative into a sustainable academic program. This will entail new thinking around credentialing and academic recognition of training programs which lead to specialized practice in non-physician health care providers which is receiving considerable attention internationally. [bib_ref] Extended roles for allied health professionals: an updated systematic review of the..., Saxon [/bib_ref] [bib_ref] The role of extended scope physiotherapists in managing patients with inflammatory arthropathies:..., Stanhope [/bib_ref] Challenges at this critical juncture include: retaining focus, drive and commitment; developing academic partnerships where the short-term success is transferred to long-term sustainability and where there is high regard for standardized, post-licensure education programming that is competency based and evaluated on an ongoing basis at the system level. Our challenge to "don't let up" requires continued: 1. Conveyance of the drive and commitment required in developing and training competent and adequately trained ERPs prepared to fill the gap in arthritis care within a sustainable, relevant academic home. 2. Monitoring and measuring progress on a consistent basis (in the form of ongoing, health services evaluation of ERPs in relevant clinical settings), as well as measuring fiscal impact of this new human health care resource. It is critical that the leaders of the ACPAC program stay the course, remain focused, and demonstrate that we are not easing the pressure to achieve the vision in which so much energy has been invested. This will require sustained effort to attract new partners (academia, industry) who will strengthen the opportunities for the ACPAC program and its graduates. # Conclusion The product of the ACPAC program (the ACPAC programtrained ERP) is aligned with the mandate of, and desired direction professed by, the Ministry of Health in Ontario as well as a key advocacy group (The Arthritis Society). The ACPAC program is a rigorous training model which aims to develop and further optimize relevant human health resources to improve arthritis care. This type of initiative aims to fulfill the education requirements that are increasingly relevant to health care regulatory bodies as their membership is challenged with rapidly changing scopes of practice which also may involve skill mixing. It is essential that partnerships are broadened, particularly in academia (credibility) and industry (fiscal), in order to achieve sustainability and maintain relevance to health care delivery. We believe that the position we are in and the processes that confront us at this time are relevant to administrators contemplating introducing new human health resource -or considering new ways of delivering care using traditional human health resources -and for whom a concern is the underling academic/clinical training necessary to fulfill these positions. The ACPAC program has realized significant shortterm wins which have propelled the change effort forward. This has enabled stakeholders to tackle some of the bigger and deeper changes that will deliver substantial benefits, including improved patient care and access, particularly in remote areas of Ontario. At this point, it is critical that change processes continue to take root and are supported, recognizing that the ACPAC Program-trained ERPS are integral to the success and design of new models of arthritis care. We need to retain a sense of urgency -don't let up! and persist in fostering collaboration between professional bodies, health care institutions, universities, and the Ministry of Health to assure continued advancement of this leading-edge, innovative, academic/clinical-based educational program. ## Key points - This is a viable model for interprofessional, post-licensure training that would markedly benefit management of other chronic disease streams (ie mental health, cardiovascular, neurology). - Graduates of the ACPAC program have demonstrated competency in advanced knowledge and skills for practising as ERPs in the arthritis/musculoskeletal field. Their presence enhances the available human health resource pool for patients with arthritis. - The ACPAC program represents a significant innovation in academic and clinical post-licensure education for allied health professionals. - In order to achieve sustainable change in healthcare system renewal, stakeholders need to be open to integrating interprofessional care, including new role development, into existing strategies. - Interprofessional healthcare models which incorporate health professionals with broadened scopes of practice will need to evolve given demographic and chronic disease trends. - A shift in traditional roles and professional boundaries by well-trained and competent new healthcare providers (ie ERPs) can narrow the gaps in healthcare to allow patients to receive the right care, at the right time, by the right person making continued development of these roles well worth the effort to overcome the challenges. ## Lessons learned ## 394 Lundon et al arthritis care to further develop as extended role (scope) practitioners within this field. 2. The development of new human health resource which is multidisciplinary in nature requires steadfast interprofessional leadership and buy-in from multiple stakeholders. 3. Changes at the system level to better accommodate interprofessional collaboration begin with interprofessional education opportunities such as the ACPAC Program initiative. [fig] 1: Formal interprofessional training opportunities and rigorous evaluation measures established at the post-licensure level supports healthcare professionals from multiple professions and disciplines who are experienced in Journal of Multidisciplinary Healthcare 2015:8 submit your manuscript | www.dovepress.com Dovepress Dovepress [/fig] [table] Table 2: Arthritis burden million canadians live with OA. this will double to 9 million in the next 20 years. 3. More than 272,000 canadians live with rA (0.9% of canadian adult population) → in the next 30 years this will increase to 1.3%. 4. the number of canadians with untreated rA is expected to increase from 117,000 to 230,000 in the next 30 years. [/table]

Digital health interventions for non-communicable disease management in primary health care in low-and middle-income countries 11,13,14,15✉ Current evidence on digital health interventions is disproportionately concerned with high-income countries and hospital settings. This scoping review evaluates the extent of use and effectiveness of digital health interventions for non-communicable disease (NCD) management in primary healthcare settings of low-and middle-income countries (LMICs) and identifies factors influencing digital health interventions' uptake. We use PubMed, Embase, and Web of Science search results from January 2010 to 2021. Of 8866 results, 52 met eligibility criteria (31 reviews, 21 trials). Benchmarked against World Health Organization's digital health classifications, only 14 out of 28 digital health intervention categories are found, suggesting critical under-use and lagging innovation. Digital health interventions' effectiveness vary across outcomes: clinical (mixed), behavioral (positively inclined), and service implementation outcomes (clear effectiveness). We further identify multiple factors influencing digital health intervention uptake, including political commitment, interactivity, user-centered design, and integration with existing systems, which points to future research and practices to invigorate digital health interventions for NCD management in primary health care of LMICs.npj Digital Medicine (2023) 6:12 ; https://doi. # Introduction Digital health interventions-known as "a discrete functionality of digital technology that is applied to achieve health objectives"have exceptional potential to promote universal health coverage and enhance health service delivery [bib_ref] Impacts of e-health on the outcomes of care in low-and middleincome countries:..., Piette [/bib_ref]. In May 2018, the World Health Assembly passed the Digital Health Resolution, recognizing the potential of digital technologies to support health systems by improving the accountability, availability, accessibility, continuity, utilization, and effectiveness of health care. The World Health Organization (WHO) further classified digital health interventions according to four types of users, including 28 categories and 87 sub-categories. These users, categories, and sub-categories of digital health interventions cover various areas of health systems with a particular focus on health service delivery. One area that has great potential for improvements through digital health interventions is the management of non-communicable disease (NCD) in primary health care. Distinct from hospital-level specialist care, primary health care emphasizes first-contact, accessible, continued, comprehensive, and coordinated patient-focused care, and is often the closest to where people live. Primary health care has been recognized as the cornerstone of combating NCDs worldwide. This is because NCDs-such as hypertension, diabetes, and cardiovascular diseases-are characterized by long disease durations and a continuous need to anticipate and mitigate risk factors through lifestyle modifications [bib_ref] A critical review of addressing cardiovascular and other non-communicable diseases through a..., Mahipala [/bib_ref] [bib_ref] Primary health care: a strategic framework for the prevention and control of..., Demaio [/bib_ref] [bib_ref] Better health and wellbeing for billion more people: integrating non-communicable diseases in..., Varghese [/bib_ref] [bib_ref] Noncommunicable diseases prevention in low-and middleincome countries: an overview of health in..., Ndubuisi [/bib_ref] , which is better addressed by primary health care than higher-level health facilities. The literature, however, has shown that substantial gaps exist in most primary healthcare systems, particularly in low-and middle-income countries (LMICs), including limited human resources and capacity, shortages in medicines and equipment, and suboptimal quality of care [bib_ref] Noncommunicable diseases prevention in low-and middleincome countries: an overview of health in..., Ndubuisi [/bib_ref] [bib_ref] Primary healthcare system performance in low-income and middle-income countries: a scoping review..., Bitton [/bib_ref]. These constraints prevent primary healthcare facilities from achieving optimal NCD management. In the past decade, many studies have explored whether and how digital health interventions can contribute to bridging such gaps. The World Heart Federation recently released a roadmap for digital cardiology, where it was acknowledged that digital health interventions had potential to help address health system challenges and achieve optimal and universal health coverage by promoting health service coverage, empowering patients and providers, and improving long-term outcomes [bib_ref] World heart federation roadmap for digital health in cardiology, Tromp [/bib_ref]. The digital cardiology roadmap considered a diversity of digital health interventions, spanning text messaging, telehealth, and electronic decision support tools [bib_ref] World heart federation roadmap for digital health in cardiology, Tromp [/bib_ref]. The CONNECT trial conducted in Australia, for example, used an interactive web-based app linked to the electronic health records (EHRs) in primary care, and found borderline improvements in blood pressure and lipids control, and significant effectiveness in increasing physical activity [bib_ref] A digital health intervention for cardiovascular disease management in primary care (CONNECT)..., Redfern [/bib_ref]. The TEXTME trial found sending text messages to people with heart disease was associated with improvements in blood pressure control, diet, physical activity, and smoking reduction [bib_ref] Effect of lifestyle-focused text messaging on risk factor modification in patients with..., Chow [/bib_ref]. Other existing reviews found text messages to be of low costs and 1 effective in addressing modifiable NCD risk factors such as medication compliance [bib_ref] Mobile phone text messaging to improve medication adherence in secondary prevention of..., Adler [/bib_ref] , and weight management [bib_ref] A systematic review with meta-analyses of text message-delivered behaviour change interventions for..., Skinner [/bib_ref]. However, most of these original studies and reviews focused on highincome countries, hospital settings, or fields other than NCDs [bib_ref] A digital health intervention for cardiovascular disease management in primary care (CONNECT)..., Redfern [/bib_ref] [bib_ref] Effect of lifestyle-focused text messaging on risk factor modification in patients with..., Chow [/bib_ref] [bib_ref] Digital interventions to promote self-management in adults with hypertension systematic review and..., Mclean [/bib_ref] [bib_ref] Effectiveness of mHealth interventions in improving medication adherence among people with hypertension:..., Xiong [/bib_ref] [bib_ref] Accelerating digital mental health research from early design and creation to successful..., Mohr [/bib_ref]. The literature on digital health interventions for NCD management in primary healthcare settings and LMICs are fragmented and sparse. This scoping review aims to synthesize evidence on the current use of digital health interventions for NCD management in the primary health care of LMICs. Specific objectives include: (1) to identify gaps in the use of digital health interventions for NCD management in primary health care of LMICs by benchmarking existing studies with the WHO digital health classification; (2) to explore the effectiveness of existing digital health interventions by different outcomes; and (3) to identify factors influencing the uptake of these digital health interventions through narrative synthesis. # Results # Search results We identified a total of 8866 records in the search from the three English databases, and 3577 duplicates were removed across the databases [fig_ref] Figure 1: Flowchart for study search and screening [/fig_ref]. After screening by title and abstract, 347 items remained for further screening. In the third round of screening by full-text, 295 items were further excluded. The primary reasons for this round of exclusion included: "not conducted in LMICs" (n = 171), "no available full-texts" (e.g. conference abstracts, n = 49), and "not conducted at primary health care settings" (n = 29). A total of 52 papers were included for final analysis. ## Study characteristics There were 31 reviews and 21 trials among the final included studies (see for data extraction tables on basic characteristics of included reviews and trials, respectively). There were three major review types in the 31 review papers: 23-53 systematic reviews (n = 22), scoping reviews (n = 3), and umbrella reviews (n = 4), in addition to two nonspecified types of literature reviews, one of which reviewed mobile phone APPs rather than papers [bib_ref] The top Chinese mobile health apps: a systematic investigation, Hsu [/bib_ref]. Among the 22 systematic reviews, six conducted meta-analysis, and most of the other papers mainly adopted narrative synthesis. Various types of publication were covered in these review studies, including randomized controlled trials (RCTs), cluster RCTs, quasi-experiments, pre-post experiments, observational studies, and literature reviews. The 21 trials were conducted in various LMICs 54-74 , including eight from Brazil, three from Thailand, three from India, two from China, and one from Kenya, Chile, Malaysia, South Africa, Turkey, and Argentina, separately. Of note, one of the trials was conducted in both China and India 71 . Regarding trial designs, there were four RCTs, five cluster RCTs, seven quasi-experiment studies, and five feasibility/pilot studies. Most of these trials had either people with NCDs or primary healthcare providers as participants, and three studies had both. There was a wide range of sample sizes (from 10 to 6979) across these trials, reflecting high heterogeneity in study designs. The most common type of participants in the trials are those with hypertension or diabetes. For quality assessment, more than half of the included trials were of suboptimal quality (n = 12), with eight of them of good quality and one with fair quality. The most common factors that compromised the studies' quality were the lack of randomization (n = 11), lack of evidence on sample size sufficiency (n = 10) and absence of pre-specification for study outcomes (n = 7), which was mainly due to the high proportions of quasi-experiment and feasibility/pilot studies. ## Digital health interventions in selected studies We identified a total of 11 types of digital health interventions for NCD management in primary health care from the selected studies, which covered 14 out of 28 categories based on WHO digital health classifications [fig_ref] Table 1: Identified digital health interventions for non-communicable disease management in primary health care [/fig_ref]. Eight of the identified digital health interventions were used by primary healthcare providers, such as EHR, decisions support systems, and telemonitoring devices. Five interventions were used by healthcare clients (i.e. people with NCDs), including short messaging services (SMS), multimedia message services, and interactive voice responses or phone calls. Two were used by both healthcare providers and clients: webbased/online telecare platforms, and smartphone applications. Of note, EHR can also be classified as being used for "data services", the fourth type of user according to the WHO classification, given the nature of EHR being collecting routine health and medical information of people using health services. We did not find any digital health interventions that were used by health system managers from the selected studies. From the perspective of the WHO digital health intervention classifications, the majority of these interventions were used for communications [fig_ref] Table 1: Identified digital health interventions for non-communicable disease management in primary health care [/fig_ref] , including targeted client communication (n = 6), untargeted client communication (n = 3), healthcare provider communication (n = 1), and client-to-client communication (n = 1). Other major types of digital health interventions were about patient information, including client health records (n = 2), on-demand information services to clients (n = 2), and personal health tracking (n = 1). Other classified digital health interventions included telemedicine (n = 3), laboratory and diagnostics imaging management (n = 2), healthcare provider decision support (n = 2), Benchmarked against World Health Organization's digital health classifications, 14 out of 28 digital health intervention categories were found, and most of them focused on improving health service efficiency and accessibility. S. Xiong et al. healthcare provider training (n = 1), client identification and registration (n = 1), and prescription and medication management (n = 1). Compared with the WHO classification, there were major under-use of digital health interventions in these studies. For example, there was no intervention in "citizen-based reporting", "client financial transactions", "referral coordination", and "health worker activity planning and scheduling", and there were no digital health interventions for healthcare managers, such as "human resource management" and "supply chain management". Regarding data services, the current intervention (i.e., EHR) was only limited to basic "data collection, management, and use", and there were no reported interventions about "data coding", "location mapping", or "data exchange and interoperability" in the selected studies. From the perspective of WHO quality of care dimensions, most of these digital health interventions focused on improving health service efficiency (n = 9) and accessibility (n = 9), followed by effectiveness (n = 9), while acceptability (n = 6), equity (n = 5), and safety (n = 4) issues were relatively less addressed in the selected studies [fig_ref] Table 1: Identified digital health interventions for non-communicable disease management in primary health care [/fig_ref]. ## Effectiveness of digital health interventions The included studies presented a variety of digital health interventions that intended to address a wide range of outcomes, making it unfeasible to conduct rigorous quantitative synthesis such as meta-analysis. Therefore, we explored the effectiveness of digital health interventions by documenting the positive and negative/ neutral findings from different of outcomes (Figs. 2 and 3). We found three major types of outcomes from all included studies: (1) eight types of clinical outcomes for individuals including control for blood pressure, blood glucose, hospitalization, and mortality; (2) six types of behavioral outcomes for individuals, including selfmanagement activities such as medication adherence, and health behaviors such as diet and physical activity; and (3) seven types of implementation outcomes for health services, which refers to factors associated with the process of health service provisions, such as service accessibility, user experience, and primary healthcare providers' capacities. In general, the effectiveness of digital health interventions differed across these three types of outcomes (Figs. 2 and 3). First, the findings for clinical outcomes, such as blood pressure and blood glucose control, were highly mixed, where both positive and negative/neutral results were common in both trials and reviews. Many of the included review papers found mixed findings within their own identified studies [bib_ref] Role of home blood pressure monitoring in overcoming therapeutic inertia and improving..., Agarwal [/bib_ref] [bib_ref] A systematic review of mHealth-based heart failure interventions, Cajita [/bib_ref] [bib_ref] Smart devices for older adults managing chronic disease: a scoping review, Kim [/bib_ref] [bib_ref] Addressing noncommunicable diseases among urban refugees in the Middle East and North..., Mcnatt [/bib_ref] [bib_ref] A systematic review of application and effectiveness of mHealth interventions for obesity..., Wang [/bib_ref] [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref]. Second, the effects of digital health interventions on individual behavioral outcomes were more positively inclined, where many studies showed a significantly positive impact on disease selfmanagement activities and healthy lifestyles, such as improved adherence to medicines, adherence to health services, and physical activity. Some of the studies, although less prevalent, also found non-significant results on behavioral outcomes. Third, for health service implementation outcomes, results were consistently positive among both trial reviews, where digital health interventions improved the accessibility and user experience in health service delivery, improved primary healthcare providers' capacities, and/or with better cost-effectiveness. Notably, several papers suggested the lack of rigorous costeffectiveness analysis in the current digital health studies [bib_ref] The impact of mobile monitoring technologies on glycosylated hemoglobin in diabetes: a..., Baron [/bib_ref] [bib_ref] Mobile phone-based behavioural interventions for health: a systematic review, Buhi [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref]. Several patterns were noticeable and informative regarding the effectiveness of different types of digital health interventions. First, the communication-related digital health interventions (i.e., SMS, multimedia message services, and online platforms, shown in different shades of blue) were the most widely explored in the included studies. Among the trials , several quasiexperimental studies documented the positive effects from these interventions in the control of blood pressure, glucose, lipids, and weight, and many RCTs also found their effectiveness in improving people's adherence to medication and health services. However, in the reviews , the effects of these interventions were highly mixed in all types of outcomes except for implementation outcomes. Second, both the trials and reviews suggested that digital health interventions that aimed to strengthen providers' The effectiveness of digital health for non-communicable disease management in primary health care in included trials. We found three major types of outcomes from all included trials, and digital health interventions' effectiveness varied among clinical (mixed), behavioral (positively inclined), and service implementation outcomes (clear effectiveness). capacities (i.e., decision support systems and online trainings, shown in different shades of purple) were substantially mixed in almost all the clinical and behavioral outcomes. Third, the digitalbased clinical practices (i.e., screening, diagnosis, prescribing, and monitoring, shown in different shades of orange) were found to be consistently effective in all types of outcomes in the trials, but in the reviews their effectiveness in clinical outcomes were mixed. Factors influencing digital health uptake Based on our inductive content analysis on the included studies, we identified multiple factors influencing the uptake of digital health interventions for NCD management in primary health care of LMICs . Based on the nature of different stakeholders, we further classified these factors into four groups: (1) factors regarding policymakers (n = 2), (2) factors regarding technological industry (n = 4), (3) factors regarding digital health designers (n = 7), and (4) factors regarding digital health users (n = 6). First, political commitment with regulations that encourage and standardize the use of digital health tools was reported as strong facilitators, while the absence of that in many settings could be barriers. Second, for the technology industry, a frequently mentioned factor that facilitated the uptake of digital health was the technological advancement and the prevalent use of information and communication technologies, such as the wide penetration of cell phones and the internet. Some less-developed places and populations such as in rural regions, however, were still faced with technical constraints and limitations. Moreover, lack of interoperability across different digital health platforms, unresolved data security, and ownership issues are barriers to further uptake of digital health interventions . The design of digital health interventions was also considered to influence their uptake . Major factors that contributed to optimal designs included the incorporation of existing behavioral science theories, tailored personalization as opposed to one-for-all contents, the emphasis on human interactivity, the consideration of users' feedback, and the involvement of digital health target users in the design phase (i.e., co-design). Finally, from the perspective of digital health users, adequate training for primary healthcare providers and guidance for people with NCDs on using digital health tools and customized incentives and motivations for sustainable use of digital health tools were reported as important factors that facilitated the uptake of digital health interventions. Lack of local capacity, on the other hand, such as technological illiteracy and suboptimal quality of data input and report, were reported as barriers to the uptake of digital health. # Discussion This study provides a holistic review on digital health interventions for NCD management in primary health care of LMICs for the past decade. We found 52 relevant studies and identified 11 digital health interventions mainly used by two types of users: primary healthcare providers and people with NCDs. This suggested the under-use of digital health interventions compared with WHO recommendations. We found the effectiveness of digital health interventions to be highly mixed for clinical outcomes, more positively inclined for behavioral outcomes, and consistently promising for service implementation outcomes. We also identified many factors that influenced the uptake of digital health interventions from policy maker, technical industry, designer, and user perspectives. Amidst the substantial and increasing global burden of NCDs 11 , our synthesis of evidence on digital health may shed light on further exploration of digital enhancements to health systems, particularly in primary health care of LMICs. The paucity of digital health interventions applied in NCD management in primary health care of LMICs is disproportionate to the high demand for health system strengthening. A recent rapid scoping review found that the vast majority of digital health interventions for NCD management during the COVID-19 pandemic were conducted in high-income countries and were mostly The effectiveness of digital health for non-communicable disease management in primary health care in included reviews. We found three major types of outcomes from all included reviews, and digital health interventions' effectiveness varied among clinical (mixed), behavioral (positively inclined), and service implementation outcomes (clear effectiveness). hospital-based [bib_ref] Digital health innovations for non-communicable disease management during the COVID-19 pandemic: a..., Murthy [/bib_ref]. An earlier study identified 12 common domains of mobile health interventions primarily used for maternal and child health, which covered almost all the digital health interventions identified in our study and more, such as human resource and supply chain management [bib_ref] mHealth innovations as health system strengthening tools: 12 common applications and a..., Labrique [/bib_ref]. The majority of the digital health interventions identified in our study, however, was focused on "communications", such as SMS and smartphone applications, aiming to improve health service efficiency and accessibility. Such digital health interventions do not require additional equipment or infrastructures except for cell phones and internet, which is already widely penetrated in the general populations and thus poses minimum additional costs. In contrast, we found very limited use for digital health interventions that entailed infrastructure updates and systemic enhancements, such as those for data collection, management, and analysis to support health administration. This is in line with findings from another review of 207 published studies, which identified major gaps in the infrastructure and information systems in the primary healthcare systems of LMICs 12 . On the other hand, regions with higher standards of digital health have been exploring and practicing the use of massive and dynamic NCD data to inform public health governance and policymaking [bib_ref] Big data for public health policy-making: policy empowerment, Mählmann [/bib_ref]. Enabled by welldeveloped digital health data services, researchers have also been using EHR systems to support the conduct of clinical trials [bib_ref] Using electronic health records for clinical trials: where do we stand and..., Mc Cord [/bib_ref]. These aspirations warranted substantive input to enhancing the infrastructure of primary healthcare systems in LMICs, particularly to strengthen the health information systems. . Factors influencing real-world uptake of digital health interventions for non-communicable disease management in primary health care. ## Factors influencing digital health uptake study numbers in references factors regarding policymakers Political commitment is key in digital health innovation and uptake, through advocate, financial support, and stakeholder engagement. Reviews: [bib_ref] The top Chinese mobile health apps: a systematic investigation, Hsu [/bib_ref] [bib_ref] Recent worldwide developments in eHealth and mHealth to more effectively manage cancer..., Lewis [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref] [bib_ref] Assessing the feasibility of eHealth and mHealth: a systematic review and analysis..., Njoroge [/bib_ref] Lack of political commitment, including regulations, standardization, monitoring, and evaluation, could be important barriers to digital health uptake. Reviews: [bib_ref] The top Chinese mobile health apps: a systematic investigation, Hsu [/bib_ref] [bib_ref] Assessing the feasibility of eHealth and mHealth: a systematic review and analysis..., Njoroge [/bib_ref] Factors regarding the technological industry The development in technologies and infrastructures provide solid ground for digital health advancement, including penetration of cell phones and the internet. Reviews: [bib_ref] Role of home blood pressure monitoring in overcoming therapeutic inertia and improving..., Agarwal [/bib_ref] [bib_ref] The effectiveness of patient activation intervention on type 2 diabetes mellitus glycemic..., Almutairi [/bib_ref] [bib_ref] Web-based interventions targeting cardiovascular risk factors in middle-aged and older people: a..., Beishuizen [/bib_ref] [bib_ref] Mobile health for non-communicable diseases in Sub-Saharan Africa: a systematic review of..., Bloomfield [/bib_ref] [bib_ref] Prescribable mHealth apps identified from an overview of systematic reviews, Byambasuren [/bib_ref] [bib_ref] The top Chinese mobile health apps: a systematic investigation, Hsu [/bib_ref] [bib_ref] Recent worldwide developments in eHealth and mHealth to more effectively manage cancer..., Lewis [/bib_ref] [bib_ref] The impact of mHealth interventions: systematic review of systematic reviews, Marcolino [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref] [bib_ref] Assessing the feasibility of eHealth and mHealth: a systematic review and analysis..., Njoroge [/bib_ref] [bib_ref] A systematic review of application and effectiveness of mHealth interventions for obesity..., Wang [/bib_ref] [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] Trials: [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] [bib_ref] Mobile phone text messages to support treatment adherence in adults with high..., Bobrow [/bib_ref] [bib_ref] InsuOnline, an electronic game for medical education on insulin therapy: a randomized..., Diehl [/bib_ref] [bib_ref] Telemonitoring and team-based management of glycemic control on people with type 2..., Lee [/bib_ref] Technical constraints (e.g., cell phone unavailability, internet instability) in marginal and remote regions limit the coverage of digital health. Reviews: [bib_ref] A systematic review of IT for diabetes self-management: are we there yet?, El-Gayar [/bib_ref] [bib_ref] The impact of mHealth interventions: systematic review of systematic reviews, Marcolino [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref] [bib_ref] Assessing the feasibility of eHealth and mHealth: a systematic review and analysis..., Njoroge [/bib_ref] Trials: [bib_ref] Use of a mobile phone app to treat depression comorbid with hypertension..., Menezes [/bib_ref] [bib_ref] Feasibility study of automated interactive voice response telephone calls with community health..., Pichayapinyo [/bib_ref] [bib_ref] Development and evaluation of a mobile decision support system for hypertension management..., Silveira [/bib_ref] Some unresolved data-related technical issues prohibit further uptake of digital health, including data security issues and data ownership issues. Reviews: [bib_ref] Mobile health applications in weight management: a systematic literature review, Dounavi [/bib_ref] [bib_ref] Recent worldwide developments in eHealth and mHealth to more effectively manage cancer..., Lewis [/bib_ref] [bib_ref] Mobile technology interventions for asthma self-management: systematic review and meta-analysis, Miller [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref] [bib_ref] Bibliometric analysis and methodological review of mobile health services and applications in..., Pai [/bib_ref] [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] Lack of interoperability across different digital health systems is an important barrier for integration, especially with EHR, APPs, and online platforms. ## Review: 41 Factors regarding digital health designers Digital health tools that enable two-way human interactions are better accepted while replacing human communications with automatic responses is less favored. Reviews: [bib_ref] Web-based interventions targeting cardiovascular risk factors in middle-aged and older people: a..., Beishuizen [/bib_ref] [bib_ref] Mobile phone messaging for facilitating self-management of long-term illnesses, De Jongh [/bib_ref] [bib_ref] A systematic review of IT for diabetes self-management: are we there yet?, El-Gayar [/bib_ref] [bib_ref] Characterizing active ingredients of eHealth interventions targeting persons with poorly controlled type..., Kebede [/bib_ref] [bib_ref] Recent worldwide developments in eHealth and mHealth to more effectively manage cancer..., Lewis [/bib_ref] [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref] Incorporating behavioral theories in digital health design is a facilitator, and the absence of that could be a barrier. Reviews: [bib_ref] Mobile phone-based behavioural interventions for health: a systematic review, Buhi [/bib_ref] [bib_ref] Characterizing active ingredients of eHealth interventions targeting persons with poorly controlled type..., Kebede [/bib_ref] [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref] Having user-centered design, such as involving target users in the design phase of digital health could be a facilitator (i.e., co-design). Reviews: [bib_ref] A systematic review of mHealth-based heart failure interventions, Cajita [/bib_ref] [bib_ref] Mobile technology interventions for asthma self-management: systematic review and meta-analysis, Miller [/bib_ref] [bib_ref] Bibliometric analysis and methodological review of mobile health services and applications in..., Pai [/bib_ref] Trial: [bib_ref] A cluster-randomized, controlled trial of a simplified multifaceted management program for individuals..., Tian [/bib_ref] Tailored design and personalized contents for patients is a facilitator, and one-for-all unified contents and designs could be a barrier. Reviews: [bib_ref] A systematic review of IT for diabetes self-management: are we there yet?, El-Gayar [/bib_ref] [bib_ref] Characterizing active ingredients of eHealth interventions targeting persons with poorly controlled type..., Kebede [/bib_ref] [bib_ref] Bibliometric analysis and methodological review of mobile health services and applications in..., Pai [/bib_ref] [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref] Incorporating users' feedback on acceptability and satisfaction is a facilitator, and lack of that could be a barrier and is currently under-addressed. Reviews: [bib_ref] Mobile health applications in weight management: a systematic literature review, Dounavi [/bib_ref] [bib_ref] Factors that promote or inhibit the implementation of e-health systems: an explanatory..., Mair [/bib_ref] Digital health designs that were integrated into existing healthcare model was a facilitator for successful uptake, and unintegrated digital health interventions that impose additional workload for primary healthcare providers is a barrier. Review: 24 Trials: [bib_ref] Development and evaluation of a mobile decision support system for hypertension management..., Silveira [/bib_ref] [bib_ref] A cluster-randomized, controlled trial of a simplified multifaceted management program for individuals..., Tian [/bib_ref] [bib_ref] Effectiveness of a primary care-based integrated mobile health intervention for stroke management..., Yan [/bib_ref] Easily navigable interface is a facilitator, and poor/complicated interface is a barrier for the uptake of digital health tools. Reviews: [bib_ref] A systematic review of IT for diabetes self-management: are we there yet?, El-Gayar [/bib_ref] [bib_ref] Smart devices for older adults managing chronic disease: a scoping review, Kim [/bib_ref] [bib_ref] The effectiveness of e-& mHealth interventions to promote physical activity and healthy..., Müller [/bib_ref] Trials: [bib_ref] Use of a mobile phone app to treat depression comorbid with hypertension..., Menezes [/bib_ref] [bib_ref] Hypertension subtypes among Thai hypertensives: an analysis of telehealth-assisted instrument in home..., Montrivade [/bib_ref] [bib_ref] Feasibility study of automated interactive voice response telephone calls with community health..., Pichayapinyo [/bib_ref] Factors regarding digital health users Adequate high-quality personnel training for primary healthcare providers is a facilitator for digital health uptake, while lack of that could be a barrier. Reviews: [bib_ref] Smart devices for older adults managing chronic disease: a scoping review, Kim [/bib_ref] [bib_ref] Empirical studies on usability of mHealth apps: a systematic literature review, Zapata [/bib_ref] [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] Trials: [bib_ref] Effectiveness of an mHealth-based electronic decision support system for integrated management of..., Prabhakaran [/bib_ref] Lack of local capacities, such as technological illiteracy, are barriers to digital health uptake. Reviews: [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref] [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] Trials: [bib_ref] Telemonitoring and team-based management of glycemic control on people with type 2..., Lee [/bib_ref] [bib_ref] Use of a mobile phone app to treat depression comorbid with hypertension..., Menezes [/bib_ref] [bib_ref] Feasibility study of automated interactive voice response telephone calls with community health..., Pichayapinyo [/bib_ref] Lack of guidance for patients is subject to low acceptance and waning interests in using digital health over time. Reviews: [bib_ref] Web-based interventions targeting cardiovascular risk factors in middle-aged and older people: a..., Beishuizen [/bib_ref] [bib_ref] Mobile phone messaging for facilitating self-management of long-term illnesses, De Jongh [/bib_ref] [bib_ref] Mobile health applications in weight management: a systematic literature review, Dounavi [/bib_ref] [bib_ref] Smart devices for older adults managing chronic disease: a scoping review, Kim [/bib_ref] [bib_ref] A systematic review of application and effectiveness of mHealth interventions for obesity..., Wang [/bib_ref] Trial: [bib_ref] Effects of using a mobile health application on the health conditions of..., Debon [/bib_ref] Designing incentives/motivations for primary healthcare providers and patients to use digital health facilitates the uptake, while lack of that could be a barrier. Reviews: [bib_ref] A systematic review of IT for diabetes self-management: are we there yet?, El-Gayar [/bib_ref] [bib_ref] Addressing noncommunicable diseases among urban refugees in the Middle East and North..., Mcnatt [/bib_ref] [bib_ref] A systematic review of application and effectiveness of mHealth interventions for obesity..., Wang [/bib_ref] [bib_ref] Using mHealth tools to improve access and coverage of people with public..., Beratarrechea [/bib_ref] Trials: [bib_ref] A telehealth strategy for increasing adherence in the treatment of hypertension in..., Santos [/bib_ref] [bib_ref] Feasibility study of automated interactive voice response telephone calls with community health..., Pichayapinyo [/bib_ref] [bib_ref] Development and evaluation of a mobile decision support system for hypertension management..., Silveira [/bib_ref] Suboptimal quality and accuracy for data input, report, and interpretation is a barrier to effective use of digital health. Reviews: [bib_ref] Mobile phone messaging for facilitating self-management of long-term illnesses, De Jongh [/bib_ref] [bib_ref] Recent worldwide developments in eHealth and mHealth to more effectively manage cancer..., Lewis [/bib_ref] [bib_ref] The impact of mHealth interventions: systematic review of systematic reviews, Marcolino [/bib_ref] Trials: [bib_ref] Hypertension subtypes among Thai hypertensives: an analysis of telehealth-assisted instrument in home..., Montrivade [/bib_ref] [bib_ref] Development and evaluation of a mobile decision support system for hypertension management..., Silveira [/bib_ref] The mobilization of local communities with local capacity improvements is a facilitator to enhance the use of digital health tools, and lack of those could be a barrier. Review: 43 Trials: [bib_ref] A telehealth strategy for increasing adherence in the treatment of hypertension in..., Santos [/bib_ref] [bib_ref] Development and evaluation of a mobile decision support system for hypertension management..., Silveira [/bib_ref] Multiple factors influenced the real-world uptake of digital health interventions, including factors regarding policymakers, factors regarding technological industry, factors regarding digital health designers, and factors regarding digital health users. Besides infrastructure constraints, another potential explanation for the under-use of digital health interventions in primary health care of LMICs is lack of local innovation. Evidence shows the best solutions are those that are responsive to local contexts [bib_ref] Effect of lifestyle-focused text messaging on risk factor modification in patients with..., Chow [/bib_ref] [bib_ref] Strengthening urban primary healthcare service delivery using electronic health technologies: a qualitative..., Bhattarai [/bib_ref]. However, current digital health interventions in LMICs relied on importing existing technologies from other settings to places they were not created for, sometimes in one-off research projects. A review of eHealth interventions in Nepal, for example, found that many were not adequately integrated into the existing health systems [bib_ref] A review of eHealth initiatives: implications for improving health service delivery in..., Kc [/bib_ref]. This limitation compromised the appropriateness, efficacy, and scale-ability of digital health interventions in LMICs. From the perspective of innovation diffusion 81 , the use of digital health interventions for NCD management in primary health care of LMICs could be classified as "late majority" or even "laggards" in some regions (as opposed to "innovators", "early adopters", and "the early majority"). This may also partially explain our mixed findings on the effectiveness of digital health interventions in LMICs, and warrants further attention to not only translate digital health interventions from high-income countries but also to encourage digital health innovations that derive from local contexts and needs. Despite the "laggardness" of digital health interventions, the included studies still presented high heterogeneity in the outcomes of using digital health, which was also found in other systematic reviews [bib_ref] Digital interventions to promote self-management in adults with hypertension systematic review and..., Mclean [/bib_ref] [bib_ref] Effectiveness of mHealth interventions in improving medication adherence among people with hypertension:..., Xiong [/bib_ref]. Such commonly acknowledged heterogeneity in digital health designs and outcome selection is informative in itself-it signals a diversity of possibilities for the role of digital health interventions in health care. However, this could also imply that digital health as an emerging field might be too broad and inclusive a concept to draw definitive conclusions at least for now, especially at settings where rigorous evidence is sparse. It might be more plausible for further research to develop more granular foci on fractions of digital health with shared homogeneity. Therefore, to mitigate concerns of such heterogeneity, our study further categorized the various types of study outcomes as either clinical, behavioral, or implementation outcomes. Among these three types of outcomes, the effectiveness of digital health varied greatly in the levels of consistency, if not direction. With respect to clinical outcomes of individuals, some studies attributed the highly mixed and neutral results to the study design limitations, such as small sample size and short follow-up durations [bib_ref] Smart devices for older adults managing chronic disease: a scoping review, Kim [/bib_ref] [bib_ref] mHealth text and voice communication for monitoring people with chronic diseases in..., Watkins [/bib_ref]. This mixture of findings on clinical outcomes added to the conflicting evidence in the existing literature, where many meta-analyses of related topics disagreed on outcomes such as blood pressure reduction [bib_ref] Digital interventions to promote self-management in adults with hypertension systematic review and..., Mclean [/bib_ref]. This disagreement may result from differences in study locations (high-income countries or LMICs), populations (general population or less advantaged populations), and study settings (hospitals or primary health care) [bib_ref] Digital interventions to promote self-management in adults with hypertension systematic review and..., Mclean [/bib_ref]. For individuals' behavioral outcomes, although still mixed, we consistently observed positively inclined results, especially when substantiated by behavioral science theories [bib_ref] Web-based interventions targeting cardiovascular risk factors in middle-aged and older people: a..., Beishuizen [/bib_ref] [bib_ref] Development of a smartphone-enabled hypertension and diabetes mellitus management package to facilitate..., Ajay [/bib_ref]. Existing studies on NCD management listed four major NCD behavioral risk factors -tobacco use, harmful alcohol consumption, unhealthy diet, and physical inactivity 11 . We found the latter two had widely documented positive improvements in our included studies. We also found positive improvements in disease management activities, such as adherence to medications and regimens, consistent with the existing literature [bib_ref] Effectiveness of mHealth interventions in improving medication adherence among people with hypertension:..., Xiong [/bib_ref] [bib_ref] Evaluating machine learning-based automated personalized daily step goals delivered through a mobile..., Zhou [/bib_ref]. Nevertheless, the long-term sustainable effects of digital health on behavioral changes remains uncertain [bib_ref] Digital interventions to promote self-management in adults with hypertension systematic review and..., Mclean [/bib_ref] [bib_ref] Motivating people to sustain healthy lifestyles using persuasive technology: a pilot study..., Kim [/bib_ref]. Further digital health evidence needs to be not only scaled up in scope but also in the duration of observation. For health service implementation outcomes, we consistently found positive effects from digital health interventions. This included improved user experience, timeliness of and accessibility to health care and information, consistent with studies in other settings [bib_ref] Patient interest in and barriers to telemedicine video visits in a multilingual..., Khoong [/bib_ref] [bib_ref] Health information seeking behaviors and preferences of a diverse multi-lingual cohort, Khoong [/bib_ref]. Although this is promising, the WHO's guideline on digital interventions expected even more on health system strengthening 3 , including improving health service coverage, service awareness and utilization, availability and capacity of human resources, availability of commodities and equipment, and service continuity and effectiveness. Following these aspirations, instead of treating health service implementation outcomes mainly as secondary outcomes or process indicators, as did in most of the included studies, a better way forward may be to focus on them more, which could produce more tangible and reliable evidence that is also greatly needed. Based on our scoping review, it could be safely inferred that digital health interventions are essentially an empowerment strategy for individuals' health and disease management and for health facilities' service delivery. In practice, however, effects are ultimately subject to digital health interventions' real-world uptake, which is influenced by many factors. A study on digital interventions for mental health mentioned an "enormous research-to-practice gap" between 15 years of evidence from efficacy trials and "virtually no successful and sustainable implementation" of digital health in real world [bib_ref] Accelerating digital mental health research from early design and creation to successful..., Mohr [/bib_ref]. Our study identified four groups of factors influencing the uptake of digital health interventions, some of which were also highlighted in the existing literature. First and foremost, having the aid of digital health or not, human interaction remains a critical ingredient to successful health service delivery, which emphasizes patient-supporter interaction as much as, if not more than, patient technology or provider technology interaction [bib_ref] Accelerating digital mental health research from early design and creation to successful..., Mohr [/bib_ref]. Second, the importance of user-centered design was repeatedly mentioned in the literature, which should entail user engagement in co-design, customization to personal needs and preferences, and feedback evaluations Our findings also agreed with existing studies in that more attention should be given to disadvantaged populations with limited capacity to use digital health tools due to technical illiteracy, and that adequate training and continuous incentives should be in place to ensure their sustainable uptake [bib_ref] Aging barriers influencing mobile health usability for older adults: a literature based..., Wildenbos [/bib_ref] [bib_ref] Exploring the barriers and facilitators for the use of digital health technologies..., Slevin [/bib_ref]. Another highly considerable facilitator to digital health uptake is the integration with existing health systems or local service delivery models, as was mentioned in two of the included studies in China [bib_ref] A cluster-randomized, controlled trial of a simplified multifaceted management program for individuals..., Tian [/bib_ref] [bib_ref] Effectiveness of a primary care-based integrated mobile health intervention for stroke management..., Yan [/bib_ref] , which was also recommended by a WHO policy brief. Finally, the optimal uptake of digital health interventions needs to be enhanced by political commitment for regulation, standardization, and support [bib_ref] Barriers and facilitators when implementing webbased disease monitoring and management as a..., Van Den Wijngaart [/bib_ref]. For future studies to optimally navigate the various factors influencing digital health uptake, we recommend the use of multiple methods such as qualitative research to preestimate local contexts and needs before implementation of digital health interventions. This study has the strength of shifting the attention for digital health interventions from high-income countries and hospitals settings to the under-represented primary healthcare settings of LMICs. Our inclusion of both trials and reviews in the study provided insights for various contexts in LMICs. However, several limitations should also be acknowledged. First, we only focused on academic publications in English, and could not include grey literatures relating to digital health, especially in non-English speaking settings. We tried to mitigate this potential loss of information by including review papers that were published in English but investigated non-English publications in their analyses. Second, given the high level of heterogeneity in the included studies, we were not able to conduct a rigorous quantitative synthesis such as meta-analysis. Instead, we conducted a narrative synthesis with the available information, which sufficed to address our research questions and also pointed to future research directions to conduct more quantitative synthesis on specific types and elements of digital health interventions. A blueprint for ideal digital health interventions should not only benefit individual health management and health facility service delivery, but also empower public health governance and policymaking through interoperable and reliable data services 77 . All these enhanced functionalities are needed, particularly in primary health care of LMICs. Our findings highlight both promises and limitations in the effectiveness of digital health interventions for NCD patients and health providers and suggest multiple factors to consider for industrial and governmental stakeholders in the initiation of digital health interventions, including political commitment, technology advancement, interactivity, integration with existing systems, and user-centered design and incentives. For future research, we call for more large-scale trials to further evaluate the real-world impact of digital health interventions in multiple aspects, particularly in the delivery of NCD services in primary health care of LMICs. # Methods ## Search strategy and selection criteria The following steps were guided by PRISMA guidelines (PRISMA-ScR, Supplementary [fig_ref] Table 1: Identified digital health interventions for non-communicable disease management in primary health care [/fig_ref] [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement (Chinese..., Moher [/bib_ref]. We searched PubMed, Embase, and Web of Science using three groups of keywords in the search syntax and required at least one keyword from each group: (1) digital health-related keywords, which included terminologies that describe subsets of digital health, such as "eHealth" (the use of information communication technologies for health [bib_ref] What is e-health?, Eysenbach [/bib_ref] , "mHealth" (a subset of digital health enabled by mobile devices [bib_ref] Emerging new era of mobile health technologies, Park [/bib_ref] , and specific digital health interventions, such as SMS and EHR; (2) NCD-related keywords, which included general terms such as "chronic conditions" and also specific conditions such as "hypertension" and "diabetes"; and (3) primary healthcare related keywords, which included synonyms of primary health care such as "basic health care". Specific search syntax with subject headings were customized based on the requirements of each database, including Pubmed (using "title/abstract"), Embase (using "title/ abstract/keywords"), and Web of Science (using "topic"). See Supplementary Notes 1 for the complete search syntax. For inclusion criteria, we included all types of review papers, such as systematic reviews, scoping reviews, and umbrella reviews, and all types of experimental studies, including RCTs, cluster RCTs, and quasi-experimental studies, that (1) focused on the use of digital health interventions for NCD management, which entailed various healthcare activities, including prevention, treatment, and rehabilitation; (2) were conducted in primary healthcare settings; (3) were conducted in LMICs, which was based on World Bank's classifications by income; and (4) were published from January 1, 2010, to ensure the timeliness of the findings. Of note, the search was completed on April 30, 2021. For exclusion criteria, we excluded: (1) studies that were conducted exclusively in high-income countries or in hospital settings; (2) study protocols, after attempting to find their completed publications; (3) papers of which the full-texts remained inaccessible (e.g., conference abstracts/proceedings) after contacting the corresponding author and seeking support from library staff; (4) qualitative evaluation studies of past trials, and (5) papers that were not written in English. Of note, we did not exclude studies that were conducted in both high-income countries and LMICs. We also did not include studies that focused on mental healththrough an important NCD issue-because of the major distinctions between mental health and other NCDs with respect to their required resources and management models. Trained researchers (S.X., H.L., E.D., A.N., R.D., and S.K.) conducted three rounds of screening, first by title, then by abstract, and finally by full-text, based on the inclusion and exclusion criteria. Reasons for exclusion were provided to studies excluded at the full-text screening stage. Each article was independently screened by at least two reviewers. Discrepancies between the reviewers were discussed in group meetings until an agreement was reached. We used EndNote X9 to manage the literature database. ## Data extraction and analysis Trained reviewers (S.X., H.L., E.D., A.N., R.D., and S.K.) independently extracted the following data and compared their results for consistency. First, we extracted the basic information of each study, including the title, year of publication, author's name, and study design. Then, in order to identify gaps in the use of digital health interventions for NCD management in primary health care of LMICs (Objective 1), we further extracted information about the specific digital health interventions that were covered by each study. Of note, the identified digital health interventions were not necessarily exclusively used for NCD management only, and some of them might also simultaneously contribute to other primary healthcare services (e.g., infectious disease management, maternal and child health services). To explore the effectiveness of the digital health interventions (Objective 2), we extracted information about the effectiveness of the digital health interventions according to the study outcomes, such as improvements in blood pressure control, patients' behavioral changes, or users' acceptance or satisfaction with health services. Finally, to identify factors influencing the uptake of the digital health interventions (Objective 3), we performed an inductive content analysis on the results, discussion, and/or implications sections of the included studies where applicable, to identify factors that influenced the uptake of the digital health interventions for NCD management at the primary healthcare level. Inductive content analysis was used because it enabled data-driven identification of themes (i.e., the "factors") in a bottom-up manner from findings of the included studies. We assessed the study quality of the included trials, following National Heart, Lung, and Blood Institute's Study Quality Assessment Tools for the quality assessment. The trials were categorized into good, fair, or suboptimal quality, considering their research practices including randomization, blinding, sample size sufficiency, pre-specification of outcomes, and intent-to-treat analysis. ## Study frameworks To identify gaps in the use of digital health interventions for NCD management in primary healthcare settings of LMICs, we utilized WHO's Classification of digital health interventions as the guiding framework for benchmarking [fig_ref] Figure 4: WHO classification of digital health interventions, adapted from WHO 2018 4 [/fig_ref]. The WHO classification categorized digital health interventions into four groups by different users: (1) clients, which refers to potential or current users of health services-in our study, they are referred to as people with NCDs; (2) healthcare providers, which refers to the health workforce to deliver health services-in our study, they are referred to as primary healthcare providers; (3) health system managers, which refers to people involved in the administration and oversight of public health systems; and (4) data services, which refers to cross-cutting functionality for data collection, synthesis, use, and exchange 4 . The health system quality of care framework provided six relevant dimensions that constitute the quality of health care, and it was previously applied in a systematic review on mobile health tools for NCD management [bib_ref] Use of mHealth systems and tools for non-communicable diseases in low-and middle-income..., Peiris [/bib_ref]. We applied these six dimensions to determine how the included studies attempted to use digital health interventions to improve NCD management in primary health care: (1) Effectiveness: being needbased, adherent to the evidence base, and resulting in improving health; (2) Efficiency: maximizing resource use and avoiding waste; (3) Accessibility: being timely, geographically reasonable, skillful, and resourceful; (4) Acceptability: considering individual preferences and aspirations as well as community cultures; (5) Equity: not varying in quality due to personal characteristics; and (6) Safety: minimizing risks and harm to service users. ## Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. ## Data availability The data underlying this manuscript is based on existing publications and is available in the referenced literature or from the corresponding authors upon reasonable request. Received: 9 June 2022; Accepted: 21 January 2023; [fig] Figure 1: Flowchart for study search and screening. The initial results were 8866 items. After duplicate removal and screening by title, abstract, and full-text, a total of 52 papers were included for final analysis.S. Xiong et al. [/fig] [fig] Figure 4: WHO classification of digital health interventions, adapted from WHO 2018 4 . The WHO classification categorized digital health interventions into four groups by different users: clients, healthcare providers, health system managers, and data services.S. Xiong et al. [/fig] [table] Table 1: Identified digital health interventions for non-communicable disease management in primary health care. [/table]

Food Behaviors, Health, and Bean Nutrition Awareness among Low-Income Men: A Pilot Study Bean consumption is known to lower blood cholesterol and postprandial blood glucose. With higher chronic disease risk, low-income men could theoretically benefit from increased bean intakes. The study objective was to explore low-income men's food behaviors, bean health benefit awareness, and bean consumption practices and preferences. Seventy-one low-income men aged 18-65 years (µ 41 ± 12.7; 53% white, 16% black, 31% Hispanic) completed a survey on health risks, food behaviors, bean health knowledge, attitudes toward dry and canned beans, and bean preferences. Eighty-seven percent had one or more health risk factors of physical inactivity, smoking, or obesity. Most men compared food prices, and thought about healthy food choices for their families, but few planned meals or read nutrition facts labels. White men had significantly higher bean health benefit knowledge than black or Hispanic men (p < 0.01). Most men liked the taste of beans, disagreed dry beans took too long to prepare, and 79% ate them at least 2-3 times per month. Forty-nine percent agreed beans caused intestinal gas. Improving men's awareness of the health benefits of beans as well as leveraging existing positive attitudes may be useful approaches to increase bean consumption among low-income and minority male populations. # Introduction The leading causes of death among adult men in the United States (US) remain heart disease (24.4%) and cancer (21.9%). The risk factors for these diseases as well as type 2 diabetes, hypertension, and obesity are mediated by lifestyle behaviors such as diet, physical activity, regular medical checkups, and not smoking. Men may be less concerned about healthy dietary patterns and lack awareness of diet-health associations in comparison to women. Along with gender, wage earnings and race/ethnicity have a considerable impact on health and disease outcomes. Low-income men have increased prevalence of health risk factors including physical inactivity, smoking, and obesity, as well as increased chronic disease rates such as heart disease, stroke, diabetes, and cancer. Disease rates are greater in blacks and Hispanics than non-Hispanic whites, yet within race/ethnicity groups, rates are linked inversely to income. Not unexpectedly, low-quality diets are associated with limited economic resources. A lack of dietary diversity accompanied with high levels of saturated fat and low vegetable, fruit, and fiber intake contributes to disease risk. Pulses, which include beans, peas, lentils and chickpeas, can improve dietary quality by providing protein, fiber, and many micronutrients associated with other vegetables, including folate, iron, zinc, magnesium, and potassium. Compared to other foods, beans have one of the best nutrient-to-price ratios, making them ideal for maximizing nutrition with economic constraints. Bean consumption is associated with both reduced risk and improved outcomes of cardiovascular disease, serum cholesterol, postprandial glucose control, type 2 diabetes, and some cancers. The dietary fiber in beans is likely a contributing component to lower risk of these chronic diseases. The 2015-2020 Dietary Guidelines for Americans (DGA), which aim to promote health, prevent chronic disease, and help Americans maintain a healthy weight, suggests 2-3 cups of cooked beans per week for men ages 19-50. On average, men currently consume 1 cup per week. Increasing bean consumption to meet the DGA could improve nutrient shortfalls such as fiber, potassium, and iron, and reduce disease-related risks. Only a handful of published studies exist on the factors that influence bean consumption among Americans, and these are mostly with minorities or women. Determining the views and current knowledge toward beans among low-income males is necessary to design tailored intervention programs to boost bean intake and improve health. There were five study objectives. These were to determine low-income men's: (1) health risk factors; (2) food behavior practices; (3) knowledge of bean health benefits; (4) attitudes and perceptions toward dry and canned beans; and (5) consumption patterns and purchasing preferences of beans. # Materials and methods ## Participants and data collection Low-income male clients, aged 18-65 years old, were recruited from an employment center, a Special Supplemental Nutrition Program for Women, Infants and Children (WIC) clinic, and classes of the Expanded Food and Nutrition Education Program (EFNEP). With agency permission, researchers set up an information table in the waiting rooms of the employment center and WIC. Signage and flyers promoted a survey on health and food behaviors, but did not specifically mention beans to reduce response bias to bean consumers only. If a man expressed interest in participating, a description of the study was read to him, and verbal consent was given before he received the survey in English or Spanish based on his choice. Participants received a $3 cash incentive at the employment center and WIC. At the EFNEP classes, men completed surveys at the end of the third session of the six-class series. Men received grocery coupons and brand-marketed notepads, pens, tote bags, etc. Data were collected in March through September 2011. The Arizona State University Institutional Review Board deemed the study exempt [#1009005462]. ## Survey instruments The survey consisted of six parts: demographics, health risk factors, food behaviors, food security, attitudes and perceptions of dry and canned beans, and bean purchasing preferences. The demographic questions and the 10-item Food Behavior Checklist from the Arizona EFNEP program enrollment form were used for all sites. Health behavior questions (smoking, physical activity, health care) were from the Behavioral Risk Factor Surveillance System (BRFSS). The six-item USDA Core Food Security Module was used to evaluate food security status. Dry and canned bean attitude and perceptions Likert statements, and bean purchasing preferences were adapted from previous research. Prior to data collection, the draft survey content was reviewed with seven Extension instructional specialists and subsequently pilot tested with two focus groups of EFNEP participants (n = 24). After modifications from this formative evaluation, 11 more agency researchers and field staff evaluated the survey for content validity. Another group of 23 EFNEP participants completed the survey and gave post-test feedback before formal data collection began. # Statistical analysis BMIs were calculated from self-reported height and weight. These raw values were classified into categories of normal, overweight, and obese. Descriptive statistics were compared by race/ethnicity categories using Chi-square and ANOVA. Likert statement responses for dry and canned bean were condensed from five to three categories of 'disagree, neutral, agree' for display in tables. Principal components analysis was applied to the five-category Likert-type responses for four groups of questions (the 10-item Food Behavior Checklist, health benefits of beans, attitudes toward dry beans, and attitudes toward canned beans) to identify thematic clustering. Reliability analysis to develop parsimonious models resulted in four scales with good reliability (Cronbach's alpha in parentheses): positive food behaviors (0.74), bean health knowledge (0.86), general bean attitudes (0.78) and canned bean attitudes (0.76). Data entry and analysis was performed with SPSS V.23.0 (IBM, Armonk, NY, USA), and a p value <0.05 was considered significant. # Results The completion rate was 91.5% (75/82). Four men (2 Native American, 2 Asian) were excluded from further analysis due to small sample size for ethnic comparisons. Sixty percent of the respondents were employment center clients, with 22% recruited from WIC and 13% from EFNEP. Over half of the men (53%) identified as white, 16% as black, and 31% as Hispanic. Hispanic men were younger (p = 0.021), had fewer years of education (p = 0.009), more children in the household (p = 0.001), and a greater percentage were born outside of the US (p < 0.001) than their peers. Eighty-seven percent of all men had one or more risk factors of physical inactivity, smoking, or obesity. There were no significant differences by race/ethnicity for food security, body mass index (BMI), exercise frequency, smoking, health care coverage, or routine checkup frequency.shows the frequency responses to the Food Behavior Checklist statements on food resource management, food safety, and nutrition practices by race/ethnicity. Seventy-three percent of the men compared prices before buying food, but black and Hispanic men did this less often (p = 0.046). Higher percentages of black and Hispanic men reported running out of food by the end of the month than white men (p = 0.001). While 70% of all men said they did not leave meat or dairy out of the refrigerator for more than 2 hours, over 60% of black and Hispanic men said they thawed foods at room temperature (p = 0.028). Hispanic men planned meals ahead of time 'sometimes' as opposed to 'always' in contrast to their peers (p = 0.039). The 'positive food behavior' scale mean was higher for white men, suggesting a trend of more frequent behaviors, than among black or Hispanic men (p = 0.056). Responses to Likert-type statements on the health benefits of beans are provided in. Close to 60% of all men agreed beans improve nutrition but about 25% of black and Hispanic men disagreed (p = 0.027). Almost 50% of all men stated a 'neutral' response regarding the ability of eating beans to lower cholesterol, lower cancer risk, or control blood sugar. Significantly more black and Hispanic men disagreed that eating beans could 'lower bad cholesterol' (p = 0.016), control blood sugar (p = 0.049), or are healthy for the gastrointestinal tract (p = 0.044). The bean health benefits scale was significantly higher for white men indicating more knowledge than black or Hispanic men (p = 0.008). The Likert-type statements for attitudes and perceptions of dry and canned beans are shown in. Over 70% of men disagreed it is difficult to make food with beans and that only 'poor people' eat beans. Few men agreed that they disliked the taste of beans, that their family will not eat beans, or that their friends do not eat beans. Almost half of the men agreed beans cause intestinal gas. Only 32% agreed that dry beans take too long to prepare. Similar positive attitudes were expressed by most men for the seven canned bean statements. There were no significant differences by race/ethnicity for the statements or the general bean or canned bean scale scores. Men's bean consumption, purchasing practices, and preferences are shown in. Significantly more black and Hispanic men purchased dry beans only (p = 0.050) compared to white men. Of the men who purchased beans, more white than black or Hispanic men indicated price was an important selection characteristic for dry beans (p = 0.036) and for canned (p = 0.017). Over 70% of Hispanic men who bought dry beans stated that they selected a dry bean based on tradition (p = 0.003) in comparison to their peers. Men were asked how often they consumed eight bean types. Sixty-three percent stated they ate pintos more than once per month, followed by baked (31%), black (27%), dark red kidney (25%), and chickpeas (21%). Fewer than 15% of men reported eating navy, lentils, or black eyed peas more than once per month (data not shown). Of the 78% of men who reported buying canned beans, 36% did not purchase a specific brand, 20% did not remember the brand name, 17% bought Bush Brothers & Company (Knoxville, TN, USA) and 14% mentioned Rosarita (Conagra, Chicago, IL, USA) (data not shown). # Discussion The purpose of the current study was to explore the food behaviors, knowledge, and attitudes toward dry and canned beans among a sample of 71 low-income men. Comprehensive details were collected on health risk factors, food behavior practices, knowledge of the health benefits of beans, attitudes and perceptions toward dry and canned beans, and consumption patterns and purchasing preferences of beans. Most men had one or more health risk factors of physical inactivity, current smoking or obesity. In comparison to other men in Arizona, study participants were physically active (76.1% vs. 74.5%) about equal as men statewide. The prevalence of overweight was about the same with participant obesity (30.4% vs. 24.9%,) and smoking (29.7% vs. 21.0%) higher than for Arizona men of all income levels (45%). The majority of men had several positive food behaviors such as comparing prices, not leaving meat or dairy unrefrigerated for long, and feeding children within 2 hours of waking. However, less than half shopped with a grocery list, planned meals ahead of time, or read the nutrition facts label on foods. Their responses on the Food Behavior Checklist are similar to Arizona women from the same time period and the pre-test results of Nebraska EFNEP adult enrollees. Over a third of the men indicated they did not add salt when preparing foods. Notably, the Arizona men's behavior toward added salt was less frequent than reported in the two other studies. Information about selecting "no salt added" canned beans, or dry beans which do not contain salt, may appeal to some low-income men. Ethnic differences were apparent, with Hispanic men having less food security and less likely to compare food prices or use a grocery list than their peers. Many also indicated their children did not always eat within 2 hours of waking. Hispanic men had more children in their households, and less income on average, yet had equivalent monthly food expenditure to their peers. In a previous study, Hispanic women in metro-Phoenix were more food secure than non-Hispanic white women, but also did not shop with a grocery list nor always feed children promptly after waking in comparison. Since food and storytelling are common themes in Hispanic and black culture, combining behavior change messages in stories and traditional cultural practices may be an appropriate way to convey healthful practices. Shopping with a grocery list has been associated with a higher dietary quality and lower BMI among high-risk adults. Since a high percentage of the low-income non-Hispanic men reported using grocery lists, and "sometimes" or "always" planning meals ahead of time, canned beans may be touted as a versatile option to add to a grocery list and to keep on hand for quick and easy meals. Most men in this study identified beans as improving nutrition and increasing satiety, but only about a third knew of the well-documented health benefits of beans to lower cholesterol, aid in blood glucose control, and reduce cancer risk. These health benefit knowledge gaps among low-income men may be mediated by race/ethnicity since more non-white men disagreed that these were known benefits. Similar studies have shown non-Hispanic whites to have greater nutrition knowledge than black, Hispanic and other non-white race/ethnic groups across income levels. However, it is difficult to extricate the influence of education from race/ethnicity when known disparities are observed. Other research suggests that those with higher education display greater nutrition knowledge scores. In response to the same bean health knowledge questions, less-acculturated Hispanic women in Arizona and in Iowa displayed lower nutrition knowledge levels than their bicultural or English dominant peers with more education. While nutrition knowledge is a necessary component for healthy food behaviors, it does not appear to be a sufficient driver of food choice on its own. Doma et al. found in a recent Canadian study of older adults that 98% felt eating beans could improve their health, but only 51.2% reported consuming beans regularly. The low-income men in this study had positive attitudes about dry and canned beans. The majority of Arizona men reported eating beans, liked their taste, and thought it was not difficult to make meals with them. However, these men were not likely to meet the DGA recommended level of 2-3 cups per week since most men reported consuming beans only 2-3 times per month. No difference was found in consumption frequencies by race or ethnicity, which was consistent with equivalent bean consumption frequencies between black and white men in another US study. People had similar positive views of beans yet low rates of consumption in other studies conducted in Mexico, Australia, France, and Canada. Food selection appears to be largely based on taste and cost, with taste cited as the biggest barrier to healthy eating. Moreover, consumers have implicit views that "healthy" foods are thought to be less filling and may have an inverse relationship to those that are "enjoyable". Since men in this pilot study perceived beans as satiating and appetizing, and taste is a leading influence on food choice, focusing on taste rather than nutrition may be a beneficial area to pursue in marketing pulses. Additional tailored nutrition education messages may highlight preferences of canned beans for white men and dry beans for black and Hispanic men. Monge et al. also found in Mexico that there was a preference among adults for dry, not canned, beans. Likewise, based on these preliminary results, messaging may be tailored to Hispanic men by focusing on aspects of tradition, and to white men by emphasizing economic value. There are several study limitations including the small sample size for the subgroups of black and Hispanic men. BMIs were calculated from self-reported height and weight and may be slightly lower than if measured directly by researchers. The actual amount of beans consumed was not asked of participants. Results cannot be generalized to populations other than those surveyed. Despite the cross-sectional convenience sample and small sample size, this study fills a research gap by providing descriptive details on health benefit knowledge, bean consumption patterns and preferences, and attitudes about beans among a diverse sample of low-income men. They are less likely to participate in health behavior research and are an understudied population. The men were recruited to answer questions about health and food rather than specifically about beans in an effort to reduce response bias to only those who ate beans. Other topics to address in future research include food environment, selection practices, shopping and preparation behaviors of the household, facilitators and barriers to bean consumption, in what manner beans are consumed (in or outside the home, types of products, cuisine, entrée/side, etc.), and the male perspective on beans as a protein source compared to meat and dairy. # Conclusions These findings suggest that low-income men practice some desirable food behaviors, have positive attitudes toward beans, and consume them, but lack knowledge of some health benefits of beans. Leveraging men's desirable food behaviors, positive views of beans, and increasing awareness of bean health benefits with clear, concise, and achievable messaging could improve health and make an increase in bean consumption more feasible.

Whisker-Mediated Touch System in Rodents: From Neuron to Behavior A key question in systems neuroscience is to identify how sensory stimuli are represented in neuronal activity, and how the activity of sensory neurons in turn is "read out" by downstream neurons and give rise to behavior. The choice of a proper model system to address these questions, is therefore a crucial step. Over the past decade, the increasingly powerful array of experimental approaches that has become available in non-primate models (e.g., optogenetics and two-photon imaging) has spurred a renewed interest for the use of rodent models in systems neuroscience research. Here, I introduce the rodent whisker-mediated touch system as a structurally well-established and well-organized model system which, despite its simplicity, gives rise to complex behaviors. This system serves as a behaviorally efficient model system; known as nocturnal animals, along with their olfaction, rodents rely on their whisker-mediated touch system to collect information about their surrounding environment. Moreover, this system represents a well-studied circuitry with a somatotopic organization. At every stage of processing, one can identify anatomical and functional topographic maps of whiskers; "barrelettes" in the brainstem nuclei, "barreloids" in the sensory thalamus, and "barrels" in the cortex. This article provides a brief review on the basic anatomy and function of the whisker system in rodents. # Introduction A fundamental goal of systems neuroscience is to identify how sensory stimuli are represented in neuronal activity, and how the activity of sensory neurons is "read out" by downstream neuronal structures to generate behavior. Researchers dissect this goal into the following questions: 1. What elemental features of sensory stimuli are encoded in the neuronal activity of sensory neurons? 2. How is each elemental feature represented in the activity of sensory neurons? 3. How do the downstream neuronal areas decode the activity of sensory neurons? 4. How does spatial and temporal context affect the efficiency with which single neurons and neuronal ensembles encode sensory stimuli? 5. How does the activity of neurons give rise to perception and ultimately behavior? Over the past decade, the increasingly powerful array of experimental approaches such as optogenetics and two-photon imaging which has become available in non-primate models, particularly in rodents, has spurred a renewed interest for the use of rodents in neuroscience research. The aim of this article is to introduce the rodent whisker-mediated touch system as a model system suitable for investigating the fundamental questions in systems neuroscience. This model serves as an anatomically well-established and behaviorally efficient system; as nocturnal animals, rodents rely on their whisker-mediated touch system to collect information about their surrounding environment. Moreover, this system represents a well-studied circuitry with an elegant structural organization. At every stage of processing, one can identify anatomical and functional topographic maps of whiskers. These clusters are referred to as "barrelettes" in the brainstem nuclei, "barreloids" in the thalamus, and "barrels" in the cortex. Mapping studies have revealed that whisker-related areas occupy a relatively large proportion of neural tissue at trigeminal medullar level (28%), at the level of thalamic sensory nuclei (27%), and at the cortical level (20%). In the following sections, I first provide a brief introduction to the basic anatomy and then the function of the whisker system in rodents. ## The whisker-mediated touch system ## Vibrissae and follicles Rat vibrissae, or whiskers, form a grid-wise layout on either side of the snout. The main distinction of the vibrissae from ordinary hairs is their large follicles which contain dense nerve terminals and sensory receptors. As mechanical transducers, the vibrissae mediate the transfer of the touch signal into these receptors. The vibrissae are categorized into two classes: (i) the micro-vibrissae, which are short and thin hairs around the nose tip, and (ii) macro-vibrissae, which are the long stiff mystacial hairs caudal to micro-vibrissae on the whisker pad. Macro-vibrissae consist of four follicles in rows A and B, seven to nine follicles in row C, D and E, and four straddlers (α, β, γ , δ) straddling between rows caudal to the mystacial pad (see . These two classes of vibrissae are believed to be functionally distinct; the macrovibrissae transmit spatial information such as localization in space, as they sweep the environment by intrinsic muscles. However micro-vibrissae are considered to be involved in acquisition of detailed tactile information for object and texture recognition. Nevertheless, there is evidence from behavioral studies demonstrating that rodents are able to perform texture and vibration discrimination tasks using their macrovibrissae. The nerve terminals and mechanoreceptors around the vibrissa shaft are of various types, morphologies and distributionsincluding Merkel cell-neurite complexes, lanceolate receptors, Ruffini corpuscles-sometimes referred to as reticular endings-and free nerve endings. Different receptors show different tuning properties and sensitivity to a variety of tactile stimulus parameters such as amplitude, frequency, duration, velocity, acceleration and direction of whisker deflections/motion . These receptors also exhibit different profiles of adaptation. Merkel cells are the most prominent mechanoreceptors. These receptors adapt slowly to sustained whisker deflections, whereas lanceolate receptors and simple corpuscles are rapidly-adapting. Each follicle is innervated by 150-200 myelinated and 100 unmyelinated distal axons of trigeminal ganglion neurons. These axons arborize around the hair shaft, sensing movements in different directions. ## Whisking Whisking is the rhythmic cyclic vibrissae sweeping action, consisting of repetitive forward (protraction) and backward (retraction) movements at an average frequency of about 8 Hz. Whisking is often synchronous to respiratory, head, and nose movements, suggesting coordination of activity among many muscle groups.observed two different patterns of whisking; the first pattern, referred to as the exploratory whisking, consists of wide-angle sweeps with a frequency range of 1 to 5 Hz in bouts of 1 to 10 s. The whisking frequency within a bout remains remarkably constant, while it changes between bouts. The second pattern of whisking consists of small-amplitude high-frequency (ranging from 15 to 25 Hz) sweeps for a period of 0.5 to 1 s while whiskers are thrust forward in a dense pattern . This pattern resembles the dense focalized arrangement of photoreceptors in the retina fovea, and is therefore referred to as "foveal whisking". Movement of the follicle is controlled by the facial motor nerve. Macrovibrissae are moved by two sets of striated musculatures; the intrinsic and extrinsic muscles. Intrinsic muscles lack a bony attachment and have their origin and insertions in the skin. They are associated with individual whiskers and generate the forward whisker motion (protraction) by pulling the base of the follicle backwards. Extrinsic muscles are located superficially in the mystacial pad with no direct connection with follicles. They move all whiskers together. On the basis of anatomical observations,andconcluded that mystacial pad muscles move the whiskers forward (protraction), whereas backward motion (retraction) is mainly a result of the elastic properties of the facial tissue, and is therefore passive. A more recent finding, however, demonstrated that retraction is under the active muscular control as well. Whisking is controlled by a neuronal oscillator located in the vibrissa-related region of intermediate reticular formation FIGURE 1 | Schematic representation of whisker-barrel system. Each whisker is identified by a unique letter-number combination corresponding to its row (A to E from dorsal to ventral) and arc (identified by numbers 1, 2 and etcetera from caudal to rostral), with α, β, γ , and δ straddlers between rows. Colors indicate rows. Barrel, barreloid and barrelets are redrawn from. PMBSF, posterior-medial barrel sub-field; PO, posterior thalamic nucleus; PrV, principal trigeminal nucleus; SC, superior colliculus; SpVi, spinal trigeminal nuclei pars interpolaris; SpVo, pars oralis; SpVc, pars caudalis; TRN, thalamic reticular nucleus; VPM, ventro-posterior medial nucleus; vMI, vibrissal primary motor cortex; vSI, vibrissal primary somatosensory cortex; vSII, secondary somatosensory cortex with the somatotopic map from; DLS, dorsolateral striatum; ZIv, ventral zona inserta. The evidence for somatotopic map in vM1 is provided inand. of the medulla (vIRt). This region includes facial premotor neurons and neurons that their spiking activity is either in phase or in antiphase with whisking protraction. Selective lesions in vIRt abolish whisking on the side of the lesion, and activation of the vIRt by iontophoretic injection of kainic acid (KA) induces long episodes of whisking under light ketamine anesthesia . Glycinergic/GABAergic neurons in vIRt rhythmically inhibit vibrissa facial motoneurons innervating the intrinsic muscles, suggesting that rhythmic whisking is driven by inhibition. During whisking, the intrinsic muscles protracting individual whiskers follow the whisking oscillation, while extrinsic muscles that move the mystacial pad follow the breathing rhythm. Both rhythms are phaselocked during sniffing (rapid rhythmic breathing). This is compatible with the unidirectional connections from the pre-Bötzinger complexthe inspiratory oscillator for respiration located in medulla adjacent to IRt-to vIRt, revealing the contribution of pre-Bötzinger complex to the mystacial pad control by driving the extrinsic muscles together with the potential contribution of putative parafacial neurons that receive their input from pre-Bötzinger complex. There are no bilateral vIRt to vIRt connections. Thus, the bilateral synchronization of whisking is mediated by the medullary commissural fibers connecting the left and right pre-Bötzinger complexes. Whiskers on the right and left sides can move asymmetrically and asynchronously. Additionally, rostral and caudal whiskers on a single side of the snout can sometimes move independently. Recently, using a three-dimensional model of the vibrissal array,quantified the search space during whisking and protraction. According to their calculations, the parabolic intrinsic curvature of the whiskers increases the volume of the search space by over 40% compared to that of the straight whiskers, while the elevation-whisker's angle relative to the horizontal plane-and torsion-torsional rotation of a whisker about its own axis-had modest effect on the search space. Elevation and torsion, however, affect the trajectory of the whisker tips. Dynamics of whisker movement reveal a rodent's expectations about the environment. During locomotion, direction and speed of running are coupled with average whisker position . The fine-scale kinematics of the whisking motion in freely moving rodents, however, is difficult to characterize. Machine learning techniques such as deep learning, visually enhanced whiskers for tracking using florescent dyesand precise controlled locomotion in virtual reality for head-fixed animalare promising future approaches for high precision characterization of whisker motion kinematics during locomotion. ## Trigeminal ganglion Trigeminal ganglion (also called semilunar ganglion) consists of the cell bodies of pseudo-unipolar neurons with their proximal axons innervating the ipsilateral brainstem trigeminal complex (BTC)and their distal axons innervating the vibrissae follicles. Each ganglion cell innervates only one whisker follicle. The trigeminal ganglion is somatotopically organized with caudal arcs represented dorsally, and dorsal rows represented medially. Early studies indicate that a great majority of the ganglion cells are slowly adapting, but see. The rapidly adapting ganglion cells have generally higher velocity thresholds. Different trigeminal ganglion units show various tuning properties, with evidence suggesting sensitivity to the following parameters: amplitude, frequency, duration, velocity, acceleration and direction of whisker deflections/motion . These neurons are highly sensitive to whisker deflection with over 50% of units responding to <1 - of whisker deflection. The spontaneous activity of these units is considered to be zero, and any discharge is potentially attributed to the high sensitivity of the units to tiny movements such as pneumatic vibrations, mechanical hysteresis of hair shaft, or tissue damage caused by microelectrode penetration. ## Brainstem trigeminal complex (btc) Traditionally, the whisker-recipient trigeminal complex in the brainstem is subdivided into the principal sensory nucleus (PrV) and the spinal nucleus (SpV). The latter is further subdivided rostro-caudally into 3 sub-nuclei: oralis (SpVo), interpolaris (SpVi) and caudalis (SpVc). Trigeminal nuclei neurons receive inputs from trigeminal ganglion cells and form discrete aggregated neuronal clusterscalled barrelettes-in each nucleus except for SpVo. Brainstem barrelettes preserve the somatotopic organization of whiskers on the mystacial pad. Each barrelette is about 55 µm in diameter and 1.2 mm long along the rostro-caudal direction and contains 160-200 neurons. The PrV and SpVi sub-nuclei provide the majority of the projections to the thalamus. Similar to first-order neurons in trigeminal ganglion, the more sensitive BTC units (with low velocity thresholds) were slowly adapting, whereas the less sensitive units (high velocity thresholds) were rapidly adapting. A majority of PrV barrelette neurons have barrelette-bounded dendritic trees. These neurons mainly project into single barreloidsneuronal aggregates representing individual whiskers-of the ventro-posterior medial nucleus (VPM) in the contralateral thalamus. Other groups of neurons in PrV with large multipolar somata and expansive dendritic branches spread over multiple barrelettes, and also respond to multiple whiskers. This population mainly projects into the posterior thalamic nucleus (POm) in thalamus, tectum, superior colliculus, zona incerta, the medial part of the medial geniculate nucleus (MGm), inferior olive and medial dorsal part of VPM (VPMdm). The electrophysiological studies identified two broad classes of neurons in PrV; tonic neurons which represent a single whisker, and phasic units which are driven by single or multiple whiskers. Neurons in SpVi spread their dendritic arbors into a broader area across multiple barrelettes, and thus respond to multiple whiskers. These neurons project to different brain areas, such as ventrobasal complex (mainly ventro-lateral VPM, VPMvl), the zona incerta, superior colliculus, medial geniculate nucleus, cerebellum and spinal cord. SpVc also projects to VPMvl similar to the thin axons of SpVi. SpVo sends a few axons only to POm. ## Thalamus VPM, POm and the intralaminar thalamic nuclei form the major thalamic targets of second-order neurons of brainstem trigeminal complex. The vibrissae representation area in VPM is somatotopically organized into discrete finger-like structures, called barreloids. Barreloids are oblong cylinder-like structures, with a length of 500-900 µm and contain 250 to 300 neurons each. The size of the barreloids is positively correlated with the length of whiskers. Cells within a barreloid have receptive fields composed of one principal and several surrounding whiskers. POm is more homogeneous than VPM, with no barreloid-like structures. However, there is evidence that POm is organized topographically. Compared to VPM cells, the receptive field of POm neurons is larger (6-8 whiskers). Moreover, POm neurons show a weaker response to single whisker deflections than VPM neurons do, and unlike VPM neurons, POm neurons exhibit less preference to a particular principal whisker. Instead, POm neurons are strongly driven by simultaneous disturbance of multiple whiskers. Thalamic barreloids receive three main inputs: 1. an ascending excitatory input from the principal trigeminal nucleus (PrV), 2. an excitatory corticothalamic input from the barrel field in the primary somatosensory cortex (SI), 3. an inhibitory input from the thalamic reticular nucleus. In all of these pathways, terminal fields of axons are mainly confined to the barreloid representing the corresponding principal whisker of their receptive field. The distal dendritic arbors of a proportion of VPM cells, however, spread in the surrounding barreloids, leading to a cross-whisker interaction . In contrast to the sensory-thalamic nuclei for other modalities, there are few, if any, dendrodendritic synapses and no local axon collaterals and inhibitory interneurons in rat VPM. Afferents of VPMdm neurons of thalamic barreloids arborize in the corresponding neuronal aggregates-barrels-in layer IV of primary somatosensory cortex and form a one-to-one connection between the VPM barreloids and cortical barrels. Multi-barrel projections of VPM neurons have never been observed. However, some axonal innervations into septal regions surrounding the barrels were found. Thalamic reticular nucleus and the upper part of layer VI of barrel field in SI are innervated by collaterals of the ascending projections from VPM. The VPMvl neurons do not directly project to the barrels. They receive presynaptic inputs from the caudal division of SpVi and branch their axons in the secondary somatosensory cortex (SII) as well as septal and dysgranular zone in SIand form the extralemniscal pathway. An additional ascending pathway parallel to lemniscal pathway originates from multi-whisker PrV neurons passing through the head of the thalamic barreloids . The neurons in the head of barreloids have multi-whisker receptive fields, innervate layer 4 septa and receive corticothalamic feedback from layer 6 of vibrissal MI. Hence it suggests this pathway is involved in relaying information related to the phase of whisking. POm projects to almost all sensory-motor areas of the neocortex, including the primary somatosensory, secondary somatosensory (SII), perirhinal, insular and motor cortices, and to a lesser extent to thalamic reticular nucleus. The laminar distribution of the terminal fields of POm projection to cortex are mainly to layers Va and I. Similarly, POm axon terminals in SI are distributed from upper layer V to layer I of the dysgranular zone and interbarrel septa, as well as in layers V and I of the barrels. The thalamic reticular nucleus (TRN) with ventrobasal thalamic nuclei forms an inhibitory feedback loop which is believed to play role in thalamic spindling, sleep-related thalamocortical oscillations, arousal, and selective attention. Optogenetic activation of TRN switches the thalamocortical firing pattern from tonic to bursting and enhances cortical spindles and delta waves. Neurons in the reticular nucleus receive vibrissae-related input from cortical Layer VI neurons in SI , collaterals from thalamocortical neurons in VPM and POm, as well as inputs from neighboring neurons in reticular nucleus. In turn, they send their GABAergic inhibitory projections back to ventrobasal nucleus and POm. These inhibitory back-projections can account for the inter-barreloid inhibition in VPM. While the topographic organization of the reticular neurons that project to VPM is somatotopic, no somatotopic map was found in the reticular neurons projecting to POm . In addition to thalamic reticular nucleus, a group of thalamic nuclei-termed extra-reticular inhibitory system-innervate POm with prominent GABAergic inhibitory projections. The extra-reticular inhibitory system includes the anterior pretectal nucleus (APT) , zona incertaand pars reticulate division of substantia nigra. Zona incerta (ZI) and APT are reciprocally connected, both project to PO and brainstem motor centers and receive layer V cortical inputs. ZI receives direct whisker input from both PrV and SpViin addition to input from SI. Neurons in the dorsal and ventral divisions of ZI exhibit multi-whisker receptive fieldswith partial somatotopy in dorsal division and a complete somatotopic organization in ventral division. The ventral division of the zona incerta (ZIv) receives the main input from SpViand serves as a relay by feed-forward GABAergic inhibition of thalamocortical neurons in higher order thalamic nuclei including the paralemniscal pathway and POm for whisker-related motor activity. The activation of vibrissal motor cortex suppresses vibrissal responses in ZIv , providing a dis-inhibition mechanism for sensory gating in higher order thalamic nuclei during whisker-related motor activity and active touch. For a thorough review refer to. ## Barrel field cortex The cortical vibrissae representation in rodents is formally referred to as the posterior-medial barrel sub-field (PMBSF) and occupies about 20% of the somatosensory cortex. The cortex is organized in 6 layers. In rodents, Layer IV of the vibrissae region of primary somatosensory cortex-referred to as the granular zone-contains anatomically distinguishable clusters of neurons called "barrels". Each elliptically shaped barrel is approximately 0.3-0.5 mm in maximal diameterand contains an average of 2500 neurons. Barrels are somatotopically arranged in an identical order as the whiskers on the snout, with the most dorsal posterior whiskers being represented by the most lateral posterior barrels. Neurons within each barrel produce their strongest and fastest response to the stimulation of the anatomically-associated whisker, also known as the "principal" whisker. There is a precise one-to-one connection between thalamic barreloids and cortical barrels, with no evidence of a multi-barrel innervation by thalamocortical axons. In rats, there are sparsecelled regions between barrels called septa. Inter-barrel septa together with regions surrounding the barrel field form the dysgranular zone. There are two main types of neurons in layer IV barrels: spiny stellate and star-pyramidal excitatory neurons, and GABAergic interneurons. Both excitatory and inhibitory neurons receive direct inputs from VPM. Neurons in layer IV heavily project into supragranular layer II/III within the same cortical column (along the barrel). Septal neurons project above septum to layer II/III and to some extent coarsely to surrounding barrels, secondary somatosensory cortex (SII) and primary motor cortex. Some layer IV barrel axons innervate into the adjacent barrels as well. The targets of layer II/III neurons include the adjacent barrel layer II/III, layer V, primary and secondary motor cortices, secondary somatosensory cortex, dysgranular zone, perirhinal temporal association cortex, dorsolateral striatum and the contralateral SI. The laminar organization of neurons along a barrel form functional barrel columns across cortical layers which mainly represent the barrel's principal whisker. Thalamic afferents innervate layer Vb and VI neurons concurrently to layer IV neurons. Their synapses with layer V pyramidal neurons reliably elicit action potentials. Axons of the layer V pyramidal neurons ramify extensively within this layer with ascending collaterals targeting the supragranular layers and descending collaterals projecting to infragranular layer VI . Layer Va is predominantly populated by slender-tufted pyramidal neurons characterized by their slender apical dendrites, while layer Vb is predominantly populated by thick-tufted pyramidal neurons characterized by pyramidal-like somas and thick apical dendrites and the untufted pyramidal cells. The pyramidal neurons in layer Va (both slender and thick-tufted) may function as integrators of lemniscal and paralemiscal thalamic pathways through monosynaptic connections with layer IV spiny stellate neurons. The layer Vb thick-tufted pyramidal neurons mainly project to anterior midbrain and thalamic nuclei, including the posterior thalamus, ZI and APT. These projections maintain the somatotopic organization beyond the cortex. For a detailed recent review of the neuroanatomy and physiology of the layer V refer to. Layer VI is the main source of corticothalamic feedback projections. Corticothalamic neurons, in addition to projections to sensory thalamic nuclei, ramify both excitatory and inhibitory neurons in layer IV as well as pyramidal neurons in layer Va. Paired whole-cell recordingand laser scanning photo-release of caged glutamaterevealed layer VI inter-laminar input and output are weak. However, repetitive optogenetic excitation of layer VI corticothalamic neurons evokes action potentials in layer Va pyramidal neurons as well as fast-spiking interneurons in both layer IV and Va by activating facilitating synapses, while the overall effect on layer IV excitatory neurons is weak excitation or disynaptic inhibition. Layer VIa corticothalamic neurons form aggregated barrel-like structures (called infrabarrels) organized somatotopically align with the layer VI barrels. Corticocortical neurons, on the other hand, predominantly populate between infrabarrels. By optogenetic stimulation of VPM and POm thalamic nuclei,found VIa corticocortical neurons receive strong synaptic input from both VPM and POm, whereas corticothalamic neurons exhibit weaker responses to VPM input and little response to POm. The receptive field properties of neurons in the barrel field are different across layers. The receptive fields in general have an excitatory center and excitatory surround structure; cortical neurons respond vigorously to the corresponding principal whisker as well as to the adjacent/surrounding whiskers with a weaker and delayed response. However, septal neurons similar to their presynaptic POm neurons, respond to multiple whiskers without preference to a certain whisker as principal. Consistent with their pattern of connectivity, layer II/III neurons, show a broader receptive field with a lower response magnitude. Synaptic integration in layer V neurons is more complex, as these neurons receive input from layers II/III, IV, from other pyramidal neurons in the infragranular layers, as well as substantial direct thalamic input. This leads to broad receptive fields and sometimes whisker non-specific response profiles. For a more detailed review on SI laminar organization refer to. Also, for a review on the functional organization of barrel cortex refer to. Across all cortical laminae, increasing the velocity/acceleration of stimuli applied to the principal whisker increased the amplitude of excitatory post synaptic potentials (EPSPs) and decreased their latency to peak. The changes in the EPSP were accompanied by a transient increase in the spiking activity of cortical neurons, typically followed by a rapid decline (within 10-20 ms of the response onset) to a lower level of tonic spiking rate. The synaptic response of supragranular (layer II/III) and infragranular (layer V and VI) neurons was on average delayed with respect to that of the granular (layer IV) neurons, but see. The peak of the spiking response of Layer IV neurons was followed by infragranular neurons' response peak and then by the response peak of layer II/III neurons. Layer IV neurons exhibit a short integration window of a few milliseconds compared to other layers. These findings suggest that layer IV neurons function as coincidence detectors, whereas supra-and infragranular circuits function as input integrators. Layer V neurons are proposed to integrate lemniscal and paralemniscal inputs in addition to inputs from most or all cortical layers. Layer IV, III and II, on the contrary, might operate as functionally segregated circuits contributing to separate lemniscal and paralemniscal processing streams. The sequence of cortical activation across layers is consistent with interlaminar interacortical local field potential recordings and current source analysis which exhibit early current sinks in layer IV followed by activation of layers II/III and V. Multi-electrode array electrophysiology from SI neurons revealed whisker deflection stimulation quenches trial-by-trial variability; the Fano factor, defined as the ratio of the variance of neuronal responses to their average, decreased as the stimulus intensity (and hence the population activity) increased. This decrease is consistent with previous findings in areas V4and MT, premotor cortex, and superior temporal sulcusof monkeys (for a detailed review see. Stimulation quenches the correlation in trial-to-trial variability between neurons (noise correlation). Noise correlation is usually characterized in terms of the correlation coefficient of the spike counts for pairs of neurons. Using principal component analysis of neuronal responses,extended this measure to neuronal populations of larger than 2 neurons (see. The functional connectivity map constructed based on the strength of pairwise correlations of ongoing spontaneous activity of urethane-anesthetized rats recorded using 10 × 10 array of electrodes predicted the anatomical arrangement of electrodes on the sensory cortex. Neurons with stronger correlations to the population during episodes of spontaneous activity, carried higher information about the sensory stimuli in their evoked response. It is, however, not clear whether this higher level of correlations is due to common input from thalamus or originates from the cortical circuitry. Moreover, the correlation profile of electrode pairs during spontaneous activity predicted both signal and noise correlations during sensory stimulation. It has been demonstrated that barrel cortex neurons in anesthetized rats robustly encode the velocity of whisker motion. The whisker motion features that these neurons encode form a common low-dimensional feature subspace of whisker motion, comprising linear combination of whisker velocity and position, and to a lesser extent whisker acceleration.recently demonstrated that the feature encoding properties of cortical neurons differ depending on the level of spatial correlation in multi-whisker sensory stimuli. In addition to velocity, cortical neurons in the whisker-related area of SI exhibit directional selectivity. The feedback projections from infragranular layers to the vibrissae-related thalamic sensory nuclei consist of three main routes: ## Neurons in the upper part of layer vi of a barrel exclusively project to the corresponding barreloid in VPMforming a reciprocal barreloidbarrel connection. 2. Neurons in the lower part of layer VI project to POm and also a major proportion of these axons make collaterals in VPM to form rostro-caudal rod-like bands representing an arc of vibrissae. 3. The corticothalamic projections of layer V cells exclusively terminate in POm . The axons originated from layer VI along the inter-barrel septa exclusively target POm . The Layer VI corticothalamic axons, but not those of layer V give off collaterals in the reticular nucleus while traversing it. The primary somatosensory cortex projects to the secondary somatosensory cortex, the primary motor cortex (MI), thalamus sensory nuclei, superior caliculus and dorsolateral neostriatum . Also, the barrel cortices on two hemispheres are linked by a callosal connection. In turn, primary somatosensory cortex receives inputs from the secondary somatosensory cortex and motor cortex. Unlike in primates, little is known about the functional properties of the secondary somatosensory cortex in rodents, and this knowledge is limited to anesthetized preparations. ## Parallel ascending subcortical routes for whisking and touch signals to cortex The whisker information from trigeminal complex is channeled to cortex through three parallel pathways, also see: 1. The lemniscal pathway is the major pathway through which the touch signal is channeled to cortex. This pathway includes ipsilateral PrV barrelettes to contralateral VPMdm barreloids to cortical barrel columns layer IV and sparsely to Layer VI in SI. The lemniscal pathway conveys a combination of touch and whisking signals and is speculated to represent the "what" pathway (analogous to the ventral stream in the visual system). 2. The paralemniscal pathway channels the sensory information from rostral part of alaminar spinal trigeminal nucleus (nucleus interpolaris or SpVi) into the thalamic posterior medial nucleus (POm), and then to the following cortical areas: layer I and Va of SI, the septal regions, SII, MI and superior colliculus. The paralemniscal pathway primarily conveys whisking signals, which can be employed to form sensory-motor coordination and positional reference signals during exploration/whisking. Hence the paralemniscal pathway is speculated to represent the "where" system in somatosensation in rodents (analogous to the dorsal stream in the visual system). ## The extralemniscal pathway conveys touch information from SpVc and caudal division of SpVi to VPMvl thalamus and then to SII and the septal regions of SI cortex. The lemniscal and paralemniscal pathways interact; the lemniscal pathway has been shown to suppress the paralemniscal pathway through cortically-activated rapid GABAergic inhibitory projections of zona incerta to POm. ## Physiology and function ## Modes of whisker-mediated sensation As in vision where controlled eye movements-saccadesenhance the efficacy of the visual system to browse the environment and extract relevant visual information, rodents sweep their mystacial vibrissae to scan the environment and collect behaviorally-relevant information. A body of literature referred to this purposively information-seeking manipulation of sensory apparatus as "active sensing". In the realm of engineering, however, "active sensing" against "passive sensing" means emitting energy (e.g., in electromagnetic form as in radar or in mechanical form as in sonar) and sensing the reflections of the emitted signal to obtain information about the medium/environment. To avoid this ambiguity, here, I follow the terminology as inwhich categorize the whisker-mediated perception in rodents into two modes: "generative" and "receptive." Whisking is the self-generated exploratory whisker motion through which rodents sense their surrounding environment in the "generative mode." This generative mode of whisking is used in the perception of surface textures, identification of objects and shapes, estimation of distances and localization of objects. As a whisker comes in contact with an object or palpates the object, its instantaneous motion changes following every contact and release from the surface with high acceleration and high velocity-stick-slip events. The sequence of these stickslip events along with the self-generated component of the whisker motion uniquely reconstructs the kinetics of surface and determines the texture of a surface, or the shape or location of an object. A body of research has focused on quantification of behavioral capacities and characterization of whisker motion and its consecutive neuronal activity in the generative mode. These include a variety of behavioral tasks or simulated conditions such as texture discrimination, identification of shape and size of objects, distance, gap and aperture width detection, object localizationand natural exploratory whisking. For other paradigms, such as width discrimination described inwhisking may not be essential. However, I categorized such behavioral tasks in the generative mode as they require controlled head positioning and movements. As in vision where fixating the gaze on a focal target provides more accurate visual information, in receptive mode, rats can immobilize their vibrissae to achieve efficient vibro-tactile signal collection from a mobile object. In vision, saccades during a fine visual task such as counting degrade the performance. Similarly, there is behavioral evidence that self-generated whisker motion reduces the rodent's performance when detecting vibrations. This aspect of whisker-mediated sensation is less investigated in the literatureand research has been mainly limited to headfixed rodents performing a go/no-go licking task . Recent studies revealed that the response dynamics of cortical neurons changes with the mode of sensation and the behavioral state. The response of cortical neurons to whisker stimuli was suppressed in the generative mode compared to the receptive mode or quiescent state. Likewise, neurons in rat auditory cortex show sensory-evoked response suppression during active behavioral states. Additionally, fluctuations in local field and membrane potentials of layer II/III cortical neurons exhibit prominent slow synchrony during receptive mode. In the generative mode during free whisking, however, membrane potential fluctuations were suppressed and desynchronized across nearby neurons. This cortical state of desynchrony was accompanied by an increase in the spiking activity of thalamocortical neurons. Cutting the sensory peripheral afferents innervating whisker follicles did not affect the generative mode response suppression and desynchrony, indicating that its origin is not peripheral. Pharmacological inactivation of thalamocortical neurons, however, halted the generativemode desynchronization. Consistently, optogenetic stimulation of thalamocortical neurons induced similar desynchronized cortical state. For further details refer to the review article by. ## Behavioral approaches to systems neuroscience: linking circuitry and function How does neuronal activity give rise to sensation and ultimately perception? To what extent does the neuronal readout match the perception of whisker vibration? In order to draw a causal link between neuronal activity and sensorimotor, perceptual, and cognitive functions, it is crucial to develop appropriate behavioral methods and combine them with requisite methods of observation and perturbation of neuronal activity. The behavioral approaches in rodent model system are either based on native forms of natural behavior such as whisking, hence require minimum training-for instance, free navigation or exploration, whisking and aperture or gap crossing-or paradigms embedded in an artificial task and require extensive training of the animal to interact with the environment and express specific behaviors in response to events and stimuliin this context, neutral tactile stimuli such as textures, vibrations or object contacts. The body of literature mainly divides into two forms of behavioral tasks: (i) go/no-go or lick/no-lick, and (ii) two-or multiple-alternative-choice tasks. Go/no-go (or lick/no-lick) tasks are often used in the headfixed preparation predominantly in mice and sometimes in rats. It provides the mechanical stability and a fixed head position ideal for precise whisker stimulation, whisker motion tracking, eye/pupil and gesture tracking, as well as electrophysiology (for instance, intracellular recording) and imaging from cortex (two-photon calcium imaging or voltage-sensitive dye imaging). To prevent learning about timing of the reward as a confounding cue, and to minimize impulsive or anticipatory responses based on the periodicity of the sensory events and reward, go/nogo tasks usually do not have a discrete trial structure, or the initiation of a trial is at random time instances with variable delays. The proportion of the trials followed by no-go should be precisely balanced in order to minimize excessive reinforcement of spontaneous incorrect go choices (false alarms) and to avoid formation of a bias toward go or no-go choices. Other limitations of the go/no-go tasks in head-fixed preparation include no re-enforcement (reward) for correct no-go choices, suppressed vestibular signals which may play a crucial role for coordination of whisking behavior and body movements, and relying on licking behavior with highly reflexive componentsas a representation of a cognitive goal-directed behavior. Using conditioned level-press responses,found that rats with only a single whisker combine touch and whisker movement to distinguish the location of objects at different angular positions along the sweep of whisker. The other limitation of go/no-go head-fixed tasks is the lack of control over motivational factors (e.g., satiation) affecting the likelihood of go choices. The motivation can be controlled by employing a self-initiation mechanism for trials. Go/no-go paradigm is commonly used to quantify the behavioral performances for detection of a stimulus or the detection of changeand discrimination of two sets of stimuli, one associated with go (and hence reward), and one associated with no-go.applied a visuo-tactile detection go/no-go task in freely moving rats with the minimum level of temporal uncertainty; upon the initiation of a trial by nose-poke into a port, the sensory cue (whisker deflection or visual flicker) appeared after a delay of either 300 or 800 ms each of which with equal likelihood. After stimulus onset, the rat had a 500 ms window of opportunity to elicit the go choice and collect the reward. For a hypothetically "logical" rat, the optimal strategy is to detect the sensory stimulus only at the time instance associated to the short delay (300 ms). Upon no detection at 300 ms, the hypothetical rat makes an anticipatory non-sensory go choice at 800 ms, as the hazard rate for stimulus presentation (and hence reward delivery) at 800 ms equals 1 (i.e., absolute certainty). This non-sensory anticipatory strategy explains the faster response time to 800-ms stimulation compared to 300-ms stimulation observed in . Additionally, this strategy predicts a higher proportion of misses for short delay stimulation and higher hit rate for the long delay stimulation (see also. Extracellular array recording from vSI neurons during this task revealed enhanced cortical activity to whisker stimulation with higher expectancy (likelihood compared to visual stimulus). This supports a plausible multiplicative gain modulation of evoked responses or alternatively an additive modulation of baseline activity. This response enhancement may be induced by expectation or attentional factors, motor preparation or sensory events related to motor output (as the task lacks a delay after stimulus presentation to withhold the go choice and to separate stimulus presentation from choice), decision processes and motor output. This is a common drawback in go/no-go, and in particular, lick/no-lick paradigms. In contrast to go/no-go tasks in which it is difficult to distinguish a lack of motivation or lapses of attention from false rejections or correct rejections, two-alternative-choice tasks provide a clear distinction of correct, incorrect, and missed trials. Two-or multiple-alternative-choice tasks can be divided into two main categories: sensory discrimination/comparison and categorization tasks . In sensory discrimination tasks, every trial includes presentation of two stimuli. Discrimination/comparison tasks take two forms depending on the association of the two choices with the stimuli. In the "comparative" discrimination , the task is to compare an attribute of the two stimuli against each other [e.g., roughness of textures , frequency, magnitude (Adibi and Arabzadeh, Adibi Rodent Whisker-Mediated Touch System FIGURE 4 | The two-alternative-choice behavioral tasks in rodents. (A) Schematic representation of the comparative discrimination paradigm. On every trial, two vibrations S i and S j were presented. (B) Four rats were trained in the detection/discrimination task to identify the vibration with the higher amplitude. The neuronal performance is the average performance (based on the area under ROC) across single-units (n = 35) and multiunit clusters (n = 58) from. For each neuron, the stimulus intensity whose detection performance was closest to 60% was chosen as detection threshold (Th). The stimuli corresponding to 1 2 −, 1 1 2 −, and 2-fold Th were then selected for estimating the discrimination performances. The same threshold of 60% defined as detection threshold for rats. The rats performed the comparison task between 0 − Th, 1 2 − 1 1 2 and Th − 2Th. Error bars indicate standard error of means across rats or neurons. (C) Schematic representation of the categorical discrimination paradigm. Stimuli were defined as either S+ or S−. In each trial, one of the two vibrations was S+ and the other was S−. Having identified the S+ vibration, the rodent expressed its choice by turning toward the corresponding drinking spout. (D) (Left) Stimulus space. Each circle represents the frequency-amplitude combination of one stimulus. Two groups of rats were trained in the task. For one group (top-left), two frequencies (f = 80 Hz and 2f = 160 Hz) and three amplitudes ( 1 2 A = 8 µm, A = 16µm, and 2A = 32 µm) were used to generate five vibrations, and for second group (bottom-left) three frequencies ( 1 2 f = 40 Hz, f = 80 Hz and 2f = 160 Hz) and two amplitudes (A = 16 µm and 2A = 32 µm) were used to generate five vibrations. Stimuli that were presented together and had to be discriminated (paired stimuli) are connected by lines. The right panel shows the proportion of correct trials (performance) for the corresponding four stimulus-pairs averaged across rats. Error bars are s.e.m. across rats. Re-plotted from. (E) The schematic representation of the categorization paradigm. The stimuli are divided into two categories of S L and S R , corresponding to left and right choices, respectively. A stimulus S was presented on every trial. The rat identifies the category which stimulus S belongs to. (F) Rats were trained to categorize the orientation of a 9.8 cm-diameter disc with alternating ridges and grooves by licking at one of the two reward spouts. Psychometric functions correspond to two rats trained to categorize orientations 0-45 - as horizontal, and 45-90 - as vertical (green), and another two rats trained to categorize orientations 0-22.5 - as horizontal, and 22.5-90 - as vertical (blue). The curves correspond to a Gaussian cumulative function fitted to data. The dots on each curve represent the perceptual decision boundary of each rat. The blue and green vertical dashed lines represent the categorization boundaries of 22.5 - and 45 - , respectively. 2011;, or duration of two vibrations]. Each outcome of the comparison is associated with one of the two reward ports. The two stimuli may present simultaneously at two distinct positions (e.g., two whiskers, or two sides of snoutor at one position but at distinct time instances. In the "categorical" discrimination , the stimuli are divided into two categories of rewarded/target (S+) vs. unrewarded/distractor (S−). Each trial comprises presentation of one stimulus from each of the two categories. The task is to select the choice associated to the position of the target/rewarded stimulus. In the categorization tasks, the stimuli are divided into two categories, each of which associated with one of the two choices . On every trial, one stimulus is presented, and the task is to identify the category to which the stimulus belongs. Categorization tasks can be considered as a discrimination/comparison task against a reference or boundary dividing the physical feature space of the stimulus into two categories. Alternatively, it can be considered as a mapping of individual stimuli with one of the two choices. Rodents can perform whisker-mediated tactile categorization tasks on sensory attributes such as textures, whisker deflection amplitude pattern, aperture width, locationand orientation (our recent data in , also seeof objects. Discrimination and detection behavioral studies quantify the psychometric response function (the likelihood of the choices as a function of stimulus attribute) which along with the acquisition of neuronal activity allows linking the behavioral function to the neuronal activity. Comparison of the neuronal and psychophysical performances started in the late 1960s in the classic electrophysiological experiments in cat retinaand in the somatosensory cortex. Thereafter, more studies have combined psychophysical and neurophysiological experiments in order to relate neuronal responses to perceptionand decision making. Instead of the traditional comparison of behavioral and neuronal thresholds or sensitivities,compared the non-linearity of the behavioral and neuronal response profiles to the amplitude of vibration. In a series of vibration detection and amplitude discrimination tasks,first quantified the detection threshold of both cortical neurons and rats (denoted by Th, . For near-threshold stimuli with identical amplitude difference, both the neuronal and behavioral discrimination performances surpassed the detection performances . This is consistent with the accelerating nonlinearity of neurometric and psychometric functions at low stimulus intensities. The results revealed the nonlinearity in the neuronal response function predicts behavioral detection and discrimination performances. This study presents the first observation of the "pedestal effect"-frequently reported in human psychophysics-in animal literature. Using the same behavioral detection task,showed rats' behavior indicated a dynamic stimulus sampling whereby stimulus sampling was continued until the stimulus was correctly identified or the rat experienced a false alarm. This is consistent with the recent evidence from texture identification task suggesting similar to primates, rats' choices are governed by bounded integration of primacy-weighted touch-by-touch evidence. Previous electrophysiology studies identified the physical features of whisker motion that are encoded in the activity of cortical neurons to be the product of elemental features of whisker motion, its frequency (f ) and amplitude (A). Consistently, behavioral studies revealed rats are unable to discriminate these elemental features independently of their product; two groups of rats were trained to discriminate either based on the frequency or based on the amplitude of the vibrations delivered to both whisker pads. The stimulus pairs with identical Af product (marked in red, were not discriminable, while the other stimulus pair with the same feature difference in the physical space (marked with orange, were highly discriminable. In both groups, rats' performance in discriminating two stimuli is accounted for by the difference in Af but not by differences in either elemental feature (A and f ) alone. This is consistent with the electrophysiological findings that neurons reduced the dimensionality of the stimulus from two features (A, f ) to a single feature: the product Af. Af defines a real physical property: the speed of whisker motion averaged over cycles. The bridge linking neuronal activity to perception is the readout mechanism of sensory neurons. The interlaced synaptic architecture of neural networks provides strong evidence for decoding by downstream neuronal structures based on "populations" of neurons rather than individual single neurons. Such a synaptic organization together with physiological properties of dendritic processes by which neurons receive information simulates an integration model in which the activity of neurons in the relevant population is summed with different weights. This provides a simple framework to investigate how a biologically plausible ideal observer of neuronal responses, a linear "decoder, " extracts information about the stimuli. Linear decoders are simple in their structure and compatible with the architecture of the brain. With optimizing the weights, it provides an upper limit to the amount of information extractable from neuronal responses. There are two limiting factors affecting the reliability of the neuronal code for sensory stimuli: the response variability of individual neurons to a given stimulus, and covariability (noise correlation) across the neurons. In our previous studies, we characterized the neuronal response statistics in terms of neuronal variability (Fano factor) and co-variability (noise correlation) and parsed out the effect of each of these components on the coding as well as decoding efficiency of cortical populations .further quantified the effect of noise correlations on the optimal linear decoder and characterize the cost of ignoring noise correlations during decoding. ## Motion detection and spatial invariancy in whisker-mediated touch system A majority of neurons across different layers of the rat barrel cortex exhibit multi-whisker receptive fields. The spatial extent of the receptive field of a cortical neuron depends on the intra-cortical connections between barrel columns. Anatomical studies revealed that intra-cortical inter-barrel connections are stronger between barrels within a row , with directionally-biased fiber projections into the anterior barrel. Additionally, intra-cortical projections from septal columns extend two to three barrels along the rows. Consistently, the activity pattern of VPM and cortical neurons to single-whisker deflections is elongated along rows. Electrophysiological studies also revealed that the multi-whisker interaction along rows and arcs is not symmetric. Suppressive two-whisker interactions have been reported to be more prominent during within-row stimulation than during within arc stimulation, while within-arc multi-whisker stimulation yields more supralinear response integration. However, multi-whisker interactions are highly dependent upon the temporal order and timing of the stimulation.demonstrated that the feature encoding properties of cortical neurons changes with the level of spatial correlation in multiwhisker sensory stimuli. In addition to its anatomical and functional importance, the rostro-caudal axis is behaviorally relevant. Through exploratory behavior, rats whisk (move their vibrissae) rostro-caudally, leading to a functional asymmetry between rows and arcs; as the whiskers palpate an object, whiskers within a row contact the object successively relative to their rostro-caudal position in the row, whereas whiskers within an arc usually contact the object nearly simultaneously. Thus, a potential function of within-arc facilitatory interactions might be to boost up the contact signal which is more likely to arise from whiskers within an arc. Alternatively, the spatiotemporal multi-whisker interactions could be an indication of crosswhisker motion detection (e.g., head relative to environment and vice versa) at the level of neurons in the rat primary somatosensory cortex or secondary somatosensory cortex. Simple biologically-plausible models such as the Reichardt model-a correlation detector based on temporal delays-or energy modelsprovide plausible frameworks underlying movement detection in barrel cortex. Such motion detectors are more likely to be identified in SII or in the infra-granular layers of SI where neurons have broad multi-whisker receptive fields. In addition to information about the velocity of moving objects or the ego motion, such motion detectors can provide information about the location of objects with respect to head during whisking or head movements. A recent studyshowed that barrel neurons provide a representation of the position of contacted objects in a coordinate frame that is normalized to the trajectory of the motor output (i.e., phase of whisking). Contact was encoded independently of the angular whisker position and was shown to be invariant with respect to the amplitude and frequency of whisking. The representation of contact in a coordinate system that is dynamically normalized by the motor output provides the basis for encoding the spatiotemporal properties of an externally induced movement. Ledemonstrated that functional principal whisker-the whisker eliciting the strongest response with the shortest latency-differed based on the direction of whisker deflection along the rostro-caudal axis. The stimulus-induced changes in the spatial structure of the receptive field of the neurons was not limited to the principal whisker, and included stimulus-dependent changes in the size, response latency and receptive field center of mass. Although the neuronal mechanisms underlying these dynamic changes are not clear, they suggest invariancy of whisker position through whisking along the rostro-caudal axis; as the rat whisks, the position of the whiskers changes along the rostro-caudal axis with respect to the head leading to potential ambiguity about the position of an object in contact with the whisker. Such a dynamic shift in the receptive fields might help to adjust the position of contact with respect to the head instead of the whisker. This position invariant information can potentially give rise to whisker-mediated coordination, and contribute to spatiotopic representations in grid cellsin the entorhinal cortex, head-direction cells in classic Papez circuitand place cellsin parahippocampal and hippocampal cortices. ## Directional selectivity in whisker-mediated touch system There are several lines of evidence that cortical neurons in the whisker area of SI exhibit directional selectivity. Direction preference is also observed in the response of thalamic and trigeminal neurons. Although the directional selectivity in the periphery and brainstem originates in the uneven arborization of nerve terminals around the follicle, directiondependent differences in the temporal profile of synaptic excitation and inhibition in barrelsand non-linear dendritic processesalso may contribute to the directional tuning in barrel cortex neurons. The directional selectivity decreases along the ascending whiskerto-barrel pathway. The functional and behavioral correlate of directional selectivity in the whisker-to-barrel system is not understood and it is not clear whether rats perceive the direction of vibro-tactile stimulus. However, several lines of research provide evidence against an angular selectivity readout such that leads to a sensation of direction. First, neurons with multiwhisker receptive fields in cortex and thalamus do not necessarily exhibit the same angular preference to different whiskers in their receptive field, but see. Second, in the visual system, orientation selectivity arises from specific convergence of directionally non-tuned thalamic inputs in layer IV of striate cortex and gives rise to selectivity to more complex features along the cortical visual hierarchy. On the contrary, in the whisker-mediated touch system, directional selectivity exists in the peripheral sensory afferents innervating vibrissae follicles and gets weaker along the ascending whiskerto-barrel pathway. Thirdly, in contrast to visual system where the arrangement of neurons across the cortical surface forms a precise "pinwheel"-like orientation preference topographic map, the evidence on a topographic directional tuning map in barrel field of SI is weak and controversial in the literature. While directional preference mapping was observed in VPM, neurons in layer IV barrels exhibit weak direction preference map. Weak correlation between the angular tuning and position of neurons with respect to the center of barrel column was observed in layer II/III of adult rats through tetrode recordingas well as two-photon calcium imaging . However, such an angular preference spatial mapping is absent in supragranular layers in juvenile rats. In layer II/III, two-photon imaging revealed orientation-specific responses were organized in a locally heterogeneous and spatially distributed manner. Additionally, neurons with similar orientation preference exhibited higher correlation in their trialto-trial response variability. Although it has been shown that rats are capable of discriminating between different orientations of an object using all of their whiskers, direction selectivity of single cortical units may or may not contribute to this discrimination. Difference in the kinematics of the contact of multiple whiskers along with the ego head motions could provide the information about the orientation of an object. Thus, the extent to which rats can extract the direction of a vibro-tactile stimulus using only one whisker is not yet known. Recent findings revealed that mice learned to detect optical microstimulation of a sparse group of supra-granular neurons in SI, as well as the difference between temporal patterns of electrical micro-stimulation. As vibrations with different orientation elicit responses in distinct populations of cortical neurons, the rat might be able to use that population information to decode orientation. A key test is to see if rats generalize the learned behavior when stimulus is presented to another whisker. ## Linking cortical function and behavioral context A given sensory stimulus may convey different meanings depending on the time and context of its occurrence, requiring the organism to take different courses of action. Sensory processing also changes with behavioral context: for example, high amplitude oscillations (known as mu rhythm) are observed in sensorimotor areas when subjects are immobile with focused attention. Similar oscillations were observed in membrane potentials recorded from layer II/III neurons of mice SI in receptive mode. In generative mode during free whisking, however, the synchronous fluctuations were suppressed and decorrelated. Beyond the spontaneous oscillations, sensory stimuli delivered to whiskers of awake rats and mice evoked a smaller response amplitude in the generative mode compared to receptive mode. Similar response suppression during active behavior was observed in rat auditory cortex, while a response enhancement was observed in visual cortex. Functional interaction between sensory and motor areas at different behavioral modesand thalamocortical synaptic depressioncould account for changes in the sensorydriven response dynamics during active behavior.reported increased hippocampal theta band oscillations during texture discrimination task compared to a memory task in rats. This was accompanied by an enhanced phaselock synchronization between whisking rhythm, SI neuronal spiking activity and hippocampal theta oscillation. Future paired recordings from primary somatosensory cortex and primary motor cortex or sensorimotor thalamic areas in awake rodents are required to understand the functional role and interaction of these areas in sensory processing and sensation. Cortical neurons process information on a background of ongoing activity with distinct spatiotemporal dynamics forming various cortical states. During wakefulness, cortical state changes constantly in relation to behavioral context, attentional level or general motor activity. A common observation in awake rodents is the rapid change in spontaneous cortical activity from high-amplitude, low-frequency fluctuations referred to as synchronized state (e.g., when animals are quiet), to faster and smaller fluctuations, referred to as desynchronized state (e.g., when animals are active).recently showed this re-organization of the activity of cortical networks strongly affects sensory processing. In the desynchronized state, cortical neurons showed lower stimulus detection threshold, higher response fidelity, and shorter response latency with a prominent enhanced late response. Interestingly, changes in the activity of a small population of locus coeruleus (LC) neurons preceded and predicted the changes in the cortical state: the cross-correlation of the LC firing profile with the cortical state was maximal at an average lag of -1.2 s. ## Link to perception It is not clear how and where in neocortex the perception of the tactile information emerges. However, a prime candidate for perceptual judgments and navigation based on tactile information is the prefrontal cortex (PFC). Somatosensory cortex projects into the dorsal part of medial prefrontal cortex (mPFC)-homolog of primate dorsolateral prefrontal cortex. There are several lines of evidence indicating that in rats, mPFC and in particular its dorsal bank is involved in memory and delayed tasks . Prefrontal cortex also projects to hippocampus both directly and indirectly through lateral entorhinal cortex. The entorhinal cortex gates sensory information to hippocampus and its lesioning impairs spatial representation. Moreover, population dynamics of place-selective grid cells in the medial entorhinal cortex predict adaptive hippocampal remapping. Somatosensory cortex projects to the lateral entorhinal cortex through indirect projections via perirhinal cortex and also via weaker direct projections. This potentially forms an additional pathway of vibrissal information to hippocampus. ## Concluding remarks Recent years have witnessed a revitalization of interest in rodent models not only in systems neuroscience, but also in the whole body of neuroscience research. This revitalization is partly due to availability of an increasingly powerful array of experimental approaches from optogenetics and two-photon imaging to whole-cell and intracellular electrophysiology and labeling that are challenging to apply to their full potential in primates. Availability of a broad range of genetically modified mouse lines offer scientists the tools to precisely target neuronal circuits and specific cell-types to study their function. The flat surface of the cortex in rodents without sulci and gyri along with its relatively small size is an asset for application of the stateof-the-art battery of techniques in observation and perturbation of neuronal activity. While the rodent somatosensory cortex is probably the most studied system in the literature, providing an immense amount of data from genome expression to cell types and neuronal circuitry, yet there is a huge gap in our understanding and knowledge about how this system functions. Filling this gap requires a comprehensive and coordinated drive from multiple disciplines including but not limited to cellular, systems, computational, behavioral and cognitive neuroscience. The somatosensory system is an expert system in rodents. This system comprises one of the major channels through which rodents as nocturnal animals collect information about their surrounding environment, making this system an ideal model system to understand the neuronal computations and their underlying cellular and neuronal mechanisms in information processing and decision making. Recent studies reveal complex cognitive functions in rodent somatosensation previously reported in humans and primates such as evidence accumulation for optimal decision making and forming abstract concepts of noisy stimulation patterns. Yet, further behavioral studies are required to unveil the cognitive abilities in rodents. The role of different connections and areas in this system (see such as vSII, vMI, TRN, ZI, and SC in different contextual and behavioral conditions is yet to be understood. Within cortical areas, the effect of different laminae and a variety of cell typeswithin this architecture on different aspects of sensory processing and behavior is not clear, and requires further investigation in future studies. # Author contributions MA drafted and wrote the manuscript. # Acknowledgments The author would like to thank the members of the Tactile Perception and Learning Lab, SISSA, Italy and the Neural Coding Lab, JCSMR, ANU, Australia, particularly Mathew Diamond and Ehsan Arabzadeh for their comments. The author would like to express gratitude to all people who supported this work at the University of New South Wales and the University of Padova. The author also would like to thank Nelly Redolfi for the inspiration and support to complete this work.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017–2019 seasons ## Analyzed influenza a(h n )pdm and A(H N ) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S N was detected in one A(H N )pdm strain. We also identified NA:I V (n = ) and NA:N K (n = ) in A(H N ) strains. In addition, we performed a molecular screening for NA:H Y in A(H N )pdm samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC values for A(H N )pdm and A(H N ) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H N ) virus presenting with PA:E D AAS. Moreover, the majority of the IAV sequences had the M :S N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis. influenza A virus, resistance, neuraminidase (NA) inhibitors, oseltamivir, baloxavir marboxil, adamantane Introduction Influenza virus (IV) is an important pathogen that causes respiratory infections and a high disease burden worldwide, often presenting as annual epidemics. The acute respiratory and febrile illness is normally self-limiting in healthy people, but it can be especially threatening in high-risk individuals such as elderly people, children, pregnant women, immunocompromised individuals, and patients with underlying chronic diseases. Per year, influenza infections are reported in 1 billion cases and up to 650,000 deaths worldwide [bib_ref] Estimates of global seasonal influenza-associated respiratorymortality: amodelling study, Iuliano [/bib_ref]. Belonging to the Orthomyxoviridae family, IVs are enveloped and contain a segmented genome composed of negative-sense single-stranded RNA. Importantly, influenza A virus (IAV) has the potential for causing pandemics, and its strains are classified according to their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, with the subtypes A(H1N1)pdm09 and A(H3N2) currently being the most relevant to human health. Influenza viruses have a well-characterized seasonal circulation pattern in some parts of the world, with peaks during the coldest seasons (autumn-winter). However, Brazil does not have a homogeneous seasonality pattern, due to its continental dimensions, and each country region can have specific patterns for IVs circulation over the year, which is influenced by the different climates present in the country (3). There are three distinct climatic patterns in Brazil, namely, equatorial, in the north and part of the northeast; tropical, in the northeast, midwest, and southeast; and subtropical (temperate), in the south of the country. In view of this, the influenza detection in the country is distinct by comparing the northern equatorial regions and the southern temperate regions. According to the Brazilian Ministry of Health, in 2019, 1,122 fatalities due to IVs infection were reported [bib_ref] Influenza: Monitoramento até a Semana Epidemiológica 52 de 2015, Secretaria De Vigilância Em Saúde [/bib_ref]. To control the high morbidity and mortality caused by IAV infections, two main strategies are currently employed, namely, the annual vaccination for target high-risk populations and the treatment with antiviral drugs. Even though the vaccination against influenza is the most effective form of prevention, it still faces some challenges, such as the absence of a universal influenza vaccine and the need to update vaccine strains included in the vaccines regularly [bib_ref] Influenza virus: dealing with a drifting and shifting pathogen, Kim [/bib_ref]. In contrast, antiviral drugs are used for the prophylaxis and control of IAV infections. These compounds act directly on some of the IV's essential proteins, interrupting its replicative cycle and presenting benefits, for instance, shortening the duration of the disease symptoms, and improving clinical outcomes if administered early in the infection [bib_ref] Developments in the treatment of severe influenza: lessons from the pandemic of..., Zambon [/bib_ref]. Antiviral drugs are also important because they can act as the first line of defense for the population at particular risk in the event of the emergence of a new IAV strain, since the mass production of an effective vaccine can take months. At present, there are several pharmacological classes of drugs with anti-IV activity approved for use. The adamantanes, such as amantadine and rimantadine, act by blocking the viral M2 ion channel [bib_ref] Shionogi announces European Commission Approval of XOFLUZA R (Baloxavir Marboxil) for the..., Hay [/bib_ref]. Neuraminidase inhibitors (NAIs), represented by oseltamivir (OST), zanamivir (ZAN), peramivir (PER), and laninamivir (LAN), bind to NA and prevent the release of the newly generated virions (9). The drug baloxavir marboxil (BXM), [formula] . /fpubh. . [/formula] which is a cap-dependent endonuclease inhibitor, targets the polymerase acid (PA) protein, one of the three subunits that constitute the IAV RNA polymerase, inhibiting its cap-snatching mechanism, hence inhibiting mRNA transcription and ending viral replication (10). BXM was initially released for use in 2018 and has been approved in over 30 countries around the world, such as Japan, the USA, and the European Union (11). However, due to the constant evolution of IAVs, through genetic drift, mutations may arise in the genes that encode viral proteins targeted by antiviral drugs, leading to amino acid substitutions (AAS), which may reduce the drug's effectiveness. This affected the adamantanes, which have not been recommended for the treatment of IAV infections since the 2005-2006 seasons. AASs such as M2:S31N, which remains fixed in IAVs, led to viral resistance to this pharmacological drug class and showed good viral fitness [bib_ref] Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza..., Bright [/bib_ref]. Despite that, in 2017, a cluster of IAV sensitive to the adamantanes was detected in Australia, but their dissemination has not been observed (13). In relation to NAIs resistance, some AASs in NA correlating with reduced inhibition (RI) to these drugs have been identified. The NA:H275Y was previously detected in high prevalence in the former A(H1N1) viruses that circulated until the end of the 2000s, causing high RI to OST, a fact that impaired the value of this drug (14) at that time. However, the emergence of the pandemic strain A(H1N1)pdm09, which did not have NA:H275Y, allowed the return of OST use to treat those infections. Since then, surveillance of NAIs-resistant strains has been intensified, with the detection of clusters of viruses carrying resistance markers in the USA, Australia, and Japan [bib_ref] Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, Takashita [/bib_ref] [bib_ref] Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors..., Lackenby [/bib_ref]. Despite that, further dissemination of these mutated viruses has not been observed. Thus, in the most recent global reports, the frequency of resistant strains has remained low (<1%) (18). Regarding BXM, which has not yet been approved for use in Brazil, resistance markers in the PA, associated with RI, have been identified during the drug initial trials and in IAVs circulating in some countries, with PA:I38T being the most relevant one (21-24). Brazilian surveillance of NAIs-resistant IAVs has identified A(H1N1)pdm09 strains carrying the markers NA:H275Y, NA:S247N, and NA:I223K, with a low frequency [bib_ref] Oseltamivir-resistant influenza A(H1N1)pdm2009 strains found in Brazil are endowed with permissive mutations,..., Lopes E Souza [/bib_ref] [bib_ref] Susceptibility of Brazilian influenza A(H1N1)pdm09 viruses to neuraminidase inhibitors in the 2014-2016..., Souza [/bib_ref]. IVs antiviral susceptibility surveillance is conducted by the World Health Organization (WHO) through the Global Influenza Surveillance and Response System (GISRS), which comprises six WHO Collaborating Centers (CCs) and 141 institutions in 111 WHO member states. Brazil is part of the GISRS and has an influenza surveillance system (ISS) with 3 national influenza centers (NICs), which are located at the Respiratory Virus and Measles Laboratory of the Oswaldo Cruz Foundation in Rio de Janeiro (Fiocruz/RJ), the Virology Laboratory of the Evandro Chagas Institute in Pará (IEC/PA), and the Respiratory Virus Laboratory of the Adolfo Lutz Institute in São Paulo (IAL/SP). Brazilian ISS comprises sentinel units for influenza-like illness (ILI) and severe acute respiratory infection (SARI) that are spread across the country collect the samples, which are further sent to the Central State Laboratories (LACENs) located in each of the 27 Brazilian states. Then, a subset of the samples received by the LACENs is sent to the NICs [bib_ref] WHO, CDC. Pyrosequencing Analysis Protocol for the Detection of the Substitution at..., Cantarino [/bib_ref]. This study presents the analysis of Brazilian IAVs susceptibility to antivirals in Brazil between 2017 and 2019. We evaluated viral genetic sequences of circulating viruses and performed functional phenotypic analysis by determining the OST concentration that inhibited 50% of NA activity (IC 50 ) of Brazilian IAV isolates from the period. # Materials and methods ## Iav genetic sequences The Brazilian IAV sequences that were available in the EpiFlu platform in the Global Initiative on Sharing Influenza database (GISAID) (https://www.gisaid.org/) were downloaded and further evaluated. For that, we used the following screening criteria: type: A, host: human, region: South America/Brazil, and collection date: from 1 January 2017 to 31 December 2019. Duplicate sequences were removed. The analysis of the presence of antivirals resistance markers was performed by using the FluSurver tool (https://www.gisaid.org/epiflu-applications/ flusurver-app/). ## Samples and data collection Our laboratory is an NIC for WHO and the Brazilian Ministry of Health and, therefore, continuously receives a subset of samples from ISS collected in 9 of 27 Brazilian states. Respiratory secretion samples, previously characterized as positive for IAV in their respective LACENs, collected from 2017 to 2019, were included in this study. These samples were collected through nasopharyngeal swabs or aspirates from patients displaying ILI or SARI. A case of ILI is defined as the presence of fever, even if reported, accompanied by cough or sore throat and, at least, one of the following symptoms: headache, myalgia, or arthralgia, in the absence of another specific diagnosis. In children aged <2 years, it is defined as the presence of fever (even if reported) and symptoms (cough, coryza, and nasal obstruction), in the absence of another specific diagnosis. SARI cases were defined as cases requiring hospitalization and presenting dyspnea or one of the following signs of severity: peripheral capillary oxygen saturation <95%, respiratory distress, or acute respiratory insufficiency. ## Ethics This study was approved by the FIOCRUZ-Oswaldo Cruz Institute (IOC) Ethics Committee under the number 68118417.6.0000.5248. . /fpubh. . ## Pyrosequencing A molecular screening was performed in influenza A(H1N1)pdm09 positive samples in order to identify the most relevant and frequently detected NAIs resistance marker, the NA:H275Y. We selected representative samples by using the following criteria: epidemiological week, Brazilian State, severe and fatal cases, OST administration, and availability of reagents. For that, the RNA of the clinical samples was extracted using a viral RNA mini kit (QIAGEN, USA), according to the manufacturer's instructions, and then an RT-PCR reaction was performed, followed by a pyrosequencing reaction and analysis using the PyroMark TM Q96 ID Platform. The run was performed in single-nucleotide polymorphism (SNP) mode, and analysis of results was performed by both SNP and allele quantification (AQ) modes, as previously described (29). The NA:H275Y substitution is characterized by the punctual exchange of the guanine (G) nucleotide by the adenine (A) nucleotide, where the GTG triplet would be changed to ATG triplet in the analyzed sequence, which was 7 bases long and is contained into NA catalytic site. ## Cell culture and viral isolation Virus isolation was performed for all IAV-positive clinical samples received at the laboratory. Madin-Darby canine kidney (MDCK) cells were cultured in Dulbecco's modified Eagle's medium (DMEM TM ) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin (Gibco, USA), and 100 µg/ml streptomycin (Gibco, USA). After removing the culture medium, 100 µl of the clinical sample was added and incubated at 37 - C in 5% CO 2 in the presence of 2 µg/ml TPCK-treated trypsin (Sigma, USA). Visualization of the cytopathic effect (CPE) for up to 72-96 h post-infection was performed. After observing CPE, the viral isolation was confirmed by using NA activity assay with the chemiluminescent kit NA-STAR (Thermofisher, USA). For the viruses with antivirals resistance markers, we also tried additional passages in cultured cells, even if they do not show any CPE after the first inoculation. ## Functional antiviral assay Determination of OST IC 50 (drug concentration to inhibit 50% of NA activity) was done by measuring the NA activity, in the presence of increasing concentrations of OST, by using the chemiluminescent kit NA-STAR TM (Thermofisher TM ). To this end, NA activity was measured in relative light units (RLUs) on the FLUOstar TM Optima equipment (BMG Labtech TM ). Classification of the susceptibility profile of the isolates was performed according to the WHO criteria for the OST IC 50 values for the IAVs, by comparing the IC 50 median to previously tested viruses (30). Thus, the classification was performed as follows: normal susceptibility, when the IC 50 value is up to 10 times the median value; RI, when the IC 50 value corresponds to an increase of 10-100 times in the median value; and high reduced inhibition (HRI), when the IC 50 value corresponds to an increase in more than 100 times of the median value. # Statistical analysis The median IC 50 of the isolates, 95% confidence interval (95% C.I.), and the analysis of the variance of the medians between isolates, clinical samples, and collection year were calculated using Kruskal-Wallis's test, followed by the Dunn's test for multiple comparisons. All analyses were carried out by using the GraphPad Prism software version 8.0. # Results ## Identification of na mutations associated with nais ri We analyzed the sequences of the NA, the gene of interest for NAIs resistance monitoring, from IAVs that circulated in Brazil in the period of 2017-2019 that were available on GISAID (Supplementary . We evaluated 280 complete NA segments from A(H1N1)pdm09 viruses as well as 450 complete NA segments from A(H3N2) . To complement the surveillance of Brazilian viruses that would pose a threat to the efficiency of the antiinfluenza treatment, a pyrosequencing molecular screening was ## Susceptibility of brazilian iav isolates to ost The gold standard for the determination of the susceptibility of IAVs to antivirals is through the functional analyses of the isolates in direct contact with the drug to determine OST IC 50 . Over the study period, we successfully . As a result, all the tested isolates showed a normal susceptibility profile to OST, according to WHO criteria . It is noteworthy that none of the viruses that presented any NAI RI mutation were successfully isolated as they did not show any CPE during isolation protocols nor had any NA activity detected after cell culture passages. ## Identification of m and pa mutations associated with adamantanes and bxm Further complementing the genetic analysis of the IAVs that circulated in Brazil during the proposed period, we reviewed their M2 and PA gene sequences available in the GISAID database with the objective to identify markers associated with RI to the adamantanes and BXM. A total of 483 M2 gene sequences were evaluated (208 from H1N1pdm09 and 275 from H3N2 viruses) and, as expected, M2:S31N AAS was predominantly found in Brazilian IAV sequences. However, one sample collected from an A(H3N2) strain (A/Brazil/358/2017) had no adamantane resistance marker. This virus was recovered in May 2017 from an 82-year-old female patient who was hospitalized with SARI in Espírito Santo State . In addition, a review of available Brazilian IAV complete PA sequences deposited at GISAID retrieved 464 results (199 from H1N1pdm09 and 265 from H3N2 viruses). Despite not identifying any A(H1N1)pdm09 virus harboring BXM RIassociated mutations, we detected one A(H3N2) PA sequence presenting the PA:E199D AAS (A/Brazil/339/2017) that was collected in May 2017 from a 21-year-old female patient who lived in Paraná State, Brazilian Southern region, and showed mild ILI symptoms . [formula] . /fpubh. . [/formula] # Discussion The availability of antiviral treatments to any emerging and reemerging infectious disease, such as caused by IVs, is critical for public health responses and control of the morbidity and mortality of these infections. In Brazil, influenza treatment is based on the NAIs OST and ZAN, of which oral OST is the most widely available drug to treat these infections, followed by the inhaled ZAN. BXM treatment, which has already been approved in countries including USA, Europe, and Asia, is still not authorized for use in Brazil (10, [bib_ref] Assessing baloxavir susceptibility of influenza viruses circulating in the United States during..., Gubareva [/bib_ref]. This study evaluated the circulation of IAV strains with genetic markers associated with RI to the antivirals NAIs, adamantanes, and BXM, in the period between 2017 and 2019, which circulated in Brazil. Among the mutations detected in this study, the NA:H275Y is the most frequent and relevant marker associated with RI to NAIs. The sample identified with this substitution showed 100% of its amino acid residues altered. Interestingly, this sample was collected on the same day that the individual started treatment with OST, which allows us to speculate that the emergence of AAS was not due to antiviral pressure. Moreover, allele quantification analysis showed that the sample had 100% of its nucleotides mutated at the NA position of AAS, which would suggest that the individual may have been initially infected with a variant virus that was spreading in the community. Despite that, we analyzed additional A(H1N1)pdm09 samples that were collected by the Brazilian ISI from the same region and period and identified no additional mutated strain. When it is present in A(H1N1)pdm09 viruses, NA:H275Y causes a high decrease in the binding affinity between NAIs and NA, which leads to a significant reduction in the effectiveness of OST and PER drugs. It is known that this mutation affects viral fitness and may influence the ability of viral transmission and cell replication in cell culture, as we and others demonstrated [bib_ref] Increased detection in Australia and Singapore of a novel influenza a(H1N1)2009 variant..., Abed [/bib_ref]. Despite that, permissive mutations, such as NA:V241I and NA:N369K, can act to compensate for this loss of fitness and are already incorporated in the circulating IAVs around the world, including Brazil [bib_ref] Oseltamivir-resistant influenza A(H1N1)pdm2009 strains found in Brazil are endowed with permissive mutations,..., Lopes E Souza [/bib_ref]. Before the 2009 pandemic, the NA:H275Y was incorporated into the genome of seasonal influenza A(H1N1) strains circulating at that time, leading to their resistance to OST. However, the pandemic strain A(H1N1)pdm09, which did not contain this mutation and was sensitive to OST treatment, emerged, spread, and replaced the previous seasonal A(H1N1) strain. Since then, the frequency of NA:H275Y identification in these viruses has remained low (18). In addition, we have identified an A(H1N1)pdm09 virus that harbored the NA:S247N marker, which exclusively causes a significant RI effect when it is associated with the NA:H275Y mutation, causing a synergistic effect, increasing the reduction in OST and PER susceptibility (36). Even though they were not jointly detected in our sample, NA:S247N should be closely monitored. In relation to the evaluated A(H3N2) viruses, the AAS NA:I222V, which is associated with the reduction of OST active site hydrophobicity, therefore, decreasing favorable drug interactions (37), was identified in six samples. However, similar to NA:S247N, this AAS has a relevant effect on the NAIs susceptibility strictly when it is associated with NA:E119V in A(H3N2) or NA:H275Y in A(H1N1)pdm09 viruses [bib_ref] Characterization of multidrugresistant influenza A/H3N2 viruses shed during 1 year by an..., Baz [/bib_ref]. Notably, sample A/Parana/340/2017, identified as carrying AAS NA:I222V, was collected 1 day after starting OST treatment, which cannot exclude antiviral pressure for the emergence of this mutation. This sample came from an 8-year-old individual with SARI. Furthermore, we show the identification of NA:N329K, already reported to cause RI to OST and ZAN [bib_ref] Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, Takashita [/bib_ref] [bib_ref] Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors..., Lackenby [/bib_ref]. The functional analysis of OST IC 50 showed that all Brazilian isolates from the period remained sensitive to the drug. We observed that median OST IC 50 was reduced in 2019, in comparison with the previous years, for both influenza A subtypes, which would be an indicator of the stabilization of the OST sensitivity of the IAVs and a further support on its use for the treatment of IAV infections. Future investigations will confirm this hypothesis. Our analyses of the M2 IAV gene sequences showed that most of them had the adamantanes resistance marker M2:S31N, as has globally been reported since the 2005-2006 season [bib_ref] Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated..., Deyde [/bib_ref]. For this reason, the adamantane drugs are no longer recommended for the treatment of IAV infections in Brazil and elsewhere for more than a decade. Despite that, one A(H3N2) strain (A/Brazil/358/2017) did not present any known adamantanes resistance markers. This sample was collected from a 2017 case, when A(H3N2) was the predominant IAV subtype circulating (40). Interestingly, in the same year, A(H3N2) clusters without any adamantanes resistance markers were also detected in Australia, despite this, their spread was not observed (13). During the emergence of IAVs with M2:231M AAS, it was suggested that their dissemination was less related to the selective pressure exerted by the use of adamantane drugs. Instead, they would have emerged spontaneously in viral variants and were fixed through their interaction with additional advantageous mutations located in other parts of the viral genome, probably through hitch-hiking effect (41). Herein, we also revealed that BXM susceptibility AASs in PA were detected in a unique A(H3N2) virus that contained PA:E199D. This substitution is in the same position of a distinct one (PA:E199G) that has been reported in association with a discrete (4.5-fold) RI to BXM [bib_ref] Assessing baloxavir susceptibility of influenza viruses circulating in the United States during..., Gubareva [/bib_ref]. The position 199 of PA is important for this drug interaction. Nonetheless, further studies need to be performed to confirm whether PA:E199D AAS also affects the effectiveness of BXM. It is worth mentioning that this marker was detected 1 year before the first approval of BXM in the world and until now, no PAI has been approved for use in Brazil, suggesting that this AAS has emerged spontaneously in the PA gene. PA is one of the 3 subunits that make up the IAV polymerase, playing a crucial role in the replication cycle of the virus. The gene encoding the PA protein is highly conserved and the emergence of mutations is a rare event. However, some markers of RI to BXM have already been identified, especially at position PA:I38X (T/F/M/S/L/V), the most frequently found (18). The monitoring of IAVs to detect reduced susceptibility to antivirals is an essential activity of the surveillance networks for providing information regarding the continuous use of these compounds. However, this study presents some limitations, such as the number of available virus sequences and isolates, which limited a robust assessment of the prevalence of the mutated viruses. In addition, there is a need for a closer monitoring of individuals in the country who are receiving treatment with these antivirals, especially among the immunocompromised groups. Therefore, there is a need to strengthen the Brazilian ISS to address these limitations. Moreover, there is less consistency in the quantity of data and numbers of samples that are collected and evaluated from distinct Brazilian regions, making it important to increase the representativeness of strain identification in some states to strengthen the planning for national vaccination campaigns. # Conclusion This study, covering IAVs that circulated in Brazil in the period of 2017-2019, reveals a low prevalence of IAVs with genetic markers associated with resistance to the antiinfluenza drugs NAIs and BXM. Our data further demonstrate that the available IAVs isolates from Brazil were sensitive to OST. In addition, the majority of IAVs from the country have the adamantanes resistance markers. Therefore, NAIs remain an option for the control of influenza infections in the country. Monitoring the susceptibility of the viruses to the available treatments is crucial for the guidance of the medical interventions. # Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. # Ethics statement This study was approved by the FIOCRUZ-Oswaldo Cruz Institute (IOC) Ethics Committee under the number 68118417.6.0000.5248. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. [fig] Funding: This project was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing a doctoral grant for JSCCM and a master grant for TCS; Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES), Fundação Oswaldo Cruz, CNPq, and Coordenação Geral de Laboratórios de Saúde Pública (CGLAB) from the Brazilian Ministry of Health. [/fig] [table] TABLE Brazilian: IAVs genetic sequences NA, PA, and M genes available at the EpiFlu database on GISAID by gene and year and amount of AAS found in each year. [/table] [table] TABLE Clinical and: epidemiological characteristics of the cases that were infected by the IAVs carrying AAS associated with antivirals RI. [/table] [table] TABLE IAVs isolates: OST IC median by year. [/table]

Effects of Hand-Washing Facilities with Water and Soap on Diarrhea Incidence among Children under Five Years in Lao People’s Democratic Republic: A Cross-Sectional Study Citation: Noguchi, Y.; Nonaka, D.; Kounnavong, S.; Kobayashi, J. Effects of Hand-Washing Facilities with Water and Soap on Diarrhea Incidence among Children under Five Years in Lao People's Democratic Republic: A Cross-Sectional Study. # Introduction Diarrhea is a leading cause of death among children under five years of age (U5), especially in low-and lower middle-income countries. According to the World Health Organization, approximately 525,000 children U5 die from diarrheal disease each year . Lao People's Democratic Republic (PDR) is a lower middle-income country located in Southeast Asia. In Lao PDR, the mortality rate of children U5 remains high compared to those in neighboring countries such as Thailand, Vietnam, and Cambodia, which is due partly to diarrhea . The Global Burden of Diseases Diarrhoeal Diseases Collaborators estimated that the mortality of diarrheal disease in children U5 was 97.1/100,000, and approximately 1,000,000 episodes occurred in children U5 in Lao PDR in 2015. Hand-washing in everyday life is effective for reducing the risk of diarrhea. A systematic review examined the impact of hand-washing with soap on the risk of diarrheal disease and reported that hand-washing with soap in community settings can reduce the risk of diarrheal disease by 42-47%; therefore, interventions to promote hand-washing can save millions of lives a year [bib_ref] Effect of washing hands with soap on diarrhoea risk in the community:..., Curtis [/bib_ref]. According to another systematic review that assessed the effects of hand-washing promotion interventions on childhood diarrhea, community-based hand-washing promotion in low-and middle-income countries can reduce the incidence of diarrhea in children by approximately a quarter [bib_ref] Hand washing promotion for preventing diarrhoea, Ejemot-Nwadiaro [/bib_ref]. Rapid observations are widely used as a proxy measure of hand-washing practice, assuming that household members practice hand-washing with soap if a specific place for hand-washing is observed in a household with available water and soap [bib_ref] Practical Guidance for Measuring Handwashing Behavior, Ram [/bib_ref]. In a nationwide survey, such as the Multiple Indicator Cluster Survey and the Demographic and Health Survey, rapid observations are widely adopted, since it can be challenging to measure hand-washing practice by direct observation because of the enormous associated costs and times. The Lao Social Indicator Survey II (LSIS II), a household-based nationwide survey that was implemented by the Lao government in 2017, also used rapid observations. In Lao PDR, expanding the coverage of water, sanitation, and hygiene (WASH) facilities has been prioritized especially in rural areas and many WASH projects including open defecation free projects have been implemented. However, very few projects measured project's impact on health outcomes including diarrhea. A comprehensive WASH in school project, which included water supply, sanitation, hand-washing, and behavior change interventions, was implemented in 492 primary schools across 13 provinces between 2013 and 2017 [bib_ref] Intervention Fidelity, and Behavioral Outcomes of a School-Based Water, Sanitation, and Hygiene..., Chard [/bib_ref]. A cluster randomized controlled trial study was done in Saravan province to measure the project's impact on pupil absence, diarrhea, respiratory infection, and soiltransmitted helminth infection. The study found, however, that even among schools with the high level of fidelity and adherence, impact of the intervention was minimal. Thus, the study concluded that WASH in school alone may not achieve significant health gains without concurrent community and household WASH improvements including the improvement of hand-washing [bib_ref] Impact of a school-based water, sanitation, and hygiene intervention on school absence,..., Chard [/bib_ref]. The association between hand-washing facilities with soap and diarrhea incidence among children U5 remains poorly understood in Lao PDR. To the best of our knowledge, two studies have assessed this association in Lao PDR. A cross-sectional study with 297 households in 50 villages in Saravan province examined associations between the presence of household hand-washing facilities and the infection status of diarrhea-causing pathogens among household members, including children U5 [bib_ref] Environmental and spatial determinants of enteric pathogen infection in rural Lao People's..., Chard [/bib_ref]. The study found that the presence of household hand-washing facilities was associated with lower infection rates of viral enteric pathogens and soil-transmitted helminths among household members. Another longitudinal study assessed diarrheal risk factors with 234 households from two villages in Saravan province, including all household members. The study found that the presence of hand-washing with soap near or in the toilet was not associated with self-reported diarrhea episodes. The LSIS II included both rapid observation of the handwashing facilities with soap and diarrhea episodes in children U5 . However, no analysis has been made on the association. Therefore, the present study aimed to assess the association between the presence of household hand-washing facilities with water and soap and diarrhea episodes among children U5 in Lao PDR using data from the LSIS II. We have long conducted research concerning community health in rural Lao PDR. The present study was conducted as a part of a larger study that aims to inform strategies to promote hand-washing with soap in rural Lao PDR. # Materials and methods ## Source of data and sample The present study used data from the LSIS II that were obtained from UNICEF (https://mics.unicef.org/surveys). Out of 11,812 children U5 eligible for the LSIS II, data were available for 11,720 children whose primary caretakers responded to the survey. From the 11,720 children, the present study excluded 316 children because of missing values or "don't know" responses to question items that were relevant to the present study. Overall, the present study included 11,404 children U5 with 9038 primary caretakers in 8640 households (spreadsheet S1: Dataset). ## Outcome variable The outcome variable was the presence of diarrhea illness episodes in children U5 in the two weeks preceding the survey. The outcome was measured by asking the following question to primary caretakers: "In the last two weeks, has (child name) had diarrhea?" ## Main predictor variable The predictor variable of interest was the presence of hand-washing facilities with water and soap. This variable had four categories: (1) hand-washing facilities with both water and soap, (2) hand-washing facilities with water alone, (3) hand-washing facilities without water and with/without soap, and (4) no hand-washing facilities. The variable was developed on the basis of three original variables of the LSIS II: "hand-washing facility," "water availability," and "soap availability." "Hand-washing facility" was measured by the following question item: "Can you please show me where members of your household most often wash their hands?" "Water availability" was measured by the following observation item: observe the presence of water at the place for hand-washing. "Soap availability" was measured by the following observation item: is soap, detergent, or ash/mud/sand present at the place for hand-washing? ## Other predictor variables Based on the LSIS II report and similar studies that examined the factors associated with childhood diarrhea, the present study included the 12 predictor variables below, which were categorized into four levels: the individual, caretaker, household, and village levels [bib_ref] Karadag-Caman, O. Determinants of childhood diarrhea among under-five year old children in..., Berde [/bib_ref] [bib_ref] Hand washing behavior and associated factors in Vietnam based on the Multiple..., To [/bib_ref] [bib_ref] Prevalence of and factors associated with diarrhoeal diseases among children under five..., Aziz [/bib_ref] [bib_ref] Diarrhoea and acute respiratory infections prevalence and risk factors among under-five children..., Siziya [/bib_ref]. The individual-level variables included the sex and age of the child U5 and the supervision by primary caretaker (adequate/inadequate). The caretaker-level variables included the age and educational attainment of the primary caretaker (no formal education, early childhood education/primary/lower secondary, or above). The household-level variables included the number of household members (six or fewer/over six), sanitation facilities (improved/unimproved/no facilities), source of drinking water (improved/unimproved), ownership of domestic animals (yes/no), and household wealth (quartiles). To assess household wealth, an asset-based index was originally built using principal component analysis. The main predictor variable was also included in the household-level variables. The village-level variables included living area (urban/rural) and source water quality determined by the level of Escherichia coli (E. coli) contamination in the three household-based samples per village (<11 colony-forming units (CFUs) in all three samples/≥11 CFUs in one or two samples/≥11 CFUs in all three samples). [fig_ref] Table 1: Characteristics of the study participants [/fig_ref] (Appendix A) presents detailed explanations of these predictor variables. # Statistical analysis Bivariate analyses were conducted to assess the association between the outcome variable and each of the predictor variables using Fisher's exact test or the chi-square test. Multivariate analyses were conducted using mixed-effect logistic regression. In the multivariate analyses, multi-level modeling was used to account for the hierarchical structure of the data: individuals (level 1) were nested within caretakers (level 2), caretakers were nested within households (level 3), and households were nested within villages (level 4). In the multivariate analyses, three models were examined: in Model 1, only the predictor of interest was included. In Model 2, the predictor of interest and the household-level variables were included. In Model 3, all of the predictor variables were included. The likelihood ratio test was used to assess the fitness of these models. The significance level was set at p < 0.05 for all tests. The presence of multicollinearity was assessed using variance inflation factor (VIF) and a VIF > 5 was considered to indicate multicollinearity. These analyses were performed using Stata 14.2 (Stata Corp LP, College Station, TX, USA). # Results ## Characteristics of the study participants Of the 11,404 children U5, 803 (7.0%) children had experienced a diarrhea episode in the two weeks preceding the survey [fig_ref] Table 1: Characteristics of the study participants [/fig_ref]. Approximately half (n = 5805, 50.9%) of the children U5 were male. The number of children in each age group did not differ greatly, ranging from 2187 (19.2%) in 1 year to 2413 (21.2%) in 3 years. The majority (n = 9990, 87.6%) of the children U5 were supervised adequately by their primary caretaker. ## Characteristics of the study participants' households The median age of the primary caretakers (interquartile range) was 28 years (range = 23-33). Of the 9038 primary caretakers, 7148 (79.1%) had completed at least primary education [fig_ref] Table 2: Characteristics of the study participants' households [/fig_ref]. The median number of household members (interquartile range) was six (range = 5-7). Among the 8640 households with at least one child U5, hand-washing facilities were observed in 7815 (90.5%) households, water was available in the hand-washing facilities of 7235 (83.7%) households, and soap was available in the hand-washing facilities of 4279 (49.5%) households. Nearly half of the households (n = 4241, 49.1%) possessed hand-washing facilities with both water and soap available, whereas 2994 (34.7%) households possessed hand-washing facilities with water alone. Meanwhile, 580 (6.7%) households possessed hand-washing facilities without water and with/without soap, and 825 (9.5%) households did not possess any hand-washing facilities. The majority (n = 6062, 70.2%) of the households possessed improved sanitation facilities, while 2350 (27.2%) households did not have any sanitation facilities. Most households had an improved source of drinking water (n = 7106, 82.2%) and owned domestic animals (n = 7122, 82.4%). The number of households in each household wealth quintile was almost the same across quintiles, ranging from 2141 (24.8%) in the fourth group (richest group) to 2176 (25.2%) in the third group (second richest group). ## Characteristics of the study participants' villages Of the 1159 villages included in the LSIS II, 792 (68.3%) were rural villages [fig_ref] Table 3: Characteristics of the study participants' villages [/fig_ref]. In terms of E. coli contamination, the quality of source water was considered to be safe in 159 villages (13.7%), whereas it was considered to be unsafe in 419 villages (36.2%). ## Bivariate analyses The factors significantly associated with diarrhea episodes among children U5 were the sex of the child, the age of the child, the supervision by the primary caretaker, the age of the primary caretaker, the educational attainment of the primary caretaker, the number of household members, soap availability, hand-washing facilities with water and soap, sanitation facilities, source of drinking water, and household wealth [fig_ref] Table 4: Bivariate analyses of the factors associated with diarrhea episodes [/fig_ref]. No significant differences were found for the rest of the variables. ## Multivariate analyses In Model 1, the children whose households possess hand-washing facilities with water alone were significantly more likely to have a diarrhea episode compared to the reference group (i.e., the children whose households possess hand-washing facilities with both water and soap) [fig_ref] Table 5: Multivariate analysis of the association between diarrhea episodes among children under five... [/fig_ref]. This difference remained significant even after adjusting for the other predictor variables in Models 2 and 3. Children whose households possess handwashing facilities without water and with/without soap were significantly more likely to have a diarrhea episode compared to the reference group in Models 1 and 2. However, the association became insignificant in Model 3. There were no significant differences in the odds ratio of diarrhea episodes between children whose households do not possess hand-washing facilities and the reference group. Additionally, in Model 3 there was a significant difference between the reference group and comparison group in the following characteristics; sex of child, age of child, supervision by primary caretaker, sanitation facilities, and household wealth. # Discussion The main finding of the present study was that children whose households possess hand-washing facilities with both water and soap were significantly less likely to experience diarrhea episodes compared to children whose households possess hand-washing facilities with water alone. This finding suggests that in the Lao setting, hand-washing with soap is more effective for preventing childhood diarrhea episodes compared to hand-washing without soap. This finding is important because hand-washing facilities with water are available in most households in Lao PDR. If soap use becomes more common, the mortality and morbidity due to diarrhea could be widely reduced. This main finding is biologically plausible. Analysis of the samples collected at Lao healthcare facilities showed that the major etiologic agents of acute childhood diarrhea are rotavirus, Escherichia coli, and Salmonella spp. [bib_ref] Etiologic agents of acute diarrhea in sentinel surveillance sites in Vientiane Capital, Houattongkham [/bib_ref] , which are transmitted from person-toperson via contaminated hands in households. A community-based study involving 1159 households in rural Lao PDR showed that enteropathogen infections are strongly correlated within members of the same household, suggesting the importance of intra-household transmission [bib_ref] Environmental and spatial determinants of enteric pathogen infection in rural Lao People's..., Chard [/bib_ref]. A randomized controlled trial with volunteers in the U.K. showed that hand-washing with plain soap is more effective for the removal of bacterial pathogens from hands than hand-washing with water alone [bib_ref] The Effect of Handwashing with Water or Soap on Bacterial Contamination of..., Burton [/bib_ref]. A community-based randomized control trial with mothers in Bangladesh also showed that hand-washing with a bar of soap is more effective for reducing the bacterial load of coliforms and Clostridium perfringens compared to hand-washing with water alone [bib_ref] Microbiological Evaluation of the Efficacy of Soapy Water to Clean Hands: A..., Amin [/bib_ref]. Additionally, an experimental study with volunteers in the U.S. showed that hand-washing with hand soap and water is effective for reducing viral contamination from finger pads [bib_ref] Reducing viral contamination from finger pads: Handwashing is more effective than alcohol-based..., Tuladhar [/bib_ref]. The main finding is also consistent with those reported from similar observational studies. A cross-sectional study involving 347 households in rural Bangladesh showed that children U5 whose family members washed their hands with soap after defecation were significantly less likely to experience a diarrhea episode in the 48 h preceding the survey compared to children whose family members washed their hands with water only [bib_ref] The Effect of Handwashing at Recommended Times with Water Alone and with..., Luby [/bib_ref]. A cross-sectional study in Malawi, which used Demographic and Health Survey data, showed that the lack of soap in hand-washing facilities was associated with higher odds of having a diarrhea episode among children U5 [bib_ref] Risk factors of diarrhea of children under five in Malawi: Based on..., Moon [/bib_ref]. In contrast, the main finding of the present study is not consistent with the findings of a study conducted in Saravan province of the Lao PDR. There are two possible reasons for this inconsistency: first, the Saravan study used all household member's diarrhea episodes as the outcome, suggesting that the reason for the discrepancy could be due to methodological differences. Second, the Saravan study included only 46 diarrhea cases as the outcome, suggesting that the study likely suffered from type II errors; i.e., false negatives. Although the effect of soap being present in hand-washing facilities on diarrhea incidence was not large (i.e., 5.9% among children in households with soap vs. 8.1% among children in households without soap), placing soap in hand-washing facilities could widely impact the health of Lao children, as more than one-third of Lao households do not have soap in their hand-washing facilities. Based on the assumption that a household has one child U5, 283,000 out of the total 786,000 children U5 in Lao could benefit from placing soap in hand-washing facilities. Additionally, the use of soap could contribute to preventing not only diarrhea, but also other illnesses including pneumonia, which is also a leading cause of death among Lao children [bib_ref] Effect of handwashing on child health: A randomised controlled trial, Luby [/bib_ref]. The reasons for the absence of soap in hand-washing facilities in many households remain poorly understood in Lao PDR, as no study has been conducted in the country to explore these reasons. The LSIS II report showed, however, that there are some household trends for the absence of soap in handwashing facilities: rural households, households whose heads have lower educational attainment, households of lower wealth quintiles, and households of minority language groups are less likely to have soap in their handwashing facilities compared to their counterparts . A study on hand-washing facilities in 51 countries reported similar trends: universally, households of higher wealth quintiles and urban households are more likely to have soap in their hand-washing facilities, compared to their counterparts [bib_ref] Handwashing in 51 Countries: Analysis of Proxy Measures of Handwashing Behavior in..., Kumar [/bib_ref]. In Lao PDR, however, soap seems to be affordable for many people: the average price of a bar of soap was 3110 kip (approximately 0.34 U.S. dollars) in 2017. Considering these trends and the price of soap, further study is necessary to identify the barriers to placing soap in hand-washing facilities in Lao PDR. The results of the present study also showed that there was no significant difference in the incidence of diarrhea between households with hand-washing facilities where soap and water are available and households without hand-washing facilities. A possible explanation for the lack of a difference is that the households without hand-washing facilities include a substantial proportion of households that live near a community well, and thus household members use the community well as a hand-washing facility. A community-shared well is commonly seen in rural villages of Lao PDR. In fact, wells are a major source of water for housework, including hand-washing, in rural Lao PDR [4]. Additionally, according to the LSIS II survey, in the 66.7% of households their toilet facilities were located not in their houses but in their yards. Thus, there is a possibility that household members have little difficulty in using a community well after defecation, if they live near a community well. Likewise, in the present study there was no significant difference in the incidence of diarrhea between households with improved sanitation facility and households with no sanitation facility. Currently, we are unable to provide a possible reason for the lack of a difference. A multi-country case control study, which assessed sanitation and hygienespecific risk factors for moderate-to-severe childhood diarrhea, also showed that there was no significant difference in the risk of diarrhea between households with private sanitation facilities and households with no sanitation facility in, for example, Bangladesh [bib_ref] Sanitation and Hygiene-Specific Risk Factors for Moderate-to-Severe Diarrhea in Young Children in..., Baker [/bib_ref]. However, the case-control study did not provide any possible reasons. A major limitation of the present study is the absence of information about actual hand-washing practices. It is of concern whether the study participants of the households where soap is available in hand-washing facilities actually use soap, as studies have shown that in settings where soap is available, people do not necessarily use soap when washing their hands before/after critical events such as fecal contact, food preparation, eating, and feeding a child. A school-based study in Lao PDR observed that of the pupils who used the school toilet, only 23.9% washed their hands with soap afterward [bib_ref] Intervention Fidelity, and Behavioral Outcomes of a School-Based Water, Sanitation, and Hygiene..., Chard [/bib_ref]. A multicountry study that evaluated the validity of rapid observation measures of hand-washing practices concluded that the observation of hand-washing materials in hand-washing facilities is a valid measure of hand-washing with soap, although the use of soap is often suboptimal: 27-82% of the primary caretakers of children U5 used soap after possible fecal contact, and overall, they used soap before 24-36% of critical events. Another limitation is that the present study was not able to incorporate all the factors which are reported to be associated with childhood diarrhea episodes in similar studies. Such factors include food preparation practices and child feces disposal practices. For example, a cross-sectional study in Viet Nam showed that the risk of childhood diarrhea was significantly higher among children whose mothers prepared food for cooking somewhere other than the table, compared to children whose mothers prepared food on the table [bib_ref] Survey of Food-hygiene Practices at Home and Childhood Diarrhoea in Hanoi, Takahashi [/bib_ref]. A cross-sectional study using the data of the 2013 Nigerian Demographic and Health Survey reported that the increased risk of childhood diarrhea was significantly associated with unsafe child feces disposal practices of caretakers [bib_ref] Karadag-Caman, O. Determinants of childhood diarrhea among under-five year old children in..., Berde [/bib_ref]. In order to maximize the effect of hand-washing on preventing communicable diseases, merely recommending hand-washing with soap before/after critical events is not enough. The Centers for Disease Control and Prevention recommends the five steps for domestic hand-washing: wetting hands with clean, running water; lathering hands by rubbing hands together with soap; scrubbing hands for at least 20 s; rinsing hands well under clean, running water; and drying hands using a clean towel or air dry hands. A communitybased study with primary caregivers of school children in Zimbabwe demonstrated the importance of these five steps in removing microbial contamination [bib_ref] but how? Microbial effectiveness of existing handwashing practices in high-density suburbs of..., Friedrich [/bib_ref]. A health education intervention study in Hong Kong showed that the five-steps hand hygiene tech-nique was effective in reducing the spread of infectious diseases in the special education school setting [bib_ref] Comparative efficacy of a simplified handwashing program for improvement in hand hygiene..., Lee [/bib_ref]. Because children learn hand-washing from their primary caretakers whose hand hygiene practices are sometimes suboptimal [bib_ref] Intestinal parasites prevalence and related factors in school children, a western city..., Okyay [/bib_ref] [bib_ref] Barriers to and motivators of handwashing behavior among mothers of neonates in..., Parveen [/bib_ref] , health education interventions on hand hygiene to children and caretakers are recommended to promote effective hand-washing. The results of the present study showed that most of the households with children U5 already had improved sanitation facilities (70.2%) and improved source of drinking water (82.2%), whereas only 49.1% of the households with children U5 had hand-washing facilities with water and soap. The results suggest that hygiene education does not keep up with the increased coverage of sanitation and water supply. Therefore, more efforts should be made in promoting hygiene education in Lao PDR. The proportion of the population that use hand-washing facilities with water and soap is one of the indicators for Target 6.2 of Sustainable Development Goal 6: "By 2030, achieve access to adequate and equitable sanitation and hygiene for all and end open defecation, paying special attention to the needs of women and girls and those in vulnerable situations". The present study showed that the proportion of households using hand-washing facilities with water and soap is 49.1%, suggesting that continued efforts are needed to achieve Target 6.2 in Lao PDR. In 2017, globally, 60% of the population had basic hand-washing facilities with water and soap, whereas 22% had limited hand-washing facilities lacking water and/or soap. Therefore, many countries, including Lao PDR, face the same challenge: trying to increase the population using basic hand-washing facilities with water and soap. # Conclusions The absence of soap in hand-washing facilities was associated with higher odds of having a diarrhea episode among children under five years of age. This suggests that hand-washing with soap is effective for preventing childhood diarrhea in Lao household settings. Households can reduce the risk of diarrhea among their children by making soap available in hand-washing facilities. Further study is necessary to inform strategies for increasing the availability of soap in hand-washing facilities in every household of Lao PDR. [fig_ref] Table 1: Characteristics of the study participants [/fig_ref]. Detailed explanation of the predictor variables. # Appendix a ## Variables category details Supervision by primary caretaker This variable has two categories: "adequate" and "inadequate." This variable is an original variable of the LSIS II, where the primary caretaker's response to the following two questions determined the category: (1) "On how many days in the past week was (child name) left alone for more than an hour?" and (2) "On how many days in the past week was (child name) left in the care of another child that was less than 10 years old for more than an hour?" When the primary caretaker's response to questions 1 and/or 2 were one day or more, the supervision was defined as "inadequate" in the LSIS II. Otherwise, the supervision was defined as "adequate." Educational attainment of primary caretaker In the LSIS II, the educational attainment of the primary caretakers was divided the following six categories: "none/early childhood education," "primary," "lower secondary," "upper secondary," "post-secondary/non-tertiary," and "higher." In the present study, "lower secondary," "upper secondary," "post-secondary/non-tertiary," and "higher" were combined into one category, titled "lower secondary or above," because "upper secondary," "post-secondary/non-tertiary," and "higher" accounted for a small proportion of less than 8% for each. Therefore, the educational attainment in the present study could be divided into the following three categories: "no formal education or early childhood education," "primary," and "lower secondary or above." ## Number of household members In the LSIS II, the number of household members was surveyed in a continuous manner. In the present study, the number of household members was dichotomized using the median, i.e., six. Therefore, this variable has two categories: "six or fewer" and "over six." ## Sanitation facilities This variable has three categories: "improved sanitation facilities," "unimproved sanitation facilities," and "no facilities." This variable is an original variable of the LSIS II, where the respondent's response to the following question determined the category: "What kind of toilet facilities do members of your household usually use?" In the LSIS II, "improved sanitation facilities" included flush/pour-flush (to a piped sewer system/septic tank/pit latrine), ventilated improved pit latrine, pit latrine with slab, and composting toilet. "Unimproved sanitation facilities" included flush/pour-flush to an open drain, pit latrine without slab or open pit, hanging toilet, and hanging latrine. ## Source of drinking water This variable has two categories: "improved" and "unimproved." This variable is an original variable of the LSIS II, where the respondent's response to the following question determined the category: "What is the main source of drinking water used by members of your household?" In the LSIS II, "improved" included piped water, tube well/borehole, protected dug well, protected spring, rainwater collection, and packaged/delivered water. "Unimproved" included unprotected well/spring and surface water. ## Ownership of domestic animals This variable has two categories: "yes" and "no." This variable is an original variable of the LSIS II, where the respondent's response to the following question determined the category: "Does this household own any livestock, herds, other farm animals, or poultry?" Household wealth This variable was measured by an asset-based wealth index. The household assets included in the present study were: television, refrigerator, fan, water pump, air conditioner, washing machine, CD/DVD player/home theatre, iron, rice cooker/steam cooker, watch, bicycle, motorcycle/scooter, animal-drawn cart, car/truck/van, boat with a motor, and Tak Tak (two-wheeled tractor with trailer). Principal component analysis was used to assess the weight of these household assets and to build an asset index by which households were divided into quartiles. The first component was used for the asset index, which explained 30.9% of the total variance. The scoring factor of the asset was 0.301 for television, 0.357 for refrigerator, 0.334 for fan, 0.225 for water pump, 0.206 for air conditioner, 0.313 for washing machine, 0.191 for CD/DVD player/home theatre, 0.327 for iron, 0.347 for rice cooker/steam cooker, 0.246 for watch, 0.211 for bicycle, 0.196 for motorcycle/scooter, 0.005 for animal-drawn cart, 0.258 for car/truck/van, 0.044 for boat with a motor, and 0.060 for Tak Tak. ## Area In the LSIS II, area was divided into three categories: "urban," "rural with road," and "rural without road." In the present study, "rural with road" and "rural without road" were combined into one category, titled "rural," according to similar studies [bib_ref] Risk factors of diarrhea of children under five in Malawi: Based on..., Moon [/bib_ref]. In the LSIS II, the definition of urban/rural followed the Lao Statistics Bureau's village register in 2015: a village was classified as urban if it met at least three out of the following five conditions: (a) the village is situated in a district or provincial center, (b) more than 70% of the total households in the village use electricity, (c) more than 70% of the total households in the village use piped water, (d) the village is accessible by road throughout year, and (e) the village has a permanent market that is operating daily. ## Source water quality This variable is a new aggregate variable that represents the quality of the village's source water. The variable was developed for the present study on the basis of the data of household-based water quality testing. In the LSIS II, water quality testing was conducted at three randomly selected households per village, and the colony-forming units (CFUs) of Escherichia coli per 100 mL were counted for each household-based sample. In the present study, the CFUs were dichotomized using a cut-off value of 11, according to a study in Cambodia which reported that diarrhea disease is significantly associated with 11 or more CFUs of E. coli/100 mL of drinking water [bib_ref] Escherichia coli in household drinking water and diarrheal disease risk: Evidence from..., Brown [/bib_ref]. When the test result was <11 CFUs in all three samples, the quality of the village's source water was considered to be of low risk for causing diarrhea. When the test result was ≥11 CFUs in one or two samples, the quality of the village's source water was considered to be of moderate risk. When the test result was ≥11 CFU in all three samples, the quality of the village's source water was considered to be of high risk. Therefore, this variable has the following three categories: <11 CFU in all three samples, ≥11 CFU in one or two samples, and ≥11 CFU in all three samples. [table] Table 1: Characteristics of the study participants (n = 11,404). [/table] [table] Table 2: Characteristics of the study participants' households (n = 8640). [/table] [table] Table 3: Characteristics of the study participants' villages (n = 1159). [/table] [table] Table 4: Bivariate analyses of the factors associated with diarrhea episodes (n = 11,404). [/table] [table] Table 5: Multivariate analysis of the association between diarrhea episodes among children under five and hand-washing facilities (n = 11404). [/table]

Implication of Hemodynamic Assessment during Durable Left Ventricular Assist Device Support Durable left ventricular assist device therapy has improved survival in patients with advanced heart failure refractory to conventional medical therapy, although the readmission rates due to device-related comorbidities remain high. Left ventricular assist devices are designed to support a failing left ventricle through relief of congestion and improvement of cardiac output. However, many patients still have abnormal hemodynamics even though they may appear to be clinically stable. Furthermore, such abnormal hemodynamics are associated with an increased risk of future adverse events including recurrent heart failure, gastrointestinal bleeding, stroke, and pump thrombosis. Correction of residual hemodynamic derangements post-implantation may be a target in improving longitudinal clinical outcomes during left ventricular assist device support. Automatic and timely device speed adjustments considering a patients' hemodynamic status (i.e., with a smart pump) are potential improvements in forthcoming devices. # Introduction Despite considerable improvement in available heart failure-specific medical therapies including beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, aldosterone antagonists, angiotensin receptor-neprilysin inhibitors, and arginine vasopressin type II receptor antagonists, morbidity and mortality in patients with advanced heart failure remain exceedingly high. In addition to mechanical circulatory support technologies including the intra-aortic balloon pump, extra-corporeal membrane oxygenation, and percutaneous axial-flow left ventricular assist device (LVAD), cardiac replacement therapy (heart transplantation and durable LVAD) remains the gold-standard therapy for those with refractory stage D heart failure. Given the considerable shortage of donors' hearts, durable LVAD therapy is a widely-used tool in bridging patients to eventual heart transplantation or as destination therapy in non-transplant candidates. LVAD therapy improves survival in patients with advanced heart failure compared to medical therapies alone; however, readmissions due to various device-related comorbidities remain unsatisfactorily high. In the MOMENTUM 3 trial (Multicenter Study of MagLev Technology in Patients Undergoing Mechanical Circulatory Support Therapy with HeartMate 3), patients with advanced heart failure were randomly assigned to the novel HeartMate 3 LVAD (Abbott, Abbott Park, IL) arm and the prior generation HeartMate II LVADs (Abbott, Abbott Park, IL). Patients in the HeartMate 3 LVAD arm experienced significantly higher survival-free from stroke compared with the HeartMate II LVAD cohort. However, the incidence of several hemocompatibility-related adverse events including gastrointestinal bleeding remained comparably high despite novel pump technology. Currently available durable left ventricular assist devices (reused with approval. Nevertheless, our group recently found that many LVAD patients had abnormal hemodynamics despite showing no apparent clinical symptomology. Here, we defined abnormal hemodynamics as central venous pressure > 12 mmHg, pulmonary capillary wedge pressure > 18 mmHg, and cardiac index < 2.2 L/min/m 2 . Notably, we can measure directly or calculate various other hemodynamic parameters using right heart catheterization, as discussed later. Particularly, many LVAD patients seem to have inappropriately elevated central venous pressure indicative of sub-clinical right heart failure. LVADs are designed to reduce the right ventricular afterload by reducing pulmonary artery pressures, whereas the preload experienced by the right ventricle increases due to the improved systemic circulation. Considerable unloading of the left ventricle and reduction in left ventricular size also modifies the geometric relationship between left ventricle and right ventricle. The increased preload experienced by the unprepared right ventricle as a result of improved systemic circulation may worsen right ventricular function in some patients acutely and others over time. As a result, central venous pressure can remain unchanged following LVAD implantation on average. Notably, the degree of hemodynamic improvements can vary greatly as governed by device speed, existing patient-related factors, and device type. For several reasons, we believe that invasive right heart catheterization should be routinely performed following LVAD implantation. First, many patients may have residual abnormal Nevertheless, our group recently found that many LVAD patients had abnormal hemodynamics despite showing no apparent clinical symptomology. Here, we defined abnormal hemodynamics as central venous pressure > 12 mmHg, pulmonary capillary wedge pressure > 18 mmHg, and cardiac index < 2.2 L/min/m 2 . Notably, we can measure directly or calculate various other hemodynamic parameters using right heart catheterization, as discussed later. Particularly, many LVAD patients seem to have inappropriately elevated central venous pressure indicative of sub-clinical right heart failure. LVADs are designed to reduce the right ventricular afterload by reducing pulmonary artery pressures, whereas the preload experienced by the right ventricle increases due to the improved systemic circulation. Considerable unloading of the left ventricle and reduction in left ventricular size also modifies the geometric relationship between left ventricle and right ventricle. The increased preload experienced by the unprepared right ventricle as a result of improved systemic circulation may worsen right ventricular function in some patients acutely and others over time. As a result, central venous pressure can remain unchanged following LVAD implantation on average. Notably, the degree of hemodynamic improvements can vary greatly as governed by device speed, existing patient-related factors, and device type. For several reasons, we believe that invasive right heart catheterization should be routinely performed following LVAD implantation. First, many patients may have residual abnormal hemodynamics following LVAD support. Second, such abnormal hemodynamics are sometimes a challenge to clinically detect by physical examination alone. Third, as discussed below, such abnormal hemodynamics are associated with future adverse events following LVAD implantation. Recently, several non-invasive procedures to estimate hemodynamic parameters have been studied and clinically implemented. Such methods might prove to be clinically useful when right heart catheterization is not readily available. ## Heart failure Our team recently demonstrated that the presence of abnormal hemodynamics post-LVAD implantation, even without heart failure symptoms, was associated with future instances of clinical volume overload and heart failure recurrence. As one would expect, abnormal hemodynamics reflect insufficient mechanical unloading of the left ventricle, causing symptoms of dyspnea and volume retention due to pulmonary and/or systemic congestion. In addition to the device speed settings, other factors also affect the hemodynamic status of patients with LVADs. The clinical implications of heart failure-specific therapies are strongly validated as a disease-modifying agent in non-LVAD populations, whereas the effect in LVAD patients has not been robustly evaluated. Nevertheless, emerging data have suggested that guideline-directed medical therapies for heart failure may improve clinical outcomes also in LVAD patients. This phenomenon may be at least partially explained by neurohormonal modulation leading to some degree of cardiac reverse remodeling and further improvement of intracardiac hemodynamics. Optimal device positioning also affects the device's ability to effectively unload the left ventricle, with malposition of the inflow cannula associated with an increased risk of future heart failure exacerbations. Lateral displacement of the inflow cannula in the HeartWare LVAD (Medtronic, Minneapolis, MN) might cause abnormal stasis and blood pooling in the left ventricle resulting in inefficient unloading. Further data are needed to better understand the relationship between implant technique and device positioning. hemodynamics following LVAD support. Second, such abnormal hemodynamics are sometimes a challenge to clinically detect by physical examination alone. Third, as discussed below, such abnormal hemodynamics are associated with future adverse events following LVAD implantation. Recently, several non-invasive procedures to estimate hemodynamic parameters have been studied and clinically implemented. Such methods might prove to be clinically useful when right heart catheterization is not readily available. ## Heart failure Our team recently demonstrated that the presence of abnormal hemodynamics post-LVAD implantation, even without heart failure symptoms, was associated with future instances of clinical volume overload and heart failure recurrence. As one would expect, abnormal hemodynamics reflect insufficient mechanical unloading of the left ventricle, causing symptoms of dyspnea and volume retention due to pulmonary and/or systemic congestion. In addition to the device speed settings, other factors also affect the hemodynamic status of patients with LVADs. The clinical implications of heart failure-specific therapies are strongly validated as a disease-modifying agent in non-LVAD populations, whereas the effect in LVAD patients has not been robustly evaluated. Nevertheless, emerging data have suggested that guideline-directed medical therapies for heart failure may improve clinical outcomes also in LVAD patients. This phenomenon may be at least partially explained by neurohormonal modulation leading to some degree of cardiac reverse remodeling and further improvement of intracardiac hemodynamics. Optimal device positioning also affects the device's ability to effectively unload the left ventricle, with malposition of the inflow cannula associated with an increased risk of future heart failure exacerbations. Lateral displacement of the inflow cannula in the HeartWare LVAD (Medtronic, Minneapolis, MN) might cause abnormal stasis and blood pooling in the left ventricle resulting in inefficient unloading. Further data are needed to better understand the relationship between implant technique and device positioning. ## Gastrointestinal bleeding Bleeding, particularly gastrointestinal bleeding, is among the most common comorbidities during LVAD support with an estimated incidence of 25%. Some gastrointestinal bleeding events are refractory to all available therapeutic strategies including octreotide, danazol, and thalidomide in addition to the termination of antiplatelet and anticoagulation therapies. The mechanism of gastrointestinal bleeding is multifactorial. In addition to antiplatelet and anticoagulation therapies, along with acquired von Willebrand disease, inappropriate ## Gastrointestinal bleeding Bleeding, particularly gastrointestinal bleeding, is among the most common comorbidities during LVAD support with an estimated incidence of 25%. Some gastrointestinal bleeding events are refractory to all available therapeutic strategies including octreotide, danazol, and thalidomide in addition to the termination of antiplatelet and anticoagulation therapies. The mechanism of gastrointestinal bleeding is multifactorial. In addition to antiplatelet and anticoagulation therapies, along with acquired von Willebrand disease, inappropriate activation of inflammatory and angiogenesis cascades triggering rises in tumor necrosis factor-alpha and angiopoietin-2 may be the primary mechanism of abnormal arteriovenous malformations in the gastrointestinal tract. This might be the most common cause of LVAD-related gastrointestinal bleeding, as opposed to polyps or gastrointestinal mucosal ulceration. It is plausible that abnormal hemodynamics, particularly systemic congestion due to elevated central venous pressures, are associated with the activation of these systems. Recently, therapies including omega-3 fatty acid and digoxin administration were found to be associated with fewer gastrointestinal bleeding events, possibly due to suppression of these maladaptive cascades. Genetic and dietary factors might also have a considerable impact on angiogenesis activity, given a considerably lower observed incidence of gastrointestinal bleeding in Japanese cohorts. Further molecular biological analyses are warranted to clarify the relationship between hemodynamics, the angiogenesis cascade, and gastrointestinal bleeding. Medicina 2020, 56, x FOR PEER REVIEW 4 of 14 activation of inflammatory and angiogenesis cascades triggering rises in tumor necrosis factor-alpha and angiopoietin-2 may be the primary mechanism of abnormal arteriovenous malformations in the gastrointestinal tract. This might be the most common cause of LVAD-related gastrointestinal bleeding, as opposed to polyps or gastrointestinal mucosal ulceration. It is plausible that abnormal hemodynamics, particularly systemic congestion due to elevated central venous pressures, are associated with the activation of these systems. Recently, therapies including omega-3 fatty acid and digoxin administration were found to be associated with fewer gastrointestinal bleeding events, possibly due to suppression of these maladaptive cascades. Genetic and dietary factors might also have a considerable impact on angiogenesis activity, given a considerably lower observed incidence of gastrointestinal bleeding in Japanese cohorts. Further molecular biological analyses are warranted to clarify the relationship between hemodynamics, the angiogenesis cascade, and gastrointestinal bleeding. ## Stroke The most apparent risk factor for LVAD-associated stroke is uncontrolled systemic blood pressure. This relationship of blood pressure to incident risk of stroke was best defined in the ENDURANCE (A Clinical Trial to Evaluate the HeartWare Ventricular Assist System) Supplemental trial, where elevated mean blood pressure was associated with a higher incidence of stroke during HeartWare LVAD support. Furthermore, abnormal hemodynamics, particularly right heart failure with elevated central venous pressure, may increase the risk of incident stroke. We postulate that an inflammatory cascade activates due to chronic and systemic congestion which may, in turn, increase cerebral vasculature vulnerability and the potential risk of stroke. Another explanation may be a decrease in blood flow through the device because of impaired right ventricular function, contributing to increased stasis of flow and potentially increased risk of clot formation in the device. ## Pump thrombosis Pump thrombosis is one of the major causes of device malfunction that requires device exchange. Abnormal LVAD hemodynamics may also increase the risk of incident pump thrombosis. Similar to other events, the mechanism of pump thrombosis is multifactorial. Sub-therapeutic anticoagulation is the most common cause, though device malposition as defined by the narrow-angle between the inflow cannula and pump body in HeartMate II LVAD, is also associated with an increased risk of pump thrombosis. With a similar pathophysiologic mechanism of clot formation which may increase stroke risk, reduced ## Stroke The most apparent risk factor for LVAD-associated stroke is uncontrolled systemic blood pressure. This relationship of blood pressure to incident risk of stroke was best defined in the ENDURANCE (A Clinical Trial to Evaluate the HeartWare Ventricular Assist System) Supplemental trial, where elevated mean blood pressure was associated with a higher incidence of stroke during HeartWare LVAD support. Furthermore, abnormal hemodynamics, particularly right heart failure with elevated central venous pressure, may increase the risk of incident stroke. We postulate that an inflammatory cascade activates due to chronic and systemic congestion which may, in turn, increase cerebral vasculature vulnerability and the potential risk of stroke. Another explanation may be a decrease in blood flow through the device because of impaired right ventricular function, contributing to increased stasis of flow and potentially increased risk of clot formation in the device. ## Pump thrombosis Pump thrombosis is one of the major causes of device malfunction that requires device exchange. Abnormal LVAD hemodynamics may also increase the risk of incident pump thrombosis. Similar to other events, the mechanism of pump thrombosis is multifactorial. Sub-therapeutic anticoagulation is the most common cause, though device malposition as defined by the narrow-angle between the inflow cannula and pump body in HeartMate II LVAD, is also associated with an increased risk of pump thrombosis. With a similar pathophysiologic mechanism of clot formation which may increase stroke risk, reduced flow through the pump motor due to right heart failure may increase also increase the risk of pump thrombosis. ## Hemodynamic patterns by disease process Several unique hemodynamic patterns vary by disease state including right heart failure, pulmonary hypertension, and aortic insufficiency. ## Right heart failure We should state at first that there is no comprehensive and consistently agreed-upon definition of right heart failure. Right heart failure clinically may manifest simply as worsening systemic congestion, whereas right ventricular function is best evaluated using echocardiography through assessment of tricuspid annular plane excursion and right ventricular fractional area change. As mentioned above, right heart failure remains a highly morbid complication of contemporary LVAD therapy. Despite the pump's purpose to restore systemic perfusion in the failing heart by unloading the left ventricle, adverse right ventricular remodeling resulting from longstanding left ventricular failure is common and challenging to correct by durable mechanical circulatory support alone. Following LVAD implantation, right ventricular preload dramatically increases due to improved systemic circulation. The right ventricle, however, is often unprepared for this drastic increase in flow due to maladaptive structural changes from the afterload of a chronically failing left heart. As a result, right heart failure can become apparent both early and in later periods following LVAD implantation. Furthermore, a decrease in the size of the left ventricle due to mechanical unloading also facilitates a geometrical unbalance between the left and the right ventricle, resulting in further impairment of normal right ventricular contractile mechanics. Elevated central venous pressures in the setting of normal pulmonary capillary wedge pressure are one of the hallmarks of right heart failure and tend to be difficult to correct alone through mechanical unloading. Right heart failure is also associated with above-discussed hemocompatibility-related adverse events including bleeding and systemic thromboembolism. Invasive right heart catheterization provides the clinician valuable information regarding right ventricular performance. Pulmonary artery pulsatility index, which is calculated as a pulse pressure of the pulmonary artery divided by the central venous pressure, is a recently proposed index of right ventricular function, with a cut-point of <1.85 associated with post-LVAD right ventricular failure. Furthermore, we showed that a deep y-descent of the right atrial waveform obtained via right heart catheterization is another surrogate of right ventricular dysfunction, and is associated with echocardiography-derived right ventricular dysfunction along with poor clinical outcomes. ## Pulmonary hypertension Many patients with advanced heart failure have secondary pulmonary (combined pre and post-capillary) hypertension due to chronically elevated left-sided filling pressures. However, many patients seemingly reverse their pre-existing pulmonary hypertension following LVAD implantation due to continuous mechanical unloading of the left ventricle. Nevertheless, there are subgroups of patients that have residual pulmonary hypertension even following LVAD implantation, which is associated with worsening long-term right ventricular dysfunction. Our group recently proposed an index to assess refractory pulmonary vasculature adverse remodeling. We described this as "decoupling", which is defined as a gradient of >5 mmHg between diastolic pulmonary artery pressure and pulmonary capillary wedge pressure. We observed that many patients have decoupling, irrespective of preoperative pulmonary hypertension. The presence of this gradient represents an afterload on the right ventricle that may contribute to progressively worsening right ventricular dysfunction. This ultimately leads to worsening congestion and an increased risk of hemocompatibility-related adverse events as discussed above. Of note, there is no general consensus about the best definition of pulmonary vasculopathy in post-capillary pulmonary hypertension, and several other indexes have been proposed, including pulmonary vascular resistance, trans-pulmonary artery pressure gradient, and pulmonary artery compliance. Further studies are warranted to investigate the detailed association among them and their clinical impacts on the LVAD cohort. pressure are one of the hallmarks of right heart failure and tend to be difficult to correct alone through mechanical unloading. Right heart failure is also associated with above-discussed hemocompatibility-related adverse events including bleeding and systemic thromboembolism. Invasive right heart catheterization provides the clinician valuable information regarding right ventricular performance. Pulmonary artery pulsatility index, which is calculated as a pulse pressure of the pulmonary artery divided by the central venous pressure, is a recently proposed index of right ventricular function, with a cut-point of <1.85 associated with post-LVAD right ventricular failure. Furthermore, we showed that a deep y-descent of the right atrial waveform obtained via right heart catheterization is another surrogate of right ventricular dysfunction, and is associated with echocardiography-derived right ventricular dysfunction along with poor clinical outcomes. . Impacts of y-descent on hemocompatibility-related adverse events during left ventricular assist device (LVAD) support (reused with permission. * p <0.05 by log-rank test. † p <0.05 by Mann-Whitney U test. HRAE, hemocompatibility-related adverse events; GIB, gastrointestinal bleeding. A deep y-descent was defined as y-descent depth >3 mmHg from the mean right atrial pressure. A deep y-descent following LVAD implantation was associated with lower freedom from HRAEs, predominantly due to GIB and stroke. ## Aortic insufficiency Aortic insufficiency is a unique and progressive comorbidity during long-term LVAD support. Continuous left ventricular unloading leads to aortic valve closure and pressure increases in the aortic root via the outflow graft. This can lead to valvular degeneration, and subsequent continuous and eccentric valvular regurgitation. Aortic insufficiency increases left ventricular end-diastolic pressure. This consequently may increase right ventricular afterload (i.e., secondary pulmonary hypertension) leading to more strain on right ventricular function over time. Aortic insufficiency, like many other LVAD-related complications, is associated with impaired functional capacity and increased mortality. Notably, aortic insufficiency develops at similar historical rates even in patients with the most contemporary devices (HeartMate 3). The severity of aortic insufficiency is assessed usually using conventional color Doppler echocardiography for visual estimation. Accurate quantification is challenging given non-physiologic continuous and eccentric regurgitant flow. Our group recently proposed several methods to more accurately quantify the severity of aortic insufficiency. First, we can estimate the degree of aortic insufficiency using a device flow monitor, which is equipped in the HeartWare LVAD. This stems from the concept that elevated left ventricular diastolic pressure results in increased device flow during diastole. Severe aortic insufficiency equalizes the pressure between the aorta and left ventricle, minimizing the pressure gradient, resulting in increased pump flow. This allows for a non-Doppler modality to estimate aortic insufficiency. Second, we can quantify the severity of aortic insufficiency using Doppler echocardiography obtained at outflow graft. The concept is similar to the above-described flow monitor, with the regurgitant fraction estimated from the slope of blood flow. Interestingly, the severity of aortic insufficiency is generally greater when we use these novel methods compared to conventional visual estimation. Using these novel methods, many LVAD patients may have significant aortic insufficiency before clinical symptomology develops. Generally, the only definitive therapies are valve replacement or urgent heart transplantation. ## Hemodynamic-guided optimization As discussed above, abnormal hemodynamics are associated with various adverse clinical outcomes during LVAD support. Furthermore, there are several hemodynamically unique comorbidities during LVAD support which have unique and specific management strategies. Our group has recently proposed a hemodynamic ramp test, in which hemodynamics are measured using right heart catheterization at each LVAD speed interval. There are two major purposes of this test. First, we can understand the hemodynamic status at each speed interval from which we can determine the need for medication adjustments. Second, we can adjust the device speed to better optimize the patient's hemodynamic profile with a primary target of right atrial pressure < 12 mmHg, pulmonary capillary wedge pressure < 18 mmHg, and cardiac index > 2.2 L/min/m 2 . We simultaneously perform echocardiography to assess for aortic valve opening, to understand the interventricular septum position, and to determine the presence of mitral valve regurgitation at each speed interval. The Ramp-it-up trial demonstrated the prognostic implications of this specific test in a prospective randomized control setting. New therapeutic targets for other LVAD-related comorbidities are being actively investigated. Data regarding therapies for LVAD-associated right heart failure including oral inotropes or newly developed vasopressin type-2 receptor antagonists are emerging. Pulmonary hypertension might be improved by pulmonary hypertension specific therapies or device speed adjustment. Some patients, however, may continue to have persistently elevated pulmonary artery pressures despite a normalized pulmonary capillary wedge pressure and despite attempts at speed optimization. For patients with severe aortic insufficiency, device speed adjustments are generally not durable as a corrective measure. Transcatheter aortic valve replacement is emerging as a potential salvage therapy in severely symptomatic patients who are not optimal surgical candidates for valve replacement or heart transplantation. ## Non-invasive assessment of hemodynamics The static nature of right heart catheterization is the principal limitation of using hemodynamics in informing hourly and daily decision making for the majority of patients with chronic heart failure. Notably, right heart catheterization testing during exercise might partially overcome such limitations. A patient's hemodynamic status is time-dependent and can change dramatically during a hospitalization. Ideally, hemodynamics should be measured repeatedly or continuously. However, routine invasive assessments in LVAD patients carry a procedural risk given the need for continuous anticoagulation. Herein, non-invasive hemodynamic assessment methods are needed to better guide clinical management of the LVAD patient. ## Cardiomems ## Hemodynamic-guided optimization As discussed above, abnormal hemodynamics are associated with various adverse clinical outcomes during LVAD support. Furthermore, there are several hemodynamically unique comorbidities during LVAD support which have unique and specific management strategies. Our group has recently proposed a hemodynamic ramp test, in which hemodynamics are measured using right heart catheterization at each LVAD speed interval. There are two major purposes of this test. First, we can understand the hemodynamic status at each speed interval from which we can determine the need for medication adjustments. Second, we can adjust the device speed to better optimize the patient's hemodynamic profile with a primary target of right atrial pressure < 12 mmHg, pulmonary capillary wedge pressure < 18 mmHg, and cardiac index > 2.2 L/min/m 2 . We simultaneously perform echocardiography to assess for aortic valve opening, to understand the interventricular septum position, and to determine the presence of mitral valve regurgitation at each speed interval. The Ramp-it-up trial demonstrated the prognostic implications of this specific test in a prospective randomized control setting. New therapeutic targets for other LVAD-related comorbidities are being actively investigated. Data regarding therapies for LVAD-associated right heart failure including oral inotropes or newly developed vasopressin type-2 receptor antagonists are emerging. Pulmonary hypertension might be improved by pulmonary hypertension specific therapies or device speed adjustment. Some patients, however, may continue to have persistently elevated pulmonary artery pressures despite a normalized pulmonary capillary wedge pressure and despite attempts at speed optimization. For patients with severe aortic insufficiency, device speed adjustments are generally not durable as a corrective measure. Transcatheter aortic valve replacement is emerging as a potential salvage therapy in severely symptomatic patients who are not optimal surgical candidates for valve replacement or heart transplantation. ## Non-invasive assessment of hemodynamics The static nature of right heart catheterization is the principal limitation of using hemodynamics in informing hourly and daily decision making for the majority of patients with chronic heart failure. Notably, right heart catheterization testing during exercise might partially overcome such limitations. A patient's hemodynamic status is time-dependent and can change dramatically during a hospitalization. Ideally, hemodynamics should be measured repeatedly or continuously. However, routine invasive assessments in LVAD patients carry a procedural risk given the need for continuous anticoagulation. Herein, non-invasive hemodynamic assessment methods are needed to better guide clinical management of the LVAD patient. ## Cardiomems CardioMEMS was originally developed to monitor pulmonary artery pressures in patients with chronic heart failure using a sensor percutaneously implanted in the main pulmonary artery . Its use is increasing in routine heart failure care, given the reduced risk of heart failure hospitalizations in patients with CardioMEMS-guided therapy as shown in prior randomized clinical trials. The device allows the clinician to remotely monitor pulmonary artery pressures, continuously allowing for precise titration of medical therapies. Pulmonary artery pressures often become abnormal before clinical symptomology develops in patients with chronic heart failure. Thus, risk of heart failure hospitalization reduction in those with the device is likely based on the adjustment of medical therapies before the development of symptomatic congestion. CardioMEMS was originally developed to monitor pulmonary artery pressures in patients with chronic heart failure using a sensor percutaneously implanted in the main pulmonary artery . Its use is increasing in routine heart failure care, given the reduced risk of heart failure hospitalizations in patients with CardioMEMS-guided therapy as shown in prior randomized clinical trials. The device allows the clinician to remotely monitor pulmonary artery pressures, continuously allowing for precise titration of medical therapies. Pulmonary artery pressures often become abnormal before clinical symptomology develops in patients with chronic heart failure. Thus, risk of heart failure hospitalization reduction in those with the device is likely based on the adjustment of medical therapies before the development of symptomatic congestion. . Examples of novel non-invasive technologies to measure hemodynamic parameters (reused with permissioin. ReDS, remote dielectric sensing. The CardioMEMS device might have the potential to be utilized in LVAD patients to monitor and adjust device parameters in response to hemodynamic status. This is of course not without considering important limitations. Right heart failure is one of the comorbidities in LVAD patients where the use of this CardioMEMS may be less helpful as concordant changes in central venous pressures and pulmonary artery pressures do not always occur. Furthermore, patients with significant secondary pulmonary hypertension may have decoupling between diastolic pulmonary artery pressure and pulmonary capillary wedge pressure, also limiting the usefulness of the sensor. ## Remote dielectric sensing (reds) ReDS is another promising tool to noninvasively estimate intra-thoracic fluid levels , which may correlate with intracardiac filling pressures. ReDS employs low-power electromagnetic signals emitted between two sensors (one each on the anterior and posterior body surfaces) embedded in a wearable vest. ReDS has been shown to have high sensitivity and moderate specificity to estimate pulmonary congestion. It may also be used to screen for pulmonary congestion at an early stage for outpatients. However, elevated ReDS value does not necessarily indicate pulmonary congestion, and more precise tests such as right heart catheterization should be considered to distinguish other potential confounders such as pleural fluid accumulation related to pneumonia. Thus far, the clinical implication of ReDS in LVAD patients remains unknown. . Examples of novel non-invasive technologies to measure hemodynamic parameters (reused with permissioin. ReDS, remote dielectric sensing. The CardioMEMS device might have the potential to be utilized in LVAD patients to monitor and adjust device parameters in response to hemodynamic status. This is of course not without considering important limitations. Right heart failure is one of the comorbidities in LVAD patients where the use of this CardioMEMS may be less helpful as concordant changes in central venous pressures and pulmonary artery pressures do not always occur. Furthermore, patients with significant secondary pulmonary hypertension may have decoupling between diastolic pulmonary artery pressure and pulmonary capillary wedge pressure, also limiting the usefulness of the sensor. ## Remote dielectric sensing (reds) ReDS is another promising tool to noninvasively estimate intra-thoracic fluid levels , which may correlate with intracardiac filling pressures. ReDS employs low-power electromagnetic signals emitted between two sensors (one each on the anterior and posterior body surfaces) embedded in a wearable vest. ReDS has been shown to have high sensitivity and moderate specificity to estimate pulmonary congestion. It may also be used to screen for pulmonary congestion at an early stage for outpatients. However, elevated ReDS value does not necessarily indicate pulmonary congestion, and more precise tests such as right heart catheterization should be considered to distinguish other potential confounders such as pleural fluid accumulation related to pneumonia. Thus far, the clinical implication of ReDS in LVAD patients remains unknown. ## Heartware lvad flow slope The HeartWare LVAD provides an estimated instantaneous flow waveform that shows insights into patients and device properties. For example, low pulsatility and low mean flow indicate hypovolemia, whereas low pulsatility and high mean flow let us detect suspected device thrombosis. High pulsatility and low mean flow might indicate continuous suction, whereas high pulsatility and high mean flow indicate volume overload. LVAD flow is determined by the pressure difference between the aorta and left ventricle at a fixed device speed. When aortic pressure is assumed to be constant at the diastole phase, LVAD flow is dependent on left ventricular pressure. When left ventricular pressure increases, LVAD flow often increases. Given this mechanism, pulmonary capillary wedge pressure can be estimated by the slope of LVAD flow at the end-diastolic phase. We showed that the estimated elevated pulmonary capillary wedge pressure calculated from LVAD flow slope was associated with higher heart failure readmission rates. Medicina 2020, 56, x FOR PEER REVIEW 9 of 14 ## Heartware lvad flow slope The HeartWare LVAD provides an estimated instantaneous flow waveform that shows insights into patients and device properties. For example, low pulsatility and low mean flow indicate hypovolemia, whereas low pulsatility and high mean flow let us detect suspected device thrombosis. High pulsatility and low mean flow might indicate continuous suction, whereas high pulsatility and high mean flow indicate volume overload. LVAD flow is determined by the pressure difference between the aorta and left ventricle at a fixed device speed. When aortic pressure is assumed to be constant at the diastole phase, LVAD flow is dependent on left ventricular pressure. When left ventricular pressure increases, LVAD flow often increases. Given this mechanism, pulmonary capillary wedge pressure can be estimated by the slope of LVAD flow at the end-diastolic phase. We showed that the estimated elevated pulmonary capillary wedge pressure calculated from LVAD flow slope was associated with higher heart failure readmission rates. ## Figure 7. Association between left ventricular filling pressure and HeartWare LVAD flow slope. Future advances in durable mechanical support may include a smart pump concept, which can automatically adjust device speed by continuously monitoring hemodynamic data points. For example, a novel smart pump might automatically measure the HeartWare LVAD flow slope and adjust its rotational speed considering estimated intra-cardiac pressure. If the intra-cardiac pressure is estimated to be increased, the device speed would be increased to better unload the left ventricle. # Conclusions Despite durable LVAD support, many patients can have abnormal hemodynamics due to a variety of clinical conditions even when clinically stable. LVAD therapy has improved survival in patients with advanced heart failure, though considerable limitations remain including unacceptably high readmission rates due to these various comorbidities, including volume overload and hemocompatibility-related adverse events. Assessment and optimization of hemodynamics might be one of the modifiable targets which could reduce the burden of these common post-implant complications. Interventions to optimize the hemodynamic status by adjustments of device speed and medications might improve clinical outcomes, though further Future advances in durable mechanical support may include a smart pump concept, which can automatically adjust device speed by continuously monitoring hemodynamic data points. For example, a novel smart pump might automatically measure the HeartWare LVAD flow slope and adjust its rotational speed considering estimated intra-cardiac pressure. If the intra-cardiac pressure is estimated to be increased, the device speed would be increased to better unload the left ventricle. # Conclusions Despite durable LVAD support, many patients can have abnormal hemodynamics due to a variety of clinical conditions even when clinically stable. LVAD therapy has improved survival in patients with advanced heart failure, though considerable limitations remain including unacceptably high readmission rates due to these various comorbidities, including volume overload and hemocompatibility-related adverse events. Assessment and optimization of hemodynamics might be one of the modifiable targets which could reduce the burden of these common post-implant complications. Interventions to optimize the hemodynamic status by adjustments of device speed and medications might improve clinical outcomes, though further large-scale prospective randomized control trials are needed to study these interventions. In the interim, noninvasive methods that estimate hemodynamics, including CardioMEMS, ReDS, and HartWare LVAD waveform analyses, may prove to be useful in more precisely guiding daily LVAD management.

The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding SUPPLEMENTARY RESULTSNucleosome entry-exit salt dependencyIn order to test whether nucleosome conformation changes when bound by TBP, we collected single-molecule traces with FRET pairs between H2B and the DNA at two locations, near the dyad (H2B-15) and adjacent to H2A-H2B (H2B-52), as described previously (63). In these experiments, a change in ionic strength slightly increases the fraction of no FRET species on the DNA-52, but not DNA-15 constructs(Fig. S11). A similar observation is made when adding TBP-TFIIA to nucleosomes at 5 mM KCl, but not at 150 mM KCl. This shows that TBP association has little influence over the nucleosome's global structure.SUPPLEMENTARY METHODSCalculation of Proximity ratio Proximity ratios were calculated by subtracting background, donor bleed-through into the transfer channel, and direct acceptor excitation, as previously described(64); all intensities were first corrected for background. Briefly:Donor bleed-through is determined from donor (D; 488 nm excitation, 500-540 nm emission) and FRET signals (F; 488 nm excitation, 655-685 nm emission) with a DNA containing only donor fluorophore.=(1)Acceptor direct excitation is then determined from acceptor (A; 633 nm excitation, 655-685 nm emission) and FRET signals with a DNA containing only acceptor fluorophore. ## = (2) The corrected intensity in the FRET channel (F corr ), for DNA carrying both donor and acceptor fluorophores, is calculated using the following equation: . TBP nonspecific binding is dependent upon double-stranded DNA. Upper: Shows a DNA construct (TATA-14I ss ) used to determine whether flaking double-stranded DNA is required for nonspecific TBP binding. This construct is 25 bp long, but contains singlestranded DNA adjacent to the TATA-box, which allows TBP to diffuse along the DNA but not compete for specific TATA binding. Lower: spFRET histograms showing unbound (red) or TBP bound (blue; 100 nM) TATA-14I ss DNA. We observe no significant broadening of the distribution widths at this high TBP concentration, indicating a dependence on flanking double-stranded DNA toward non-specific binding. [formula] = ( ) ( )(3 [/formula] [fig] Figure S1, Figure S2, Figure S3: D., Chua, E.Y. and Davey, C.A. (2010) Crystal structures of nucleosome core particles containing the '601' strong positioning sequence. J Mol Biol, 403, 1-10. TATA DNA constructs used for TBP binding to DNA. A) and B) Short 14 bp constructs for testing TBP binding to DNA. Each is end-labeled with donor (green) and acceptor (red) fluorophores. Upon binding by TBP, the DNA is bent giving rise to an increase in FRET signal. (A) TATA-14E and (B) TATA-14E* differ by one sequence change outside of the TBP binding region. C) and D) 25 bp DNA constructs which are 5' end-labeled with a donor fluorophore and internally-labeled with an acceptor fluorophore about the TATA-box. TATA-14I (C) and TATA-18I (D) have 14 bp and 18 bp between FRET pairs with total DNA lengths of 25 bp and 27 bp, respectively. E) Cartoon representation showing how the FRET pair dyes come closer together upon TBP binding. Data from globally plotting and fitting TBP binding to DNA. A-B) showcurves for TBP binding to TATA-14E and TATA-14I, respectively. Data points are averages and error bars represent one standard deviation or a range of two values. Notably, some error bars are too small to be seen. Prepared human TBP binds TATA-14E* stoichiometrically. Shown is a plot of proximity ratio for TATA-14E* upon binding and bending by hTBP, shown as the mole ratio of TBP:DNA. By performing linear regression of each phase of the curve, an intersection point of 1.1 was obtained, signifying 1.1 TBP molecules per DNA fragment. This indicates the TBP is of high spectific activity and only one TBP molecule binds this short DNA fragment. [/fig] [fig] Figure S5, Figure S6, Figure S7: Non-specific contacts are the reason for assymmetric binding curves. (A-C) Binding curves showing TBP binding to TATA-14I DNA at 0 (A), 50 (B), and 150 (C) mM KCl in the absence (-) or presence (+) of unlabeled 35 bp competitor DNA co-titrated in equal molar concentration with TBP. Upon binding, proximity ratio decreases at eleveated TBP concentrations in the absence of competitor, but in the presence of competitor, the curve reaches a saturatable plateau. These data show at high ionic strength that non-specific TBP association is highly subdued and the addition of competitor adds no extra benefit. The sequence of the double-stranded competitor DNA is GGTGCCGAGGCCGTCAATTGCTCG-TAGACAGC, containing no apparent TATA-box sequence. Data are fit to the hill equation for single-site binding. Comparative analysis of TBP kinetic dissociation. A) A comparison of data for the 1 minute time-point shown inFig. 4Afit to either a single or double exponential decay. Residuals are shown for each curve-fit relative to the data are shown above. Residuals highlight the deviation from a single-exponential after short incubation times, indicative of a heterogenious populaiton of molecules. B) Same as in (A) for the 20 minute timepoint. Residuals show comparable results for both single and double exponential, indicating a large majority of the population is in a uniform state. Construct used to test TBP access to nucleosomal DNA A) The 601 positioning sequence was used as a template for TBP binding, where a TATA-box was inserted approximately +5 bp from the last contact point with the nucleosome. The DNA was dual labeled on the end with donor and internally 14 bp away with acceptor fluorophores. An additional 10 bp of DNA flanks the linker arm opposite of the TATA-box. B) A schematic showing the location of the TATA-box based upon the crystal structure of a nucleosome containing 601 DNA from pdb accession code 3LZ0 (65). As can be seen, the minor groove of the TATA-box (magenta) spans the region of first contacts with the nucleosome near the histone H3 N-terminal -helix. Shown are H2A (yellow), H2B (red), H3 (cyan), H4 (green), and DNA (grey). [/fig] [fig] Figure S8: Data from globally plotting and fitting TBP binding to nucleosomes. (A-G) Shown are curves for TBP binding to 601-TATA nucleosomes under different salt and acetylation conditions; (-) and (+) indicate nonacetylated and hyper-acetylated histones, respectively. Unless indicated, histone octamer was acetylated. Data points are averages and error bars represent one standard deviation or a range of two values. [/fig] [fig] Figure S9, Figure S10, Figure S11: Native PAGE gels showing nucleosome preparations used for study. Samples were incubated in RB and loaded onto a 5% native page 0.5X TBE and run at 300 V for 3 hrs. Gels were fluorescently imaged using a typhoon imager with settings for the Atto 633 dye. Samples show high quality with little unassociated DNA and alternative nucleosome species. Abbreviations: Nuc. -Nucleosome, Alt. PCR. -Alternative PCR product. A plot showing the salt-dependent apparent binding affinity of TBP and TFIIA to TATA-601 nucleosomes. Specific TATA-box binding is repressed at elevated salt concentrations. Error bars represent the range of two independent binding experiments. Nucleosome conformation is independent of TBP binding. Nucleosomes were constructed which contained FRET pairs on H2B and positions 15 bp (A,C,E) and 52 bp (B,D,F) from the nucleosome dyad. DNA contained the TATA-box in the identical site as TATA-601 DNA. (A-B) spFRET was measured and histograms generated under 5 mM or 150 mM KCl in the absence of TBP and TFIIA for H2B-15 and H2B-52, respectively. (C-F) spFRET measured on H2B-15 and H2B-52 in the absence or presence of TBP and TFIIA at 5 mM KCl (C,D) and 150 mM KCl (E,F). [/fig]

BCG vaccination strategy implemented to reduce the impact of COVID-19: Hype or Hope? # Introduction Currently, the whole world is busy with combating the coronavirus disease 2019 (COVID- [bib_ref] Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of..., Kleinnijenhuis [/bib_ref] , whose causative agent is Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). This virus poses a significant threat to both the economy and health across the globe. The novel virus spreads primarily via physical/close contact or via respiratory droplets released while coughing or sneezing by an infected individual. The new coronavirus has an irregular and non-uniform pattern of development. Due to this fact, it has infected more than 300,000 individuals in the first three months of the outbreak and has led to more than 13,000 deaths within the same period of time. These figures are still on the rise. SARS-CoV-2 consists of a single-stranded positive-sense RNA genome. It belongs to the Coronaviridae family and is the seventh of its kind known to have infected humans leading to ailments. The other six members of the family, including the SARS-CoV-1 and MERS-CoV (the Middle East Respiratory Syndrome Coronavirus), have also been encountered by the mankind previously, and have displayed relatively higher morbidity than of the novel SARS-virus. SARS-CoV-2 is comprised of a genome that is approximately 30 kb long [bib_ref] Considering BCG vaccination to reduce the impact of COVID-19, Curtis [/bib_ref] nucleotides to be precise). Orf1ab gene known to encode for the non-structural proteins (NSPs) are present at the 5′ end of the genome. The genome also consists of genes encoding structural proteins like a spike (S), membrane (M), nucleocapsid (N), and envelope (E) along with genes encoding the accessory proteins like orf3a, orf6, orf7, and orf10 etc. [bib_ref] A new coronavirus associated with human respiratory disease in China, Wu [/bib_ref] [bib_ref] The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines, Shang [/bib_ref]. Till date, any antiviral or antimicrobial treatments have not shown promising results or been proven to be useful to prevent SARS-CoV-2 infections. WHO does not recommends a treatment involving a combination of more than three antivirals. For now, the treatment options primarily rely on the promising results obtained from studies during previous encounters with SARS, Ebola, MERS, influenza, and other viral infections. ## What is the bcg vaccine? Bacillus Calmette-Guerin (BCG) vaccination has been used against tuberculosis from long back in many countires via their national tuberculosis programs. It holds an inconsistent position of being amongst the most broadly used and most controversial vaccines today. BCG vaccines were first used on man in the year 1921 [bib_ref] BCG vaccination against tuberculosis and leprosy, Fine [/bib_ref]. The protective effect of BCG vaccine against meningitis and disseminated TB in children has been very well documented. The primary use of the BCG vaccine, obtained from Mycobacterium bovis isolate, is the treatment of tuberculosis (TB). BCG vaccine is generally administered to infants intradermally after their birth. The most paradoxical and contended aspect of this vaccine is the varied efficacy found in several clinical trials. BCG vaccination was found to be protective in the case of young children who were not previously infected by the severe forms of tuberculosis [bib_ref] BCG vaccination in India and tuberculosis in children: newer facets, Udani [/bib_ref]. ## Epidemiological studies In a study carried out in Guinea Bissau, a country in West Africa, a 50% reduction in overall morbidity was observed in BCG vaccinated children. It was deduced that this curtailment in overall mortality rate was because of the administration of BCG vaccination, which reduced sepsis and subsequent respiratory infections [bib_ref] Routine vaccinations and child survival: follow up study in, Kristensen [/bib_ref]. Several epidemiological studies reached a parallel conclusion that the countries that adopted the universal policy of BCG vaccination had relatively a reduced death rate caused by COVID-19 to that of the countries which did not put this policy into effect. It was conjectured that BCG vaccination rendering protection against COVID-19 was because of the vaccine's non-specific effects. Reduction in respiratory tract infections in children, antiviral effects, and reduced viremia in experimental animals were attributed to the non-specific effects of the BCG vaccine . 4. BCG vaccination: why is it hope against COVID-19? ## Antiviral effects of bcg vaccine Appurtenant results retrieved from clinicaltrials.gov and PubMed propounded that apart from preventing the propagated types of TB, some strains of BCG vaccine also induced protection against infection caused by other non-mycobacterial pathogens and non related causative agents [bib_ref] Non-specific effects of BCG vaccine on viral infections, Moorlag [/bib_ref]. Evidence obtained from clinical studies and laboratory experiments suggested that BCG vaccination may have non-specific preventive results against viral infections in the case of humans [bib_ref] Randomized trial of BCG vaccination at birth to low-birth-weight children: beneficial nonspecific..., Aaby [/bib_ref]. For example, in order to prevent the infection from the vaccinia virus, enhanced production of interferon-gamma (IFN-γ) from CD4 + cells took place in the BCG vaccinated mice [bib_ref] CD4 T-cell-mediated heterologous immunity between mycobacteria and poxviruses, Mathurin [/bib_ref]. This phenomenon of overproduction of IFN-γ via stimulating the CD4 + cells for the prevention of viral infection is essentially a form of 'adaptive immunity. Several epigenetic, as well as metabolic changes, are responsible for building adaptive immunity. Many genetic regions are promoted for encoding pro-inflammatory cytokines due to the build-up of adaptive immunity [bib_ref] Trained immunity: A program of innate immune memory in health and disease, Netea [/bib_ref]. There is an enhanced secretion of pro-inflammatory cytokines like Interleukin-1β (IL-1β), which is also known as leukocytic pyrogen, upon BCG vaccination. IL-1β plays a crucial role in immunity against viruses [bib_ref] Long-lasting effects of BCG vaccination on both heterologous Th1/Th17 responses and innate..., Kleinnijenhuis [/bib_ref]. ## Prime immune response and non-specific immune response attributed to bcg vaccine BCG mediated prime immune response usually involves the release of chemokines and cytokines which further activates the immune cells at the site in the microenvironment. These mediators released are considered to function in a non-specific pattern or manner. The primary cytokines stimulated by BCG include Interleukin-2 (IL-2), TNF-α (tumor necrosis factor), and IFN-γ (interferon-γ), which are released upon the activation of CD4 + T cells [bib_ref] The immune responses of central and effector memory BCG-specific CD4+ T cells..., Li [/bib_ref]. Antigen presentation to the APCs (antigen presenting cells) is the first and the most crucial step for BCG to act as a powerful therapeutic agent/vaccine resulting in an effective immune response. Activation of T helper cells occurs upon the internalization of BCG by APCs. The activation is carried out with the help of MHC class II molecules which are expressed on the surface of APCs and are recognized by the CD4 + T cells via the T cell receptor (TCR). The interaction between MHC II molecules and TCR are regulated by the binding of co-stimulatory molecules (CD28) to B7-1 on the T cells. The binding of co-stimulatory molecules results in an upregulation of adhesion molecules like LFA-1 (lymphocytes function associated antigens-1) which binds to the macrophages via ICAM-1 (intracellular adhesion molecule-1) [bib_ref] Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of..., Desjardin [/bib_ref]. BCG vaccines are also known to have off-target effects since the 1970s. These non-specified immunomodulatory effects of BCG showed improved immunity in murine models against listeria and influenza [bib_ref] Nonspecific protection of mice against influenza virus infection by local or systemic..., Spencer [/bib_ref] [bib_ref] Correlation of increased metabolic activity, resistance to infection, enhanced phagocytosis, and inhibition..., Ratzan [/bib_ref]. The molecular mechanism of BCG vaccination and its non-specific effects were unveiled in recent studies. These non-specific advantages of the vaccine are mainly due to mTOR and NOD2 mediated changes in the epigenetic landscape of immune cells [bib_ref] Trained immunity: A program of innate immune memory in health and disease, Netea [/bib_ref] [bib_ref] Immunometabolic Pathways in BCG-Induced Trained Immunity, Arts [/bib_ref] [bib_ref] Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of..., Kleinnijenhuis [/bib_ref] [bib_ref] Training innate immunity: the changing concept of immunological memory in innate host..., Netea [/bib_ref]. BCG priming induces a conformational change in the chromatin in adaptive as well as innate immune cells. Immune cells are assisted by these changes and augment immunity against fungal, mycobacterial, bacterial, and viral infections [bib_ref] Trained immunity: A program of innate immune memory in health and disease, Netea [/bib_ref] [bib_ref] Immunometabolic Pathways in BCG-Induced Trained Immunity, Arts [/bib_ref] [bib_ref] Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of..., Kleinnijenhuis [/bib_ref] [bib_ref] Long-term in vitro and in vivo effects of gamma-irradiated BCG on innate..., Arts [/bib_ref] [bib_ref] BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction..., Arts [/bib_ref] [bib_ref] Mycobacterial growth inhibition is associated with trained innate immunity, Joosten [/bib_ref] [bib_ref] Anti-mycobacterial activity correlates with altered DNA methylation pattern in immune cells from..., Verma [/bib_ref]. The production of pro-inflammatory cytokines like TNF-α and IL-1β (interleukin-1β) from mononuclear cells in the peripheral blood was increased strongly upon BCG vaccination in healthy human volunteers. This enhancement in the cytokine production continued for up to 3 months, even when exposed to unrelated pathogens in vitro [bib_ref] Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of..., Kleinnijenhuis [/bib_ref]. As a result of this, human monocytes at the promoter sites of genes which encodes for proinflammatory cytokines underwent epigenetic reprogramming as well as the activation markers like CD11b, TLR4 and CD14 also increased. On the basis of these outcomes, it was hypothesized that increased cytokine production in monocytes induced by BCG may give rise to some better clinical results during a subsequent viral infection [bib_ref] BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction..., Arts [/bib_ref]. It has been demonstrated in various controlled trials that BCG vaccination reduces the severity of infections by several viruses having a somewhat similar structure to that of SARS-CoV-2. For instance, yellow fever vaccine viremia was reduced by 71% due to the administration of the BCG vaccine in volunteers in the Netherlands [bib_ref] BCG Vaccination Protects against Experimental Viral Infection in Humans through the Induction..., Arts [/bib_ref]. The severity of encephalomyocarditis virus (mengovirus) was also significantly reduced in two studies conducted on mice [bib_ref] The Role of the Reticuloendothelial System in the Host Reaction to Neoplasia, Old [/bib_ref]. A study was conducted on BCG vaccinated healthcare workers appointed to take care of the patients suffering from COVID-19. Several clinical trials were run on the healthcare workers using two registered protocols that aimed at perceiving the fact whether the BCG vaccination was enhancing their immunity against SARS-CoV-2 [bib_ref] Anti-mycobacterial activity correlates with altered DNA methylation pattern in immune cells from..., Verma [/bib_ref] [bib_ref] The Role of the Reticuloendothelial System in the Host Reaction to Neoplasia, Old [/bib_ref]. ## Clinical evidences supporting the strategy ## Random trials The impact of BCG vaccine in reducing the chances of respiratory infections in old individuals as well as their prevention was tested in two intended studies. These studies revealed that the risk of pneumonia in old individuals, above the age of 65, was reduced due to BCG vaccination [bib_ref] Prevention of elderly pneumonia by pneumococcal, influenza and BCG vaccinations, Ohrui [/bib_ref]. The acute infections in the upper respiratory tracts were prevented remarkably, when the elderly were administered with BCG vaccine, once a month for 3 months [bib_ref] The efficacy of Bacillus Calmette-Guerin vaccinations for the prevention of acute upper..., Wardhana [/bib_ref]. To study the potential non-specific effects of BCG vaccine in prevention from respiratory infections, four different randomized trials were carried out. In a randomized trial, conducted in West Africa, it was found that the infant mortality due to neonatal respiratory sepsis in low birth weight children, underwent a reduction by 17% at 12 months of age after receiving early BCG vaccination in comparison to the delayed vaccinated children [bib_ref] Randomized trial of BCG vaccination at birth to low-birth-weight children: beneficial nonspecific..., Aaby [/bib_ref]. Another randomized trial in the year 2012 compared two groups (vaccine administered and control), and concluded that BCG vaccination may contribute towards reduced mortality because of lesser deaths from respiratory sepsis/ pneumonia [bib_ref] The efficacy of Bacillus Calmette-Guerin vaccinations for the prevention of acute upper..., Wardhana [/bib_ref]. In 2015, a placebo-controlled randomized trial suggested that the immunogenicity of H1N1 (2009 influenza A pandemic) vaccine was augmented in healthy adults due to BCG vaccination [bib_ref] BCG Vaccination Enhances the Immunogenicity of Subsequent Influenza Vaccination in Healthy Volunteers:..., Leentjens [/bib_ref]. ## Advancing and ongoing clinical trials At present, there are 3 active clinical trials going on to find out whether the BCG vaccination prevents SARS-CoV-2 infection in healthcare workers involved in the care of COVID-19 patients. A registered study on clinicaltrials.gov, entitled "BCG Vaccination to Protect Healthcare Workers Against COVID-19 (BRACE)" (clinical trial number:NCT04327206) is a two-group, multicentre, phase III randomised controlled trial involving 10,078 healthcare workers to find out whether BCG vaccination reduces the severity and occurrence of COVID-19. Another registered study "Reducing Health Care Workers Absenteeism in COVID-19 Pandemic Through BCG Vaccine (BCG-CORONA)" (clinical trial number: NCT04328441) primarily aims to reduce the absenteeism among healthcare workers with direct patient contacts during COVID-19's epidemic phase and to reduce ICU/hospital admission or death in healthcare workers with direct contacts with patients during COVID-19's epidemic phase. In the third registered study, entitled "BCG Vaccine for Health Care Workers as Defense Against COVID-19 (BADAS)" (clinical trial number: NCT04348370), researchers hypothesize that BCG vaccination can reduce infection and severity of disease in healthcare workers during the epidemic phase of SARS-CoV-2. Their hypothesis was based on the findings of several in vivo and in vitro studies, according to which, the BCG vaccine was able to prevent several respiratory tract infections and reduced the mortality and morbidity by percentage as high as 70%. ## Bcg vaccination against covid-19: a presumed hype? Presently, WHO does not recommend the usage of BCG vaccination to treat COVID-19 because of the absence of any concrete evidence suggesting that the vaccine prevents the SARS-CoV-2 infection. Although, it has been observed in the studies and experiments conducted both on animal and human subjects that the vaccine has off-target or non-specific effects (NSEs), the proper classification and clinical relevance of these effects are yet to be discovered. Epidemiological studies reporting less occurrence of COVID-19 in BCG vaccinated infants are regulated by various influential factors such as demographical differences and burden of disease in the country, pandemic phase in each country, and testing rates for SARS-CoV-2 infection in each country. Thus these epidemiological findings are weak evidences and prone to confounding as they are based on population rather than individual data. The fact that a BCG vaccine administered decades ago in childhood will alleviate COVID-19 now is also implausible and questionable because the favorable NSEs of the vaccine might get altered by the subsequent infliction of a different vaccine. One of the main reasons, why it is essential to stick to WHO's recommendation to not use BCG vaccine is that there might be a bare possibility that up-regulation of the immune system by BCG vaccination and its NSEs will aggravate COVID-19 in a minority of patients with severe ailments [bib_ref] Considering BCG vaccination to reduce the impact of COVID-19, Curtis [/bib_ref] [bib_ref] Non-specific effects of vaccines: plausible and potentially important, but implications uncertain, Pollard [/bib_ref]. # Conclusion Due to the lack of strong clinical evidence corroborating the fact that BCG vaccine, possessing various NSEs and anti-viral effects enhancing the immunity against viral infection, might be a possible hope to combat COVID-19. Several randomized controlled trials are in progress in order to evaluate whether the BCG vaccine reduces the severity and occurrence of COVID-19 in healthcare workers. Unless a SARS-CoV-2 specific vaccine is not developed, it is crucial to stick to the WHO guidelines. Administration and of BCG vaccine should be done only under scientific supervision and the vaccine to cure COVID-19 should be only in randomized controlled trials. Acknowledgements KR and SR thanks IIEST Shibpur and Mahatma Gandhi Central University Motihari, Bihar, respectively.Disclosure statementThe authors report no conflicts of interest.

Long non-coding RNA NORAD promotes pancreatic cancer stem cell proliferation and self-renewal by blocking microRNA-202-5p-mediated ANP32E inhibition Background: Cancer stem cells (CSCs) are key regulators in the processes of tumor initiation, progression, and recurrence. The mechanism that maintains their stemness remains enigmatic, although the role of several long noncoding RNAs (lncRNAs) has been highlighted in the pancreatic cancer stem cells (PCSCs). In this study, we first established that PCSCs overexpressing lncRNA NORAD, and then investigated the effects of NORAD on the maintenance of PCSC stemness.Methods: Expression of lncRNA NORAD, miR-202-5p and ANP32E in PC tissues and cell lines was quantified after RNA isolation. Dual-luciferase reporter assay, RNA pull-down and RIP assays were performed to verify the interactions among NORAD, miR-202-5p and ANP32E. We then carried out gain-and loss-of function of miR-202-5p, ANP32E and NORAD in PANC-1 cell line, followed by measurement of the aldehyde dehydrogenase activity, cell viability, apoptosis, cell cycle distribution, colony formation, self-renewal ability and tumorigenicity of PC cells.Results:LncRNA NORAD and ANP32E were upregulated in PC tissues and cells, whereas the miR-202-5p level was down-regulated. LncRNA NORAD competitively bound to miR-202-5p, and promoted the expression of the miR-202-5p target gene ANP32E thereby promoting PC cell viability, proliferation, and self-renewal ability in vitro, as well as facilitating tumorigenesis of PCSCs in vivo.Conclusion:Overall, lncRNA NORAD upregulates ANP32E expression by competitively binding to miR-202-5, which accelerates the proliferation and self-renewal of PCSCs. # Background Pancreatic cancer (PC), a highly fatal disease causing over 200,000 deaths worldwide every year. This high morbidity is due to the tumor's aggressiveness and the lack of markers or symptoms enabling timely diagnosis and treatment. Therefore, the majority of PC patients are diagnosed at a late stage, when tumors have already metastasized towards distant organs. The main causes of the high mortality of PC are cancer resistance to existing therapies, as well as the occurrence of metastasis that precedes diagnosis. In this regard, it is important is to probe the mechanism of PC progression, if we are to develop more effective early diagnosis and treatment methods. As previously demonstrated, the lncRNAs are engaged in a wide range of processes, including proliferation, migration and apoptosis. Besides, the mechanism whereby lncRNAs participate in cancer growth, cancer stem cells (CSCs), and chemoresistance in PC has recently been illustrated. More specifically, the upregulation of Cdc2 by lncRNA SPRY4-IT1 promoted cell proliferative and invasive capabilities in PC, and conversely, the knockdown of lncRNA MIR115HG regulated miR-802 expression to inhibit PC cell viability, and promote cell cycle arrest, and apoptosis. Recently, it has emerged that NORAD can promote the expression of SIP1, thereby inducing the promotion of cell proliferative and invasive abilities in cervical cancer. Moreover, NORAD boosts colorectal cancer cell proliferation, migration, and invasion by means of inhibiting microRNA-202-5p (miR-202-5p) expression. However, much remains to be learned about the effects and biological mechanisms of NORAD in PC According to recent research, miR-202-5p acts as a tumor-suppressor in the context of breast cancerand colorectal carcinoma. Besides, enforced expression of miR-202 is capable of significantly reducing the epithelialto-mesenchymal phenotypic characteristics of parenchymal PC cells. According to the bioinformatics website, miR-202-5p emerged a downstream miRNA for NORAD, while itself potentially targeting ANP32E. Consequently, we designed our investigation with the purpose of verifying the role of the NORAD/miR-202-5p/ANP32E axis in regulating the biology of PC stem cells (PCSCs). # Methods # Ethics statement The Ethics Committee of Shanghai Tenth People's Hospital, Tongji University School of Medicine ratified our study. Written informed consents were acquired from patients before their participation in this study. All experimental methods abided by the Declaration of Helsinki. All animal studies were undertaken in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals issued by US National Institutes of Health. ## Clinical sample collection Cancer and adjacent normal tissues (more than 2 cm away from tumor margins) were surgically acquired from 28 patients (18 males, 10 females; at the age of 39-72 years with a mean age of 54 years) who were pathologically confirmed PC at Shanghai Tenth People's Hospital, Tongji University School of Medicine from May 2016 to December 2017. The patients enrolled had not received either local or systemic treatment prior to the operation. There were nine cases at the stage I, five cases at the stage II and 14 cases at the stage III. The specimens were assessed histopathologically by the hospital pathology department with detailed clinical data were recorded. All specimens were frozen in liquid nitrogen in a quick manner and stored at controlled temperature of − 80 °C for later analysis. ## Cell culture One normal human pancreatic ductal epithelial cell line HPDE6-C7 (HZ-H296; Shanghai Huzhen Biotechnology Co., Ltd., Shanghai, China) and three PC cell lines BxPC-3, MIAPaCa-2, and PANC-1 (ATCC, Manassas, VA, USA; www. atcc. org) were used. These cells were subjected to culture with the RPMI 1640 medium (consisted of 10% FBS, 100 U/mL streptomycin and 100 U/mL penicillin) at controlled temperature of 37 °C under 5% CO 2 , with the medium being renewed every 2 days. Upon growing to 80-90% confluence, cells were passaged, and exponentially growing cells were used for subsequent experiments. The expression of NORAD in cell lines was corresponding negative controls (NCs) by using pCMV-Flag-N/C plasmid vector. The third generation of cells were trypsinized and seeded in plates with 24 wells to form monolayer cells. The cells were divided into two parts and subjected to transfection using Lipofectamine 2000 reagent. One portion of the cells was transduced using miR-202-5p mimic, si-ANP32E and ANP32E, alone or in combination. The other portion of cells was treated with si-NORAD, NORAD, miR-202-5p mimic, either alone or in combination. Finally, after 48-h transfection, to screen stablytransfected cells, cells were maintained for 4 weeks under standard condition in G418 (1000-2000 μg/mL) medium, which was renewed every 3-5 days. ## Bioinformatics prediction and dual-luciferase reporter assay WT luciferase reporter plasmid ANP32E (ANP32E-WT-Luc) containing WT ANP32E sequence and the MUT luciferase reporter plasmid ANP32E (ANP32E-Mut-Luc) containing MUT ANP32E sequence were all from Shanghai Genechem Co., Ltd. (Shanghai, China). Thereafter, 293T cells underwent cotransfection with miR-202-5p mimic or miR-202-5p mimic-NC and reporter vectors with the use of Lipofectamine ™ 2000. After incubation for 24 h, luciferase activity was assessed at 560 nm by Dual-Luciferase Reporter Assay Kit and a microplate reader (Thermo MK3). WT and MUT primers of NORAD were designed and synthesized by Sangon. Total RNA content of PANC-1 cells was extracted and amplified by means of PCR with WT and MUT primers. Hind III and Bgl II enzyme endonuclease sites were added at both ends of the amplified products. pGL3-Basic luciferase reporter vector (Promega) was then digested by restriction endonuclease Hind III/Bgl II, and the large fragments were recovered by electrophoresis. Ligase 4 was linked the amplified target gene and vector to obtain NORAD-WT-Luc and NORAD-MUT-Luc plasmids, which were subsequently transformed into the E. coli competence sequence. After colony identification by PCR, the plasmids were extracted from the colony shaker kit containing the target fragment and sequenced. Other procedures were the same as described above. ## Rna-pull down assay Cells were subjected to transfection with the use of 50 nM biotin-labeled Bio-miR-202-5p-WT and Bio-miR-202-5p-MUT. The cell lysates were incubated with RNase-free BSA and yeast tRNA precoated Dynabeads Streptavidin Magnetic Beads. The enrichment of NORAD was measured by RT-qPCR. ## Rip assay PANC-1 cells were initially lysed in RIP lysis solution and centrifuged at 14,000 rpm at 4 °C for 10 min. The supernatant was harvested, a portion of which was removed as input while the other was probed with antibodies against rabbit anti-human Ago2 (ab186733, 1: 50, Abcam) and rabbit anti-human IgG (ab109489, 1: 100, Abcam, taken as NC), with SNRNP70 (Millipore) used as a positive control, for co-precipitation. At last, the immunoprecipitated RNA was isolated and analyzed by means of RT-qPCR. ## Fluorescence in situ hybridization (fish) Cells post 24-h transfection in each group were detached with trypsin by shaking for 5 min, and then centrifuged for 2-3 min in 1.5 mL Eppendorf (EP) tubes. The cells were fully mixed with the pre-cooled CER I and lysed on ice for 10 min. Additional spinning was conducted with precooled CER II, followed by incubation and centrifugation. Next, the supernatant containing cytoplasmic components was transferred into a new EP tube and then stored at − 80 °C for later analysis. Afterwards, the supernatant was spun with pre-cooled nuclear extraction reagents and incubated. Then, it was remixed for 15 s at intervals of 10 min and finally centrifuged for 10 min at 4 °C. Finally, the supernatant containing the nuclear fraction was put into fresh EP tubes, and stored at − 80 °C. The coverslips were dried and fixed, after which cells were subsequently treated with protease K, DEPC-4% paraformaldehyde, then acetic acid, and incubated with 200 μL pre-hybridization solution for 1 h at room temperature. After that treatment, 250 μL hybridization solution containing 0.1-0.2 ng/μL probe was added for a further incubation at 65 °C for 14 h. The cells were washed, sealed and finally incubated with anti-DIG-AP Fab antibody (diluted 1:5000 din Buffer B2) overnight at 4 °C. Thereafter, the coverslips were developed with freshly prepared BCIP/NBT solution for 3-24 h in the dark. ## Rt-qpcr Total RNA content of PC cells was extracted by TRIzol reagent, and the purity and concentration of the extracted RNA were determined by NanoDrop ND-1000. Thereafter, cDNA was synthesized with a PrimeScript RT reagent Kit, while RNA was converted to cDNA by a One Step PrimeScript MicroRNA Gene Synthesis Kit. RT-qPCR of the product was implemented using a Quanti-Tect SYBR Green PCR kit on the ABI7500 quantitative PCR system. With U6 and GAPDH serving as the internal reference, relative expression pattern of each target gene was measured by means of 2 −ΔΔCt method. PCR primer sequences are listed in Additional file 1: . # Western blot analysis After 72 h of transfection, total protein was extracted and the concentration was assessed by a bicinchoninic acid kit. All the protein lysates were separated using 10% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane, and sealed by 5% skimmed milk powder. After that, the membrane underwent overnight probing at 4 °C with primary antibodies, namely rabbit anti-human antibodies to cleaved-caspase 3 (1:1000, #9665, Cell Signaling Technology, Beverly, MA, USA), cleaved-caspase 9 (1:1000, #9508, Cell Signaling Technology), PARP1 (1:1000, ab32064, Abcam), Oct4 (1:1000, ab181557, Abcam), Nanog (1:200, ab21624, Abcam), and Sox2 (1:1000, #14962, Cell Signaling Technology). Thereafter, the membrane underwent re-probing with HRP-conjugated secondary goat anti-rabbit for 1 h at 37 °C. Finally, the membrane was visualized with enhanced chemiluminescence reagent (Pierce). Ratio of the gray value of target bands to that of the internal reference GAPDH (1:2500, ab9485, Abcam) band represents the relative protein expression. ## Aldefluor assay Cells post 24-h transfection were resuspended in Aldefluor buffer to adjust the density to 1 × 10 6 cells/mL. The activity of aldehyde dehydrogenase (ALDH), a stem cell marker, was detected by an Aldefluor kit according to the instructions. The cells were subjected to incubation at 37 °C for 25 min with 15 μM ALDH specific inhibitor exogenous 4-(diethylamino)benzaldehyde (DEAB) and 0.15 μM ALDH substrate. Then the activity of ALDH was measured by a flow cytometer. ## Mtt assay Exponentially growing cells were cultured with 20 μL of MTT (5 mg/mL) at 0, 12, 24, 48 and 72 h after transfection for 4 h in the dark. Cells of each well were supplemented with 150 μL dimethylsulfoxide and placed on a shaking table for 10 min, and the OD value was then measured by means of a microplate reader (DG5031, Shanghai Kehuai Instruments Co., Ltd., Shanghai, China) at 490 nm. ## Flow cytometry After 24 h of transfection, cells were detached with trypsin without EDTA and centrifuged. After that, collected cells were fixed by addition of 3 mL pre-cooled 70% ethanol, centrifuged, and stained with 0.5 mg/mL propidium iodide (PI) staining solution, followed by detection by a flow cytometer at more than 575 nm. Apoptosis rate of PC cells was assessed by an Annexin V-FITC/PI double staining kit (556547, Shanghai Solja Technology Co., Ltd., China). After centrifugation, cells were resuspended in pre-cooled 1× phosphate buffer saline, centrifuged at 200 rpm for 5-10 min and resuspended in 300 µL 1× binding buffer. Next, the cells were incubated with 5 µL of Annexin V-FITC and stained with 5 µL PI, followed by analysis with a flow cytometer (Cube6, Sysmex Partec, Am Flugplatz, Görlitz, Germany). FITC was detected at 480 and 530 nm, while PI at a wavelength greater than 575 nm. The proportion of stem cell markers CD24+ and CD44+ cells was then calculated. Cells were incubated with FITC-conjugated CD44 (mouse anti-human, BD Biosciences, 555478), and phycoerythrin (PE)-conjugated CD24 (mouse anti-human, BD Biosciences, 555428), along with their corresponding isotype controls (BD Biosciences, 555742 and 55554) for cell surface staining, washed twice with the use of PBS, and resuspended in PBS for analysis/sorting. ## Colony formation assay PC cells post 24-h transfection were detached with 0.25% trypsin and triturated into single cell suspension. This single-cell suspension was plated in plates with 6 wells (1 × 10 4 cells/mL) and grown for 2 weeks under standard condition. When cell colonies were observed by naked eye, the culture was halted and the cells underwent 3.7% methanol fixation for 10 min and 0.1% crystal violet staining for 10-30 min. After staining and washing, the cells were photographed and the number of clones (> 50 cells) per well was counted using the Image J software for statistical analysis. ## Sphere formation assay Cells post 24-h transfection were plated in ultralow attachment plates with 24 wells at a density of 1000 cells/well in serum-free DMEM/F-12 medium containing B27 (1:50), 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor. The number of microspheres formed within 7 days was counted, and the colony formation ratio was calculated based on a factor of 1000. ## Xenograft tumors in nude mice BALB/c mice aged 5 weeks (equal numbers of male and female) were randomly grouped into 13 groups (12 for each group). The mice were housed under room temperature conditions at a stable humidity of 50-60% under a 12-h light/dark cycle with free access to drinking water. For tumor propagation analysis, 1.5 × 10 6 cells resuspended in 0.1 mL serumfree DMEM was mixed with 0.1 mL Matrigel and injected subcutaneously into the back of nude mice. After 3 days, a second cell suspension of the same volume was injected at the same site. Tumor formation and volume were observed every 2 days after injection. Four weeks later, mice were euthanized, thus at 5 weeks after tumor innoculation. The weight and volume of the tumors were measured. The volume was then calculated as the calculation (length × width 2 )/2. # Statistical analysis The measurement data described as mean ± standard deviation and SPSS 21.0 software was used to analyze the data. The statistical significance was measured using paired t-test, unpaired t-test, one-way ANOVA with Tukey's multiple comparisons test and two-way ANOVA or repeated measures ANOVA, followed by Bonferroni post hoc test for multiple comparisons. A value of p < 0.05 denotes statistical significance. # Results ## Bioinformatics analysis predicts that norad competitively binds to mir-202-5p to increase anp32e expression, thus indicating an involvement in pc development In an attempt to identify eligible lncRNAs in PC, we analyzed data in microarray expression profiles. According to the GEPIA database (http:// gepia. cancerpku. cn/), NORAD was found to have expression in PC , and in most other cancers . Following Venn diagram analysis of the downstream miRNAs of NORAD predicted by the starBase (http:// StarB ase. sysu. edu. cn/ index. php), RNA22 (https:// cm. jeffe rson. edu/ rna22/) and DIANA (http:// carol ina. imis. athenainnov ation. gr/ diana_ tools/ web/ index. php?r= lncba sev2/ index) databases, three miRNAs were found at the intersection, including miR-202-5p, miR-496, and miR-485-3p . Existing literature has shown that miR-202-5p is involved in the occurrence and development of PC, and that NORAD overexpression can inhibit the expression pattern of miR-202-5p LncRNA, miRNA and mRNA expression profiles in PC. A Expression of NORAD in PC where the X axis represents the grouping and the Y axis represents the NORAD expression; B Expression of NORAD in all patient tumor samples and paired normal tissues (black represents normal tissues, and red represents tumor samples); C Prediction of downstream miRNAs of NORAD (three circles in the figure represent the prediction results of the three databases, respectively, and the middle part represents their intersection); D Volcano plot of expression of differentially expressed genes in PC-related datasets, where the X axis denotes differential log10 p value and the Y axis denotes log FoldChange. Each point in the plot represents a gene, where red dots represent upregulated genes while green dots represent down-regulated genes; E Prediction of target genes of miR-202-5p (the three circles in the figure represent the prediction results of the three databases respectively, and the middle part represents their intersection); F Expression of ANP32E in GSE107610 (the X axis represents the tumor samples and normal tissues and the Y axis represents the level of ANP32E). Therefore, miR-202-5p was selected as the target gene for follow-up research. Additionally, the downstream targets of miR-202-5p were predicted by means of mirDIP, DIANA, TargetScan and starBase databases. At the same time, through the PC-relevant dataset GSE107610 of the GEO database (https:// www. ncbi. nlm. nih. gov/ geo/), 514 differentially expressed genes were obtained . Intersection analysis on the predicted results of the target genes of miR-202-5p and differentially expressed genes yielded ANP32E , which was highly expressed in the GSE107610 dataset . The aforementioned results indicate NORAD may competitively inhibit miR-202-5p expression and consequently promote the expression of ANP32E, thus participating in PC. ## Upregulated norad and anp32e and downregulated mir-202-5p are determined in pc tissues and cells To investigate the role of NORAD, miR-202-5p, and ANP32E in PC, we measured their expression in PC samples and cell lines. In relation to adjacent normal tissues, PC tissues exhibited higher expression of NORAD and ANP32E and lower miR-202-5p expression. In addition, NORAD and ANP32E exhibited significantly high expression in the three PC cell lines (BxPC-3, MIAPaCa-2, and PANC-1) relative to HPDE6-C7 cell line, but the expression of miR-202-5p was reduced. Among the three PC cell lines, PANC-1 showed the highest expression of NORAD, and was thus selected for subsequent experiments. Furthermore, PANC-1 cells with CD24 + CD44 + ESA + were selected as PCSCs (80%). ## Mir-202-5p suppresses the viability, proliferation and stemness of pcscs by inhibiting anp32e expression The. Meantime, the luciferase activity of cells co-transfected with ANP32E-WT-Luc and miR-202-5p mimic was remarkably inhibited, while no alteration occurred in that of cells co-transfected with ANP32E-MUT-Luc and miR-202-5p mimic. Transfection efficiency of cells with miR-202-5p mimic and si-ANP32E was confirmed by RT-qPCR, the results of which illustrated a decreasing trend in the cellular ANP32E expression upon miR-202-5p mimic treatment, while the expression of miR-202-5p was dramatically increased; the expression of ANP32E in the cells transfected with si-ANP32E was remarkably reduced, but was elevated in the cells treated with ANP32E. Based on the effects of DEAB treatment, the ALDHpositive cell population was divided (Additional file 2: , B), and then analyzed by Aldefluor, MTT assay, flow cytometry, colony formation assay, sphere formation assay, and Western blot analysis. Relative to, B). In summary, miR-202-5p retarded the viability, proliferation, and self-renewal of PCSCs, and accelerated their apoptosis by binding to ANP32E. ## Norad downregulates mir-202-5p expression in pcscs FISH data illustrated that NORAD was concentrated in the cytoplasm. The starBase database predicted miR-202-5p as a downstream gene of NORAD and the presence of binding sites between miR-202-5p and NORAD. Meantime, the luciferase activity of the cells co-transfected with NORAD-WT-Luc and miR-202-5p mimic was suppressed. The results of RIP showed an enhancement in the expression of NORAD and miR-202-5p in Ago2-pulled samples in comparison with IgG-pulled samples. NORAD was found enriched in samples pulled down by the miR-202-5p probe relative to the samples pulled down by the NC probe, as illustrated by RNA pull-down assay data. Transfection efficiency was determined (Additional file 5:. The RT-qPCR results indicated that the expression of NORAD and ANP32E was remarkably decreased in cells after si-NORAD or miR-202-5p mimic treatment, but that of miR-202-5p was increased; the expression of NORAD and ANP32E was augmented in cells transduced with NORAD, while that of miR-202-5p was dramatically reduced. No difference appeared in the expression of NORAD, mIR-202-5p and ANP32E in cells co-transfected with NORAD and miR-202-5p mimic. Based on the above results, NORAD competitively inhibited miR-202-5p expression in PCSCs. ## Norad promotes the viability, proliferation, and stemness of pcscs through down-regulation of mir-202-5p We then aimed to evaluate the role of miR-202-5p and NORAD in PC. Transfection with si-NORAD or miR-202-5p mimic decreased proportion of ALDH high-activity cells, inhibited proliferation, accelerated apoptosis, increased G0/G1 phase-arrested cells and decreased S phase-arrested cells, increased ratios of cleaved-caspase3 to pro-caspase3 and of cleaved-cas-pase9 to pro-caspase9, upregulated protein expression of PARP1, reduced cell colony and cell sphere formation, and downregulated expression of Oct4, Nanog and Sox2. However, the cells transfected with NORAD exhibited opposite results. Interestingly, the stimulating effects of NORAD on the stemness of PCSCs were partially rescued by miR-202-5p mimic-G; Additional file 6:. In addition, flow cytometric data indicated that overexpression of miR-202-5p or knockdown of NORAD reduced the proportion of CD24+ and CD44+ cells, while overexpression of NORAD increased the proportion. Overexpression of miR-202-5p abolished the promoting effect of overexpression of NORAD on the proportion of CD24+ and CD44+ cells (Additional file 5:. It is notable that we verified the overexpression efficiency of NORAD by RT-qPCR in MIAPaCa-2 cells (Additional . Measurement data were expressed as mean ± standard derivation. Data between two groups were compared by unpaired t-test, and data among multiple groups were analyzed by one-way analysis of variance with Tukey's post hoc test. The experiment was repeated three times file 7:. As shown in Additional file 7:-H, NORAD overexpression increased the proportion of ALDH high-activity cells, accelerated proliferation, reduced apoptosis, colony, and sphere formation, and G0/G1 phase-arrested cells, increased S phasearrested cells, and elevated the ratios of cleaved-cas-pase3 to pro-caspase3 and that of cleaved-caspase9 to pro-caspase9, but downregulated protein expression of PARP1, and upregulated the expression of Oct4, Nanog, and Sox2. To summarize, NORAD induced the viability, proliferation, and self-renewal while suppressing apoptosis of PCSCs through inhibiting miR-202-5p expression. ## Norad upregulates anp32e expression to enhance tumorigenicity of pcscs by competitively inhibiting mir-202-5p in vivo To investigate whether and how miR-202-5p overexpression or knockdown of NORAD and ANP32E affected the tumor formation ability, in vivo experiments were applied in nude mice. The tumor volume and weight decreased in the mice treated with miR-202-5p mimic, si-ANP32E and si-NORAD but a contrasting trend appeared following treatments with ANP32E or NORAD. Meanwhile, no alteration occurred in tumor weight and volume of mice injected with PC cells that had been transfected with miR-202-5p mimic-NC, si-ANP32E-NC, ANP32E-NC, NORAD-NC and co-transfected with miR-202-5p mimic and ANP32E or NORAD and miR-202-5p mimic. The above data conclude that NORAD promoted ANP32E to enhance tumorigenicity of PCSCs via competitive inhibition of miR-202-5p in vivo. [formula] D A R O N C N - D A R O N - i s D A R O N - i s k n [/formula] # Discussion PC denotes a lethal human malignancy around the world in which many patients diagnosed at a late stage. Patients, clinicians, and researchers are depressed by the slow progress being made, suggesting that new ideas and solutions to the disease are urgent needed. Due to their ability to interact with different structures and molecules, lncRNAs have high heterogeneity and functional diversity. As demonstrated previously, lncRNAs function importantly in regulating the cell fate determination, disease occurrence, and tumor progression. Thus, this study focused on exploring the regulatory role of NORAD (also known as LINC00657)in the stemness of PCSCs. According to Li et al.'s report, NORAD promotes the tumor cell migratory and invasive abilities in pancreatic cancer through modulation on the hsa-miR-125a-3p-metiated RhoA axis. Similarly, as our experiments turned out, NORAD accelerates the viability, proliferation and self-renewal and inhibits apoptosis of PCSCs by impairing expression of miR-202-5p. Recent investigations uncovered the essential roles of NORAD in biological processes, which also exerts oncogenic functions among various cancers. For instance, as Sun et al. discovered, in esophageal squamous cell carcinoma, the acceleration of upregulated NORAD on the cancerous cell invasive, migratory, and proliferative abilities. Wang et al. has shed light on the promotive effects of NORAD overexpression on colorectal cancer cell proliferation, migration, and invasion. Based on these researches, aberrantly upregulated NORAD was identified in several human cancers and affect the development of cancers. In our study, the result turned out that NORAD exhibited a significantly high expression in PC cells, and accelerates the cells viability and proliferation, which were in consistency with the research by Li et al.. Furthermore, lncRNA could sequester miRNAs to modulate the gene expression. Specifically, Tong et al. suggested that NORAD downregulation could suppress the cell function of epithelial ovarian cancer by endogenously binding to miR-155-5p. indicated the function of NORAD in promoting proliferative ability and glycolysis in non-small cell lung cancer by working as a competing endogenous RNA for miR-136-5p. In addition, it has been found that overexpression of NORAD enhances the invasive and migratory capabilities of melanoma cells via competitive inhibition of miR-205. Therefore, with the attempt to understand the possible mechanism by which NORAD affecting the PC development, based on the microarray-based analysis, miR-202-5p was screened as a downstream miRNA for NORAD, with specific binding sites identified between them. Meanwhile, based on the mirDIP, DIANA, TargetScan and starbase databases, ANP32E was revealed as a target gene of miR-202-5p. According to has the potency to induce the tumor formation capacity of triple-negative breast cancer cells by transcriptionally potentiating E2F1. Additionally, knockdown of ANP32E by siRNA lentivirus inhibits the cancerous cell proliferative, migratory, and invasive capabilities in breast cancer. For the purpose evaluating the function of ANP32E in PCSCs, we conducted a wide range of experiments, revealing that NORAD upregulated the ANP32E expression to accelerate the proliferation, self-renewal, and tumorigenic abilities of PCSCs through competitive inhibition of miR-202-5p. In addition, as Chen et al. demonstrated in their report, the miR-202 knockout exerts inhibition on the activity of spermatogonial stem cells. Our study suggested that through competitive inhibition of miR-202-5p, NORAD could negatively regulate Oct4, Nanog and Sox2, thus promoting the self-renewal ability of stem cells. Self-renewal is the process of giving rise to indefinitely more cells of the same cell type, perpetuating the stem cell pool throughout life. The stem cells' self-renew potency is under the regulation of the interaction between intrinsic proteins it expresses and extrinsic signals which it receives from the niche microenvironment. Self-renewal program involves the balance among gate-keeping tumor suppressors (limiting self-renewal), proto-oncogenes (promoting self-renewal), and care-taking tumor suppressors (maintaining genomic integrity). # Conclusions In a word, our investigations offered a new insight into the association between NORAD and PC. In this study, we identified that the promotion of NORAD on the viability, proliferation, and self-renewal of PCSCs, and the inhibition on the apoptosis. Mechanisms suggested that NORAD upregulates ANP32E expression by competitive inhibition of miR-202-5p expression. Our findings not only highlighted the role of NORAD in PC, but also provided clues for underlying clinical applications. Still, further studies with larger sample size and the presence of metastatic patients are needed to confirm the clinical application of the biomarker of NORAD for the treatment and diagnosis of PC.

Identification of shared genetic variants between schizophrenia and lung cancer Epidemiology studies suggest associations between schizophrenia and cancer. However, the underlying genetic mechanisms are not well understood, and difficult to identify from epidemiological data. We investigated if there is a shared genetic architecture between schizophrenia and cancer, with the aim to identify specific overlapping genetic loci. First, we performed genome-wide enrichment analysis and second, we analyzed specific loci jointly associated with schizophrenia and cancer by the conjunction false discovery rate. We analyzed the largest genome-wide association studies of schizophrenia and lung, breast, prostate, ovary, and colon-rectum cancer including more than 220,000 subjects, and included genetic association with smoking behavior. Polygenic enrichment of associations with lung cancer was observed in schizophrenia, and weak enrichment for the remaining cancer sites. After excluding the major histocompatibility complex region, we identified three independent loci jointly associated with schizophrenia and lung cancer. The strongest association included nicotinic acetylcholine receptors and is an established pleiotropic locus shared between lung cancer and smoking. The two other loci were independent of genetic association with smoking. Functional analysis identified downstream pleiotropic effects on epigenetics and gene-expression in lung and brain tissue. These findings suggest that genetic factors may explain partly the observed epidemiological association of lung cancer and schizophrenia.Schizophrenia (SCZ) is a mental disorder that greatly impacts the life of the affected individuals and ranks globally among the leading causes of disability. Genetic factors are important for development of SCZ, and heritability estimates range up to 0.8 1 . Large genome-wide association studies (GWAS) suggest that SCZ is a polygenic disease with many genetic variants associated, each with a small effect 2 . Recently, several lines of evidence indicate genetic overlap between SCZ and other brain disorders 3 as well as cardiovascular risk factors [bib_ref] Improved detection of common variants associated with schizophrenia by leveraging pleiotropy with..., Andreassen [/bib_ref]. Due to the polygenic nature of SCZ, it is possible that shared genetic factors may also underlie other diseases or traits associated with SCZ. Epidemiological studies report both inverse and direct co-morbidity between SCZ and some cancer types. For example, a meta-analysis of cancer incidence in more than 500,000 participants showed an increased risk for breast cancer and decreased risk for melanoma and lung cancer [bib_ref] Inverse and direct cancer comorbidity in people with central nervous system disorders:..., Catala-Lopez [/bib_ref]. Similarly, a prospective cohort study found increased risk of breast cancer for women and lung cancer for men [bib_ref] Cancer mortality in patients with schizophrenia: an 11-year prospective cohort study, Tran [/bib_ref]. Additional support for comorbidity between SCZ and lung cancer was given by a Danish nation-wide registry study [bib_ref] The risk of schizophrenia and child psychiatric disorders in offspring of mothers..., Benros [/bib_ref]. In contrast, a large UK cohort study did not show any significant difference in incidence of colorectal cancer, breast cancer and lung cancer between SCZ and controls [bib_ref] Relative incidence of common cancers in people with severe mental illness. Cohort..., Osborn [/bib_ref]. Another study investigating parents of patients with SCZ did not find any significantly reduced risk for overall cancer types, although it reported an increased risk for lung cancer in mothers of patients with SCZ [bib_ref] Risk for cancer in parents of patients with schizophrenia, Dalton [/bib_ref]. Furthermore, first-degree relatives of patients with SCZ showed significantly reduced overall cancer risk [bib_ref] Cancer incidence in patients with schizophrenia and their first-degree relatives -a meta-analysis, Catts [/bib_ref]. In summary, the literature seems to provide inconsistent results. This can be due to study design, as well as confounders including lifestyle factors, such as smoking or diet, antipsychotic medication, and different approaches to cancer screening and treatment. Additionally, cancer is a disease of the older ages, while patients with SCZ have a decreased life expectancy of 10-25 years. Combining GWAS from multiple disorders provides insights into genetic pleiotropy, a single genetic variant associated with more than one distinct phenotype, and could elucidate shared pathophysiology. We used a genetic epidemiology framework based on the conjunction false discovery rate (FDR), which enables identification of specific loci of cross-phenotype association independent of direction, thus making it particularly useful to test overlap between different diseases where directions of effects are unknown 4 . Since the FDR framework requires only summary statistics we were able to integrate GWAS data from SCZ and cancer sites from more than 220,000 subjects . Our first aim was to visualize polygenic overlap between SCZ and cancer in a genome-wide enrichment analysis and if this varies depending on cancer sites. Secondly, we aimed at identifying specific loci sharing association between SCZ and cancer using conjunction FDR, a two-dimensional extension of the FDR. Finally, we functionally characterized the shared loci using epigenetic and expression data in relevant tissue types to better understand joint disease etiologies. # Results Enrichment pattern between schizophrenia (SCZ) and cancer. A stratified quantile-quantile (Q-Q) plot showed a strong enrichment pattern for SCZ given lung cancer [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref]. While the blue line shows the standard enrichment of the main trait of interest (SCZ) including all SNPs irrespective of their association with the secondary trait (lung cancer), we observe a stronger leftward deflection from the dashed line of no association with increasingly stronger association with lung cancer. We did not see any similar enrichment pattern for any other cancer sites. Breast cancer showed weak enrichment (Supplementary [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref] , i.e. strata conditional on association with breast cancer did not diverge from the line of all SNPs. Conditioning on prostate cancer did not result in any deflection (Supplementary [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref] from the Q-Q line of all SNPs. Furthermore, there was no substantial enrichment given strata defined by ovarian cancer (Supplementary [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref] or colon cancer (Supplementary [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref] , which might be due to the comparatively small sample sizes of these GWAS. To test for statistical significance of enrichment for the Q-Q plot strata we used LD-score regression [bib_ref] Partitioning heritability by functional annotation using genome-wide association summary statistics, Finucane [/bib_ref]. After adjusting for multiple testing (four cancer traits and three strata) we detected an increase in the enrichment parameter for SCZ given lung cancer ranging from 1.424 (−log10pval >1) to 2.190 (−log10pval >2), and 6.512 (-log10pval >3) of which the first stratum is significantly enriched after multiple testing correction, and the second stratum is nominally significant (Supplementary . None of the other traits showed significant enrichment of any strata. The prostate cancer study was excluded from the enrichment analysis since its coverage (211,155 SNPs) using a customised genotyping platform was too low. All analysis was performed after excluding SNPs mapping to the major histocompatibility complex (MHC, genomic position (hg 19): chr6:29,528,318-33,373,649 12 ) since the MHC has been shown to be one of the key driving factors for enrichment of genetic association in SCZ [bib_ref] Genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential..., Andreassen [/bib_ref]. In order to check involvement of the MHC region, we repeated the stratified Q-Q plot for SCZ given lung cancer (Supplementary [fig_ref] Figure 2: Manhattan plot for independent [/fig_ref] including all SNPs mapping to the MHC, but we did not find substantial changes in enrichment as seen in the stratified Q-Q plots between analysis including the MHC (Supplementary [fig_ref] Figure 2: Manhattan plot for independent [/fig_ref] and excluding the MHC [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref]. Further we note the symmetry of the observed enrichment and show the stratified Q-Q plot for lung cancer given SCZ in . Shared risk loci between schizophrenia (SCZ) and lung cancer. Three independent (r 2 < 0.2) loci shared between SCZ and lung cancer passed the conjunctional FDR < 0.01 threshold. See for p-values and effect directions and [fig_ref] Figure 2: Manhattan plot for independent [/fig_ref] for the conjunctional FDR Manhattan plot. Variants mapping to the MHC have been removed prior to fitting the conjunction FDR. The three loci with joint association between SCZ and lung cancer were explored with functional follow-up studies. The strongest association was found for the locus on 15q25.1 mapping to genes of the nicotinic acetylcholine receptors, which has been previously implicated for cross-phenotype association between lung cancer and smoking [bib_ref] A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit..., Hung [/bib_ref]. The 15q25.1 locus showed a concordant effect direction between SCZ and lung cancer. There was one LD proxy (rs2904130, r 2 = 0.89) of the lead SNP rs8042374, which is an expression quantitative trait loci (eQTL) with neuronal acetylcholine receptor subunit alpha-5 (CHRNA5) and in both in lung and brain (caudate) tissue (Genotype-Tissue Expression (GTEx) [bib_ref] Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in..., Consortium [/bib_ref] Supplementary [fig_ref] Table 3: A [/fig_ref]. The locus on 6p22.1, has been identified and replicated as a cross-phenotype association between lung cancer and blood triglycerides [bib_ref] Pleiotropic Analysis of Lung Cancer and Blood Triglycerides, Zuber [/bib_ref]. This locus harbors two SNPs (rs28360634 and rs72839477) in strong LD (r 2 = 1) with the lead SNP rs7749305, which are eQTL (GTEx 15 ) in both brain and lung tissue with the same gene butyrophilin subfamily 3 member A2 (BTN3A2). . Independent (r 2 < 0.2) loci associated with both schizophrenia (SCZ) and lung cancer (LgCa) as defined by conjunction false discovery rates (ConjFDR < 0.01). In addition, we include cross-phenotype association of SCZ and smoking status (measured by number of cigarettes per day (CPD)). For each locus we report the lead single nucleotide polymorphism (SNP), closest annotated gene (Gene), genomic position (Band), p-values and z-scores with A1 (reference allele) and A2 (effect allele) for the specific traits. The major histocompatibility complex (MHC) was excluded from the analyses. The SNP rs7749305 on band 6p22.1 has the genomic position (hg19) chr6:27,446,566 and is thus outside the physical boundaries of the MHC. Still, it is an eQTL with a MHC-related gene (BTN3A2, Supplementary [fig_ref] Table 3: A [/fig_ref]. Not available number (NaN) if not available in the summary data file. The eQTL in brain tissue was confirmed in the independent Brain eQTL dataset (Braineac 17 , Supplementary [fig_ref] Table 3: A [/fig_ref]. The lead SNP rs7749305 is outside of the physical boundaries of the MHC, but it is an eQTL with BTN3A2, a MHC-related gene, underscoring the complicated and extensive LD structure in this region. The third association was on 11q12.1 and included the lead SNP rs2081361, which was an eQTL (GTEx) in lung tissue with the gene translocase of inner mitochondrial membrane 10 homolog (TIMM10) and with the leucine-rich repeat-containing protein 55 (LRRCP55) which is an auxiliary protein of the large-conductance, voltage and calcium-activated potassium channel. Further we found evidence for rs2081361 to be a moderate eQTL with TIMM10 in brain tissue in the Braineac database [fig_ref] Table 3: A [/fig_ref]. We found epigenetic evidence for rs2081361 in lung tissue (normal human lung fibroblast (NHLF) and adenocarcinomic human alveolar basal epithelial cells (A549)), and Henrietta Lacks (HeLa) S3 cells (Supplementary . In particular, for A549 and HeLa S3 we found CCCTC-binding factor (CTCF) binding and for NHLF open chromatin as characterized by DNase1 was discovered. A summary of the eQTL data is given in Supplementary ## Genetic overlap and shared risk loci between schizophrenia (scz) and smoking. smoking is the main risk factor for lung cancer, and there is a higher prevalence of smoking among patients with SCZ than controls. There is also one reported cross-phenotype association between lung cancer and smoking [bib_ref] A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit..., Hung [/bib_ref] [bib_ref] A variant associated with nicotine dependence, lung cancer and peripheral arterial disease, Thorgeirsson [/bib_ref]. Thus, we investigated if association with smoking behavior measured by cigarettes per day (CPD), correlated with the polygenic overlap between SCZ and lung cancer. As shown in the stratified Q-Q plot (Supplementary , there is an enrichment of SCZ association given CPD (after removing MHC region). After removing SNPs mapping to the nicotinic acetylcholine receptors (genomic position (hg 19) chr15: 78,686,690-79,231,478) the enrichment of SCZ given CPD disappears (Supplementary which suggests that the shared signal between SCZ and CPD is driven by genetic variation within the nicotinic acetylcholine receptors. To detect cross-phenotype association between SCZ and smoking behavior we computed the conjunction FDR for joint association between SCZ and CPD. There is only one locus, 15q25.1, with conjunction FDR < 0.01 between SCZ and CPD. This has a concordant association between lung cancer and smoking, as reported earlier [bib_ref] A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit..., Hung [/bib_ref] [bib_ref] A variant associated with nicotine dependence, lung cancer and peripheral arterial disease, Thorgeirsson [/bib_ref] , and the effect direction is also concordant for SCZ. Remarkably, the other two loci shared by SCZ and lung cancer had a conjunction FDR for SCZ and CPD close to one, which indicates no association between SCZ and CPD apart from the locus on 15q25.1 . We included further smoking traits such as onset, cessation, and initiation into the analysis, but except for the locus on 15q25.1 none of the cross-phenotype associated SNPs shows any association with any other smoking trait (Supplementary . ## Shared risk loci between schizophrenia (scz) and squamous cell carcinoma type of lung cancer. Furthermore, we refined the definition of shared genetic variants between SCZ and lung cancer to subtypes of lung cancer, adenocarcinoma (ADENO) and squamous cell carcinoma (SQUAM). We analyzed the two subtypes and presented stratified Q-Q plots for SCZ given SQUAM (Supplementary and for SCZ given ADENO (Supplementary . Noteworthy, we observed a strong enrichment for SCZ for SQUAM, and a weaker enrichment for ADENO. This is in line with previous findings of a different genetic architecture of the two cancer sub-types [bib_ref] Influence of common genetic variation on lung cancer risk: meta-analysis of 14..., Timofeeva [/bib_ref]. We found three independent loci with conjunction FDR < 0.01 for SCZ&SQUAM, and one locus with conjunction FDR < 0.01 for SCZ&ADENO (Supplementary . The locus shared between SCZ, SQUAM and ADENO is the locus on 15q25.1, which was the strongest association in the general lung cancer analysis. # Discussion We report polygenic enrichment between SCZ and lung cancer, but not for any other cancer site. This suggests that shared genetic risk factors may underlie the association between SCZ and lung cancer shown in epidemiological studies. Smoking is strongly associated with both SCZ and lung cancer, and here we show that variants mapping to the nicotinic acetylcholine receptors may contribute to this overlap. The current findings of shared variants associated with these three phenotypes have implications for the underlying pathophysiological processes, and interpretation of epidemiological findings. In particular, the finding of partly genetic causes for the high smoking prevalence in SCZ are of clinical relevance. It underscores the importance of preventive measures against smoking initiation and smoking cessation programs in mental health care, and suggests evaluation of lung cancer screening programs in SCZ. The conjunction FDR is a genome-wide approach and it is possible that inclusion of larger LD blocks such as the MHC can impact the model fit and confound the results. Therefore, the main results are based on the analysis after excluding the MHC and re-fitting the FDR estimate, which showed associations of three loci [fig_ref] Figure 1: Stratified Q-Q plot for schizophrenia [/fig_ref]. The statistical framework we used has the advantage of pinpointing loci of cross-phenotype associations even when the effect directions are mixed as it is the case for the three loci we identified here . In contrast, LD score regression 11 , a useful approach for genome-wide co-heritability analysis as presented in Supplementary Table 2, is neither able to identify specific genetic regions nor pleiotropic traits with mixed effect direction [bib_ref] New statistical approaches exploit the polygenic architecture of schizophrenia-implications for the underlying..., Schork [/bib_ref]. The locus on chromosome 15q25.1, including the nicotinic acetylcholine receptors CHRNA3, CHRNA5 and CHRNB4, showed concordant effect direction between SCZ, lung cancer, and smoking behavior. When the two lung cancer sub-types were analyzed, the associations with SCZ were in same direction. The locus on chromosome 11q12.1 showed concordant effect direction for SCZ and lung cancer. It harbors several variants that are moderate eQTL in both lung and brain tissue with the gene translocate of inner mitochondrial membrane 10 (TIMM10). The protein encoded by TIMM10 functions as a preprotein translocase for the import of proteins into inner and outer membranes, particularly inner membrane metabolite carriers [bib_ref] Mitochondrial protein translocases for survival and wellbeing, Sokol [/bib_ref]. Under-expression of genes of the TIMM family has been associated with neurodegenerative diseases [bib_ref] Alzheimer's disease is associated with reduced expression of energy metabolism genes in..., Liang [/bib_ref] recently identified as significantly down-regulated in dorsolateral prefrontal cortex layer 3 pyramidal cells isolated from tissue from SCZ patients [bib_ref] Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder, Arion [/bib_ref]. However, the present evidence for involvement of TIMM10 is moderate and replication and further investigations are needed. We found associations between SCZ and both histological types of lung cancer, squamous cell carcinoma and adenocarcinoma. The enrichment was stronger and more extensive in the squamous cell type, which had three loci associated with SCZ, and only one with adenocarcinoma (the CHRNA3/CHRNA5/CHRNB4 cluster on chromosome 15q25.1). It was reported that more than 90% of patients with squamous cell carcinoma were or had been smokers, as compared to about 55% of those suffering from adenocarcinoma [bib_ref] Never-smokers with lung cancer: epidemiologic evidence of a distinct disease entity, Toh [/bib_ref]. We expect that the present findings will form the basis for future studies of the role of the 15q25.1 in smoking behavior, lung cancer, and SCZ. Our approach aimed at identifying cross-phenotype associations and cannot distinguish between biological and mediated pleiotropy [bib_ref] Pleiotropy in complex traits: challenges and strategies, Solovieff [/bib_ref]. The present findings demonstrate the importance of further functional follow-up studies and further investigations using other approaches such as Mendelian Randomisation, which can help to distinguish between biological and mediated pleiotropy. Recent epidemiological studies, including a Mendelian Randomisation study [bib_ref] Investigating causality in associations between smoking initiation and schizophrenia using Mendelian randomization, Gage [/bib_ref] and a prospective co-relative control study [bib_ref] Smoking and schizophrenia in population cohorts of Swedish women and men: a..., Kendler [/bib_ref] have found evidence for smoking initiation as putative risk factor for SCZ. The present findings suggest pleiotropic downstream effects of the cross-phenotype associations. Especially eQTL studies in relevant tissue types provide important insights how genetic variants exert downstream effects on gene-expression [bib_ref] The role of regulatory variation in complex traits and disease, Albert [/bib_ref]. The evidence that all three cross-phenotype associations from our pleiotropic analysis are eQTL with the same gene (nicotinic acetylcholine receptors, BTN3A2, TIMM10) in relevant tissue types including lung and brain further support the claim of downstream pleiotropy between SCZ and lung cancer and complement observed associations from epidemiology studies. Further analyses of the molecular downstream consequences of these genetic variants are beyond the scope of this manuscript. One should be cautious with interpretation, as the relationship between SCZ and lung cancer is complex. Cancer risk in SCZ seems to vary with age, with higher than expected frequencies during young ages and lower than expected frequencies later in life [bib_ref] Inverse association between cancer risks and age in schizophrenic patients: a 12-year..., Lin [/bib_ref]. Also lung cancer followed this pattern, with higher standardized incidence ratios at ages less than 60 years, and lower incidences at higher ages [bib_ref] Inverse association between cancer risks and age in schizophrenic patients: a 12-year..., Lin [/bib_ref]. We do not have data stratified for age in the present study. In conclusion, we identified shared genetic variation between SCZ and lung cancer in the CHRNA3/CHRNA5/ CHRNB4 cluster on chromosome 15q25.1, and two other loci (6p22.1, 11q12.1) show cross-phenotype association and downstream pleiotropic effects on gene-expression in relevant tissue types for lung cancer and SCZ. The genetic effects are however complex, giving rise to both increased and decreased risk of the disorders. Further efforts into fine-mapping, causal analysis, and functional annotation are needed to clarify how these cross-phenotype associations exert their pleiotropic effects. Especially of interest is the role of the nicotinic acetylcholine receptors in the synthesis of smoking behavior, lung cancer and SCZ. # Methods Genome-wide association studies (GWAS) Samples. GWAS summary statistics on SCZ were provided by the Psychiatric Genomic Consortium (PGC) and comprised association analyses of 32,405 cases and 42,221 controls 2 . The summary statistics on five cancer sites were obtained from the Genetic Associations and Mechanisms in Oncology (GAME-ON) consortium and included lung cancer (13,373 cases and 26,014 controls) [bib_ref] Influence of common genetic variation on lung cancer risk: meta-analysis of 14..., Timofeeva [/bib_ref] (including sub-types referred to as adenocarcinoma (ADENO) and squamous cell carcinoma (SQUAM)), breast cancer (15,863 cases and 40,022 controls) [bib_ref] Large-scale genotyping identifies 41 new loci associated with breast cancer risk, Michailidou [/bib_ref] , prostate cancer (25,074 cases and 24,272 controls) [bib_ref] Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom..., Eeles [/bib_ref] , colon cancer (5,100 cases and 7,529 controls) [bib_ref] Genome-wide association study of colorectal cancer identifies six new susceptibility loci, Schumacher [/bib_ref] , and ovarian cancer (3,995 cases and 3,277 controls) [bib_ref] GWAS meta-analysis and replication identifies three new susceptibility loci for ovarian cancer, Pharoah [/bib_ref]. Additionally, we included GWAS data on smoking behavior measured by cigarettes per day (CPD) (74,503 individuals) [bib_ref] wide meta-analyses identify multiple loci associated with smoking behavior, Tobacco &amp; Genetics [/bib_ref]. For more details see . Pre-processing. As a first pre-processing step we aligned all summary statistics to a common set of reference single nucleotide polymorphisms (SNPs) (of size d = 2,558,411) generated from the 1000 genomes project. As summary statistics we saved for each reference SNP and each trait one p-value and one z-score. Next we performed genomic control [bib_ref] All SNPs Are Not Created Equal: Genome-Wide Association Studies Reveal a Consistent..., Schork [/bib_ref] , and finally we adjusted for overlap between samples 36 . There were overlaps between controls of the PGC study on SCZ and controls of the cancer studies, i.e. n = 3,179 individuals for lung cancer, n = 4,834 for breast cancer, and n = 713 for colon cancer. All p-values reported are adjusted for genomic control, all false discovery rates reported are adjusted for genomic control and sample overlap. As reference panel for the computation of the linkage disequilibrium (LD) structure between SNPs we use the European populations from the 1000 genomes project. The European population best reflects the mainly European composition of the PGC study on SCZ and the lung cancer GWAS. Quantile-Quantile (Q-Q) plots. Q-Q plots are standard tools in genomics to visualize the distribution of the observed p-values with the expected distribution of p-values under the null hypothesis, or in other words under no association of the tagged SNPs with the phenotype of interest. Q-Q plots depict the quantiles of the observed p-values on the y-axis against the theoretical quantiles under no association on the x-axis. In order to focus on the tails, Q-Q plots are often displayed on the −log10 scale. In case of no association a Q-Q plot follows a straight line. Deflection from this null line describes enrichment, i.e. the presence of lower p-values as expected by chance. Stratified Q-Q plots investigate differential enrichment between pre-specified strata of SNPs [bib_ref] Improved detection of common variants associated with schizophrenia and bipolar disorder using..., Andreassen [/bib_ref] [bib_ref] Dense genotyping of immune-related disease regions identifies nine new risk loci for..., Liu [/bib_ref]. When investigating polygenic shared architecture between two traits we focused on the p-values of trait 1 (SCZ), and defined the strata based on trait 2 (cancer). More specifically we plotted the p-values of trait 1 given or conditional on different strength of association with trait 2 (i.e. p-value > −log10 p-values of 1, 2, or 3). Thus, we were able to visualize if conditioning on a secondary trait leads to stronger enrichment in the primary trait of interest. A strong enrichment increasing with association on the secondary trait is an indicator of a shared polygenic architecture between the two traits. Large blocks of linkage disequilibrium (LD) may confound the results. To account for this we applied a random pruning approach, where one random SNP per LD block (defined by an r 2 of 0.8) were used and averaged over 100 random pruning runs. The impact of differing correlation parameters (from 0.7 to 0.3) on the Q-Q plots is displayed in . Further we focus the Q-Q plots on the region below genome-wide significance (−log10 p-values < 7.3) in order to highlight the polygenic component of the cross-phenotype association. In order to test for differential enrichment of the Q-Q plot strata we use LD score regression [bib_ref] Partitioning heritability by functional annotation using genome-wide association summary statistics, Finucane [/bib_ref] to test for fold enrichment. We assess the fold enrichment of each of the three strata (i.e. p-value > −log10 p-values of 1, 2, or 3) represented in the stratified Q-Q plots with the total LD score as covariate. The prostate cancer study was excluded from the analysis since its coverage (211,155 SNPs) using a customised genotyping platform was too low. Multiple-testing correction is performed for four cancer traits and for the three strata (p_adjusted = pvalue × 4 cancer types × 3 strata). Conditional and conjunction false discovery rate (FDR). The second part of our genetic epidemiology framework aimed at pinpointing shared cross-phenotype associations using the conjunction false discovery rate (FDR). The basic FDR framework is based on the assumption that the distribution of p-values follows a mixture distribution where SNPs are either associated (non-null) or not associated (null) with the phenotype [bib_ref] power and false discovery rates, Efron [/bib_ref]. The (tail-area based) FDR is defined as the probability that a given SNP is null given that its p-value is as small as or smaller than the observed one. Note that in context of the FDR all modeling is done on the summary statistic level, and no access to genotype data is needed. The conditional FDR is a simple extension of the standard FDR that allows including additional information on the association of a SNP with a secondary trait or more precisely, with the p-value of the same SNP in a secondary trait. It is defined as the probability that a specific SNP is null given that the p-values for both, trait 1 and trait 2, are as small as or smaller than the observed ones [bib_ref] Improved detection of common variants associated with schizophrenia and bipolar disorder using..., Andreassen [/bib_ref] [bib_ref] Dense genotyping of immune-related disease regions identifies nine new risk loci for..., Liu [/bib_ref]. Low values of conditional FDR can be driven by the first trait only. To detect SNPs associated jointly with both traits at the same time we employed the conjunction FDR. It is defined as the probability of being null for either trait, or for both traits simultaneously given that the p-values for the two traits are as small as or smaller than the observed ones. Thus, a true discovery is only the case when a SNP is non-null for both traits jointly. This symmetric behavior of the conjunction FDR weights both traits equal. Low values in conjunction FDR can only be found when a SNP is associated with both traits jointly. For example, for lung cancer and SCZ this symmetric behavior is best demonstrated by a stratified Q-Q plot of SCZ given lung cancer and then vice-versa lung cancer given SCZ (Supplementary . For more information on conditional and conjunction FDR we refer to [bib_ref] Shared common variants in prostate cancer and blood lipids, Andreassen [/bib_ref]. We set a conservative FDR level of 0.01 per pair-wise comparison, which relates to one expected false positive finding within 100 reported findings. The conjunction FDR provides a genome-wide unbiased scan and is thus a suitable technique to discover novel associations that are not detected by a univariate conservative Bonferroni threshold. ## Functional follow up. To investigate downstream effects of the cross-phenotype associated genetic loci we looked up expression quantitative trait loci (eQTL) in relevant tissue types (especially lung and brain) in the Genotype-Tissue Expression (GTEx) database [bib_ref] Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in..., Consortium [/bib_ref] , and the UK Brain Expression Consortium (Braineac) [bib_ref] Genetic variability in the regulation of gene expression in ten regions of..., Ramasamy [/bib_ref]. [fig] Figure 1: Stratified Q-Q plot for schizophrenia (SCZ) given lung cancer (LgCa). Stratified Q-Q plot of theoretical vs empirical −log 10 p-values (corrected for genomic control) in schizophrenia (SCZ) below the standard GWAS threshold of -log10 p-values equal to 7.3 (equals p-values above 5 × 10 −8 ) as a function of significance of association with lung cancer (LgCa) at the level of p < 1 (all SNPs), p < 0.1, p < 0.01, p < 0.001 respectively. Dotted lines indicate the theoretical line in case of no association. Prior to this analysis single nucleotide polymorphisms (SNPs) in the major histocompatibility complex (MHC) have been excluded. SCIENTIFIC RepoRTs | (2018) 8:674 | DOI:10.1038/s41598-017-16481-4 [/fig] [fig] Figure 2: Manhattan plot for independent (r 2 < 0.2) loci associated with both schizophrenia (SCZ) and lung cancer (LgCa) as defined by conjunction false discovery rates (ConjFDR) < 0.01 after excluding single nucleotide polymorphisms in the major histocompatibility complex.SCIENTIFIC RepoRTs | (2018) 8:674 | DOI:10.1038/s41598-017-16481-4 [/fig] [fig] SCIENTIFIC: RepoRTs | (2018) 8:674 | DOI:10.1038/s41598-017-16481-4 [/fig] [table] Table 3: A. GTEx 15 , B. Braineac 17 ). [/table]

Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma Background: Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. Methods: Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni-and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). Results: RCC with and without miR-21 up-and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease.Conclusions:A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis. # Background The incidence of RCC is increasing annually by about 2%. Over 200 000 new cases are diagnosed per year and more than 100 000 related deaths occur globally [bib_ref] Global cancer statistics, Parkin [/bib_ref]. RCC is marked by adverse tumour biology and its CSS ranks lowest among urological malignancies. RCC is clinically demanding due to its prognostic heterogeneity. The establishment of concepts of adjuvant therapy has been hindered by lacking reliability of prediction of outcome by both clinical and molecular parameters. Therefore, identification of novel markers is warranted for tailoring therapy and follow-up. One current approach for molecular tumour characterization is profiling of microRNA (miR) expression [bib_ref] Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies, Hui [/bib_ref]. MiRs are small noncoding RNA strands posttranscriptionally regulating gene expression and appearing to be modulators of urologic cancers [bib_ref] MicroRNA in prostate, bladder, and kidney cancer: a systematic review, Catto [/bib_ref]. Among the large number of miRs, miR-21 and miR-126 have received special attention because of their relationship with multiple cancer entities. Upregulation of miR-21 has been reported e.g. in breast, gastric and lung cancer [bib_ref] Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies, Hui [/bib_ref]. A respective role has also been suggested for urological malignancies. In prostate cancer, elevated expression of miR-21 alone was shown to convey castration resistance [bib_ref] miR-21: an androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer..., Ribas [/bib_ref]. In RCC, several studies describe upregulation of miR-21 [bib_ref] The clinical utility of miR-21 as a diagnostic and prognostic marker for..., Faragalla [/bib_ref] [bib_ref] Identification of a microRNA panel for clear-cell kidney cancer, Juan [/bib_ref] and recently, association with reduced survival [bib_ref] Up-regulation of microRNA-21 correlates with lower kidney cancer survival, Zaman [/bib_ref] , indicating a pathogenetical role of miR-21 as a so-called oncomiR. Such role has also been suggested for miRNA-126, which is mapped within its host gene EGFL-7 (epidermal growth factor like-7) and is highly expressed in vascular endothelial cells. By regulating the VEGF (vascular endothelial growth factor) pathway miR-126 plays an important role in angiogenesis, lymphangiogenesis and vessel integrity in endothelial cells as well as in cancer cells [bib_ref] miR-126 and miR-126*: new players in cancer, Meister [/bib_ref]. In several studies miR-126 was reported to act as a tumour suppressor and was shown to be downregulated in various cancer types including breast, gastric, prostate cancer and RCC [bib_ref] miR-126 and miR-126*: new players in cancer, Meister [/bib_ref] [bib_ref] Accurate molecular classification of kidney cancer subtypes using microRNA signature, Youssef [/bib_ref]. In non-small cell lung and oral squamous cell cancer downregulation of miR-126 was related to poor survival suggesting miR-126 to be prognostic [bib_ref] Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in..., Sasahira [/bib_ref] [bib_ref] MicroRNA-126 inhibits tumor cell growth and its expression level correlates with poor..., Yang [/bib_ref]. In metastatic colorectal cancer miR-126 was related to the response of treatment with capecitabine and oxaliplatin [bib_ref] The predictive value of microRNA-126 in relation to first line treatment with..., Hansen [/bib_ref]. In RCC miR-126 is described to play a role in molecular classification of different subtypes [bib_ref] Accurate molecular classification of kidney cancer subtypes using microRNA signature, Youssef [/bib_ref] and recently, association of downregulation with progression was supposed [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] [bib_ref] Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell..., Slaby [/bib_ref]. While dysregulation of miR-21 and miR-126 has been linked to metastasis and progression in many cancer types, to date data on RCC are scarce. To assess a potential role of these miRs as prognostic molecular markers in RCC we analysed the expression of both miRs in 139 clear cell RCC specimens aiming at clinical application as molecular markers. # Methods ## Patients and tissue sample preparation Fresh frozen samples of clear-cell RCC and adjacent histologically benign renal tissue of patients undergoing radical nephrectomy or nephron-sparing surgery at the Department of Urology and Paediatric Urology of the Julius-Maximilians-University Medical Centre Würzburg between 2006-2010 were included in the study. Fresh specimens were collected, snap frozen in liquid nitrogen immediately after resection, and directly stored at -80C until RNA extraction was performed. Samples from cancerous areas were isolated from non-necrotic parts of the tumour tissue. Tumour classification and staging were performed according to the 2004 World Health classification and the 2002 TNM System. The study was approved by the Ethical Review Board of the Julius-Maximilian-University Würzburg (no. 136/08) and written informed consent was obtained from all patients. ## Rna extraction and quantitative real time pcr Total RNA from frozen tissue was isolated using the miRNAeasy kit (Quiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration and A260/280 ratio were analysed with a Nano Drop ND-100 spectrometer (NanoDrop Technologies, Wilmington) and RIN (RNA Integrity Numbers) and calculated with a Bioanalyzer. RNA samples showing RIN < 6.0 were excluded from further analysis. The resulting miRNA was retained for quantitative Real Time PCR (qRT-PCR). QRT-PCR was performed using TaqMan Micro Array assays (Applied Biosystems) as described previously. 5 ng total RNA was used for microRNA-specific reverse transcription as recommended by the manufacturer for miR-21 and miR-126. Cycling conditions were chosen according to manufacturer's protocols. All reactions were performed in triplicates and samples showing SD > 0.5 were excluded. Relative expression values of miRs were normalized to small nuclear RNA (RNU6b) previously described as reference gene [bib_ref] Expression of microRNA-221 is progressively reduced in aggressive prostate cancer and metastasis..., Spahn [/bib_ref]. ΔC t for tumour samples and adjacent non neoplastic tissue of the two miRs were performed by the comparative C t method. Relative over-or underexpression of miRs in tumours compared to the normal adjacent kidney tissue was obtained by the ΔΔC t method assuming equal RNA concentrations and complete efficiency of qRT-PCR as described previously [bib_ref] Expression of microRNA-221 is progressively reduced in aggressive prostate cancer and metastasis..., Spahn [/bib_ref]. All samples characterized by expression levels of RNU6B > 30 C t were excluded from further analysis (Additional file 1: [fig_ref] Table 1: Clinical and pathological patient characteristics [/fig_ref]. ## Statistics, computational analysis and combined risk score calculation Thresholds for dichotomising relative expressions of miR-21 and miR-126 were determined by receiver operating characteristic (ROC) curve, based on CSS. Impact of clinic-pathological parameters, miR-21 and miR-126 on CSS was assessed by uni-and multivariate COX regression analysis (R package, Thernaux, 2000). Calculation of a CRS of miR-21 and miR-126 was implemented as proposed by Lossos et al. [bib_ref] Prediction of survival in diffuse large-B-cell lymphoma based on the expression of..., Lossos [/bib_ref]. Therefore, a factor derived from the z-score, resulting from the COX model, was determined for both miRs separately and the relative expression multiplied by this factor resulting in the formula (−2.1 × miR-126) + (2.6 × miR-21). The negative factor indicates that higher expression correlates with longer survival, whereas the positive factor correlates with shorter survival. A cut-off for the risk score was again determined by ROC curve. The best fitting COX model was selected by measuring the relative good ness-of-fit with the Akaike information criterion (AIC). Differences in mean between miR-expression and clinical parameters were analysed by Student's t-test and ANOVA. # Results Expression of miR-21 and miR-126 in RCC As previously described by other studies miR-21 and miR-126 was expressed in normal kidney tissue and in RCC samples (Additional file 1: [fig_ref] Table 1: Clinical and pathological patient characteristics [/fig_ref]. Moreover, we found a significant upregulation of miR-21 in RCC [fig_ref] Figure 1: Expression of miR-126 and miR-21 in RCC [/fig_ref] compared to adjacent renal tissue by qRT-PCR. Clinical and pathological characteristics of the used collective are summarized in [fig_ref] Table 1: Clinical and pathological patient characteristics [/fig_ref]. The ΔΔCt method demonstrated miR-21 to be upregulated more than two-fold in 65% of the RCC cases. In addition to the known oncomiR miR-21 we analysed expression of miR-126 in our study collective. As shown in [fig_ref] Figure 1: Expression of miR-126 and miR-21 in RCC [/fig_ref] no significantly different overall expression of miR-126 between malign and benign samples was observed. However, the standard deviation (SD) of miR-126 expression in RCC samples showed greater variation compared to the SD in histologically benign tissue [fig_ref] Figure 1: Expression of miR-126 and miR-21 in RCC [/fig_ref]. The comparative ΔΔCt method showed a more than twofold downregulation and a more than two-fold upregulation of miR-126 in 36 (35%) and 22 (22%) RCC samples, respectively [fig_ref] Figure 1: Expression of miR-126 and miR-21 in RCC [/fig_ref]. From these results we concluded that miR-126 was silenced or upregulated in different subgroups of the RCC study cohort resulting in comparable mean expression between RCC cases and adjacent normal renal tissue. ## Association of mir-21 and mir-126 expression with clinical parameters in rcc To test a potential clinical relevance of miR-21 or miR-126, we analysed their expression in different risk groups stratified by conventional clinical parameters. Expression of miR-21 tended to be reduced in lower compared to higher tumour stages and grades. Conversely, miR-126 tended to be higher in lower compared to higher tumour stages and grades. Both trends missed the level of statistical significance [fig_ref] Figure 2: MiR-21 and miR-126 expression in association to clinical parameters [/fig_ref] and B). Of 103 RCC patients in the present series, 16 showed synchronous metastasis [fig_ref] Table 1: Clinical and pathological patient characteristics [/fig_ref]. In cases with synchronous metastasis versus those without, significant upregulation of miR-21 (p=0,02) and a trend towards downregulation of miR-126 missing the level of statistical significance was found (p = 0.08) [fig_ref] Figure 2: MiR-21 and miR-126 expression in association to clinical parameters [/fig_ref]. But three patients with synchronous metastasis showed low miR-21 expression and notably two of these three cases had CSS exceeding three years. We concluded from these results that dysregulation of both miRs might be involved in metastasis and progression of RCC. ## Association of mir-21 and mir-126 expression with overall survival in rcc We found upregulation of miR-21 and downregulation of miR-126 to be associated with CSS, respectively [fig_ref] Figure 2: MiR-21 and miR-126 expression in association to clinical parameters [/fig_ref]. The study group was dichotomized by a ROC curve and sensitivity and specificity were calculated [fig_ref] Figure 3: ROC curve of miRNA risk scores [/fig_ref]. A threshold ΔΔC t = 1.61 for miR-21 and ΔΔC t = 0.57 for miR-126 provided a sensitivity and specificity of 66% and 81% and of 36% and 100%, respectively [fig_ref] Figure 4: Risk stratification of patients with or without cancer related death [/fig_ref]. In Kaplan-Meier analysis upregulation of miR-21 and downregulation of miR-126 were related to adverse outcome (log rank p < 0.001 for miR-21 and p < 0.01 for miR-126, [fig_ref] Figure 3: ROC curve of miRNA risk scores [/fig_ref]. Expression of both miRs was combined to assess potential improvement of prediction, sensitivity and specificity. Using a previously described PCR-based risk score model CRS for CSS was calculated [bib_ref] Prediction of survival in diffuse large-B-cell lymphoma based on the expression of..., Lossos [/bib_ref]. A cut-off of the CRS of 6.82 stratified 36 (35%) cases in the high and 67 (65%) in the low risk group, respectively. Out of 16 cases with cancer-related death (CRD) during follow-up 14 were grouped in the predicted high risk and 65 out of 87 cases without CRD throughout follow-up in the low risk group, respectively. As shown in [fig_ref] Figure 4: Risk stratification of patients with or without cancer related death [/fig_ref] and [fig_ref] Table 2: Specificity and sensitivity for the CRS in the learning [/fig_ref] the CRS correctively classified 75% low risk cases and 88% high risk cases. Compared to singular miRNAs, sensitivity (88%) and specificity (75%) were increased by CRS [fig_ref] Figure 4: Risk stratification of patients with or without cancer related death [/fig_ref] and B). Kaplan-Meier estimates showed that CRS correlated significantly with CSS (log rank p < 0.0001); predicted 5 year CSS rates were 96% for low and 48% for high risk patients, respectively. ## Prognostic model combining mir-21 and mir-126 expression The potential of the two dichotomised miRNAs and the combined risk score (CRS) to predict overall survival in comparison to clinicopathological factors like tumour stage or grade was evaluated by uni-and multivariate Cox regression analysis. In Univariate Cox regression analysis CRS [fig_ref] Table 3: Univariate and multivariate Cox regression analysis determined by relative goodness of fit... [/fig_ref] and both single miRs (Additional file 2: [fig_ref] Table 2: Specificity and sensitivity for the CRS in the learning [/fig_ref] were significantly prognostic for CSS (p < 0.0001 for CRS, p < 0.0002 for miR-126 and p < 0.0008 for miR-21; the estimate of a coefficient for miR-126 was infinity since there were no events in one group) as well as tumour stage and grade, and in contrast to age and gender, which were not significant. By stepwise regression analysis the best model for predicting CSS (tested by AIC) contained the CRS (HR: 19,37; p < 0,0002) and the clinicopathological factor tumour grade (HR: 13.88; p < 0.001) indicating that both of these factors were independent predictors of CSS in the study cohort. To internally validate this result we performed bootstrap analysis of our regression model. The bootstrap estimates generated comparable hazard ratios and confidence intervals for both factors, suggesting a robust regression model and excellent internal validation [fig_ref] Table 3: Univariate and multivariate Cox regression analysis determined by relative goodness of fit... [/fig_ref]. To evaluate the ability of miR-21 and miR-126 to predict survival we calculated a regression model by substituting the CRS with the separate dichotomised miR-21 and miR-126 expression data (Additional file 2: [fig_ref] Table 2: Specificity and sensitivity for the CRS in the learning [/fig_ref]. Again, tumor grade and both miRs were shown to be independent factors in the regression model. Comparison of both regression models indicated that the combined risk score was a better predictor than the individual miRs. Validation of the combined risk score (CRS) as prognostic factor in RCC To validate the predictive potential of the model the determined high risk cut-off level of CRS (CRS > 6.82) was used to classify a test cohort for prognostic risk stratification. The test dataset contained 16 high risk patients with early disease progression and cancer-specific death (<48 months) and 20 low risk patients characterized by progression-free survival for over 48 months and no cancer-specific death in the follow-up time. We determined miR-21 and miR-126 expression and subsequently the CRS in samples of the validation cohort and calculated the predictive power of the CRS. Among the high risk group 13 of 15 (86.7%) and among the low risk group 17 of 20 (85%) cases were classified correctly by CRS [fig_ref] Figure 4: Risk stratification of patients with or without cancer related death [/fig_ref] and D, [fig_ref] Table 2: Specificity and sensitivity for the CRS in the learning [/fig_ref]. Samples of the validation cohort with CRS over the previously determined cut-off level were found to be associated with CRD by univariant Cox regression analysis (HR (95% CI) =1.39 (1.18-1.62); p < 0,0001). # Discussion Clinical management of RCC has changed in recent years with increased incidental diagnosis and by initiating therapy in localized stages and the establishment of antiangiogenic agents. While tumour size at time of diagnosis has decreased, mortality rate for RCC has not, suggesting an impact of differential tumour biology in morphologically similar tumours [bib_ref] Molecular profiling of renal cancer: the journey to clinical application, Ribal [/bib_ref]. Therefore, identification of patients at high risk for cancer progression is warranted to tailor adjuvant treatment. While numerous articles have recently studied associations between miRs and carcinogenesis and tumour progression proposing several so-called oncomiRs as regulators in carcinogenetic pathways and biomarkers in many cancer entities, considerably fewer data are available on such roles of specific miRs in RCC. Whereas recent expression studies in metastatic or progressive RCC revealed a large number of different miRs potentially linked to progression [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] [bib_ref] Specific miRNA signatures are associated with metastasis and poor prognosis in clear..., Heinzelmann [/bib_ref] [bib_ref] Expression of miRNA-106b in conventional renal cell carcinoma is a potential marker..., Slaby [/bib_ref] [bib_ref] Identification of metastamirs as metastasis-associated microRNAs in clear cell renal cell carcinomas, Wotschofsky [/bib_ref] , surprisingly only a small number of miRs have been found to be concordantly differentially expressed in metastasised or progressive RCC. The small overlap between the different studies and largely contradictory results remains to be explained. Therefore, we selected the oncomiRs, miR-21 and miR-126, based on literature search and own unpublished expression analyses as promising markers for CSS in RCC. As expected, we identified differential expression of miR-21 in our RCC study cohort, but, surprisingly, we could not find changes in the overall miR-126 expression in our study cohort. However, miR-126 was highly up or down-regulated in subgroups of the study collective, balancing the expression changes of miR-126 in the total RCC collective. Nevertheless, the dysregulation of miR-126 in subgroups of the RCC collective might indicate a role of miR-126 dysregulation in the development of RCC. This suggestion is supported by previous studies describing an impact of miR-126 in molecular classification of different RCC subtypes [bib_ref] Accurate molecular classification of kidney cancer subtypes using microRNA signature, Youssef [/bib_ref] [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] [bib_ref] Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell..., Slaby [/bib_ref]. Among oncogenetic miRNAs, miR-21 may be one of the most attractive for clinical use. MiR-21 is upregulated in various human cancers [bib_ref] Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies, Hui [/bib_ref] [bib_ref] miR-21: an androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer..., Ribas [/bib_ref] [bib_ref] Serum miRNA-21: elevated levels in patients with metastatic hormone-refractory prostate cancer and..., Zhang [/bib_ref] support such notion; cell lines with miR-21 overexpression increase cell proliferation, migration and invasion [bib_ref] MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene, Lu [/bib_ref]. Zhang et al. showed that knockdown of miR-21 inhibited cell proliferation and induced cell apoptosis by targeting multiple genes in RCC cells [bib_ref] modulates cell apoptosis by targeting multiple genes in renal cell carcinoma, Zhang [/bib_ref]. Association of miR-21 expression with adverse outcome was reported for various cancer entities, such as breast and gastric cancer [bib_ref] Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies, Hui [/bib_ref]. Such results recently also have been reported by two previous studies using smaller RCC study cohorts [bib_ref] The clinical utility of miR-21 as a diagnostic and prognostic marker for..., Faragalla [/bib_ref] [bib_ref] Up-regulation of microRNA-21 correlates with lower kidney cancer survival, Zaman [/bib_ref]. Our present results stemming from a considerably larger and unselected series representative of tertiary cancer care are in line with these data demonstrating marked upregulation of miR-21 in RCC and significant association with synchronous metastasis and CSS. Our results show miR-21 to be an independent predictor. In one of the two previous and smaller studies in RCC Faragalla et al. found miR-21 upregulation to be associated with CSS, although it was not independent of tumour stage and grade. While Faragalla used relative miR-21 expression levels, in the present study levels of the RCC samples were normalized to adjacent benign tissue. Thus, the present study is the first to date that identifies miR-21 as an independent marker for CSS in a large and representative series using such normalization mode. Recently miR-126 has been reported to be a tumour suppressor in various cancer types including RCC [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] [bib_ref] Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell..., Slaby [/bib_ref] regulating target genes like CRK, VEGF and EGFL7 in cancer cells [bib_ref] miR-126 and miR-126*: new players in cancer, Meister [/bib_ref]. Lately, regulation of pro-angiogenic genes has been demonstrated in metastatic breast cancer [bib_ref] A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells, Png [/bib_ref]. Inhibition of apoptosis in leukaemia and promotion of cancer development in NSCLC or prostate cancer have also been reported [bib_ref] miR-126 and miR-126*: new players in cancer, Meister [/bib_ref] [bib_ref] Independent and tissue-specific prognostic impact of miR-126 in nonsmall cell lung cancer:..., Donnem [/bib_ref]. In several miR expression studies downregulation of miR-126 was associated with metastatic disease and early relapse after nephrectomy in smaller series of RCC [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] [bib_ref] Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell..., Slaby [/bib_ref]. The present series is the largest assessing miR-126 as prognostic factor in RCC to date and the first analysing expression levels normalized with benign tissue. Finding no overall downregulation of miR-126 expression we conclude no pivotal role in the initiation of RCC. Downregulation was significantly related to synchronous metastasis and independently predicted CSS. The predominant downregulation of miR-126 in progressive RCC suggest miR-126 to act as a tumour suppressor, which is supported by the recent description of miR-126 regulating VEGF-A in RCC [bib_ref] Exploring the role of miRNAs in renal cell carcinoma progression and metastasis..., Khella [/bib_ref] , one of the pivotal factors of angiogenesis and tumour progression. Currently, no clinically applicable molecular marker of CSS is available in RCC. To develop an accurate prediction system, we generated a dual-factorial marker model based on a CRS using the expression levels of miR-21 and miR-126. The determined CRS provided higher sensitivity and specificity compared to risk stratification, which was based on expression of each single miR. The CRS is associated with disease prognosis and predicts CSS independently from other clinicopathological factors in the analysed RCC cohort. Validation in an independent study cohort has shown that the CRS is able to classify robustly RCC samples into relevant risk groups with high sensitivity and specificity suggesting that it might have potential as a prognostic molecular assay in a clinical setting. However, the current validation is limited by various factors, like size of the validation cohort or applicability of qRT-PCR based expression analysis in clinical routine. To further test the effectiveness of this molecular marker model, we are planning to evaluate it on expanded validation cohorts in the future. Recently, a miR signature based on the expression of miR-10b, miR-139, miR-130b and miR-199b was found to be associated with synchronous metastasis and CSS in RCC [bib_ref] Identification of a 4-microRNA signature for clear cell renal cell carcinoma metastasis..., Wu [/bib_ref] providing similar sensitivity (76%) and specificity (100%) as our study. Although no direct comparison can be made and the present series is considerably larger and assessed two miRs, the 5-year CSS rates predicted by both miR signatures of 32% and 84% for the high and low risk cases in the 4-miR signature compared with 48% and 96% in the current study support the robustness of such models and the impact of certain miRs on RCC. Several studies have evaluated the prognostic value of clinicopathological features like performance status, metastatic status, lymph node involvement, sarcomatoid features, perinephritic fat invasion, Fuhrman grade and histological subtype in RCC patients [bib_ref] Prognostic factors and predictive models in renal cell carcinoma: a contemporary review, Sun [/bib_ref]. We have found that only tumour grade independently predicts survival in our study cohort. Tumour stage, sex or age of the patients did not significantly correlate with prognosis and survival in our regression model. This might depend on the limited sample size and follow-up time of our RCC collective and has to be further validated in larger cohorts. Also certain additional limitations of the present analysis need to be taken into account. For one, only clearcell RCC was assessed limiting our conclusions to this entity. Since it represents the largest and clinically most relevant subtype, however, the clinical significance of our data is not diminished and inclusion of clear-cell RCC only added to the homogeneity of the data. Secondly, while the present series is among the largest reported to date and mode of diagnosis, surgical treatment and pathological processing are homogeneous, the data acquisition was retrospective and the exact use and regimens of anti-angiogenic medication and its impact on CSS could not be assessed. The overall use of antiangiogenic medication was homogeneously distributed over the study group and among cases with differential miR expression limiting respective bias. A further limitation is the lack of functional data; such was not the focus of the present study however, since we aimed at establishing clinical evaluation of miR as prognostic tools rather than adding basic knowledge on the role of miR in RCC tumorbiology. # Conclusion We found a significant correlation of miR-21-upregulation and miR-126-downregulation with metastasis and CSS in clear cell RCC. While tumour grade was the only clinicopathological parameter independently predicting CSS in the used study cohort in multivariate analysis, miR-21, miR-126 and a signature combining expression of both miRs (CRS), were independent prognosticators and might add to the limited assessment of prognosis based on clinicopathological parameters only. The determined CRS was validated in an independent test cohort showing high sensitivity and specificity in predicting CRD. The presently described miR signature appears apt to predict CSS in RCC justifying validation in larger cohorts and subsequent implication in clinical management. ## Additional files Additional file 1: [fig_ref] Table 1: Clinical and pathological patient characteristics [/fig_ref]. Ct levels of mir-21, miR126 und RNU6b. [fig] Figure 1: Expression of miR-126 and miR-21 in RCC. Box-Whisker-Plot : A) Relative expression of miR-21 and miR-126 in RCC and normal renal tissue (n = 103). MiR-21 expression is significantly higher in RCC (p < 0,001), but overall miR-126 expression is not changed (p = 0.57) in RCC compared to the expression of control tissue. Expression was analysed by qRT-PCR and normalized against RNU6b. P-values were calculated by unpaired students t-test. B) Proportion of RCC patients with dysregulation of miR-21 and miR-126: Expression of miRs in tumour and control tissue was verified by qRT-PCR in triplicates. MiR expression ratio of each tumour specimen compared to the expression in corresponding adjacent normal tissue was calculated by the ΔΔC t method. All patients were divided into three groups by > < 2 fold up-or down-regulation of miR-21 or miR-126 in RCC. [/fig] [fig] Figure 2: MiR-21 and miR-126 expression in association to clinical parameters. Box-Whisker-Plot: relative expression of miR-21 and miR −126 was analysed by the ΔΔC t method in RCC cases and subsequently the cases were divided into subgroups based on tumor grade (A), T stage (B) the presence of synchrone metastasis (C) or cancer related death (CRD) (D). P-values were calculated by students paired t-test (C and D) or by ANOVA (A and B). [/fig] [fig] Figure 3: ROC curve of miRNA risk scores (miR-21, miR-126 and CRS) and Kaplan Meier survival analysis of cancer specific survival (CSS) in RCC patients stratified by miR-21, miR-126 and CRS expression data. A) ROC curves; the cross indicates the selected cutoff score for miR-21, miR126 or CRS resulting in highest sensitivity and specificity. The used cut-off scores were indicated in the graphs. B) Kaplan Meier curves with log rank test and numbers of patients stratified by the calculated risk scores of miR-21, miR-126 and CRS. [/fig] [fig] Figure 4: Risk stratification of patients with or without cancer related death (CRD). Proportion of patients without (A and C) or with (B and D) cancer related death stratified by the risk score of miR-21 (high risk: miR-21 expression > 1.61), miR-126 (high risk miR-126 expression <0.57) or CRS (combined high risk score > 6.82). The proportion of correctly or incorrectly classified patients of the learning cohort (A and B) and the test cohort (C and D) are shown. The proportion of correctly classified patients at low risk (A and B) is shown as grey bars and indicates the sensitivity of the different risk scores. B and D show the proportion of correctly classified patients at high risk as black bars and indicate the specificity of the different risk scores. For both cohorts the CRS shows higher true positive rate and lower false positive rate as the separated miR-21 and miR-126 risk scores. [/fig] [table] Table 1: Clinical and pathological patient characteristics (n = 103) [/table] [table] Table 3: Univariate and multivariate Cox regression analysis determined by relative goodness of fit with AIC (p < 0.00001; Wald-Test) including the combined risk score (CRS) as variable [/table] [table] Table 2: Specificity and sensitivity for the CRS in the learning (A) and test data set (B)Learning data set CRS > 6.8 (n) CRS ≤ 6.8 (n) % correct classified CRD: cancer related death; CRS: combined risk score for miR-21 and miR-126. [/table]

N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status Human cervicovaginal fluid (CVF) is a complex, functionally important and glycan rich biological fluid, fundamental in mediating physiological events associated with reproductive health. Using a comprehensive glycomic strategy we reveal an extremely rich and complex N-glycome in CVF of pregnant and non-pregnant women, abundant in paucimannose and high mannose glycans, complex glycans with 2-4 N-Acetyllactosamine (LacNAc) antennae, and Poly-LacNAc glycans decorated with fucosylation and sialylation. N-glycosylation profiles were observed to differ in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Compared to CVF from women experiencing term birth, CVF from women who subsequently experienced preterm birth showed lower sialylation, which correlated to the presence of a diverse microbiome, and higher fucosylation, which correlated positively to pro-inflammatory cytokine concentration. This study is the first step towards better understanding the role of cervicovaginal glycans in reproductive health, their contribution to the mechanism of microbial driven preterm birth, and their potential for preventative therapy. # Results N-glycan structural profiling. CVF samples from a total of 10 donors with diverse ethnicities were collected for analysis. 4 of the donors were non-pregnant women, while 6 of the 10 donors were pregnant women defined at high risk of spontaneous preterm delivery (sPTB). Of these, 3 delivered at term, while 3 delivered preterm (< 37 weeks of pregnancy) [fig_ref] Table 1: Summary of CVF samples collected for glycomic analysis [/fig_ref]. A detailed MALDI-MS based glycomic characterisation of permethylated N-glycans was undertaken. This produces [M + Na] + molecular ions. An overall relative quantitation of paucimannose, high mannose, hybrid and complex glycans is shown in [fig_ref] Figure 1: MALDI-B who delivered at term in panel [/fig_ref]. More detailed structural characterization is illustrated in [fig_ref] Figure 1: MALDI-B who delivered at term in panel [/fig_ref]. Paucimannose gycans were identified in the low mass range at m/z 1141, 1171, 1345 and 1375, (Man 2-4 GlcNAc 2 Fuc 0-1 ). The glycans in the mid mass range were dominated by high mannose glycans (m/z 1579-2396, Man [bib_ref] Glycans and glycan-binding proteins as regulators and potential targets in leukocyte recruitment, Krautter [/bib_ref] [bib_ref] Biological Functions of Glycans, Gagneux [/bib_ref] [bib_ref] The human female urogenital microbiome: Complexity in normality, Macintyre [/bib_ref] [bib_ref] The pregnancy microbiome and preterm birth, Bayar [/bib_ref] GlcNAc 2 ) and bi-antennary complex glycans with fucosylation and/or sialylation (m/z 1835-3211, NeuAc 0-2 Gal 0-2 Man 3 GlcNAc 4-5 Fuc 0-1 ). The glycans at higher mass range contained multiple LacNAc units (Gal-GlcNAc) modified by different numbers of fucose and sialic acid (m/z 3402-4650, NeuAc 0-3 Gal 3-6 Man 3 GlcNAc 4-8 Fuc 0-5 ). The largest complex N-glycan observed was at m/z 6259 corresponding to a composition of Gal 9 Man 3 GlcNAc 11 Fuc 6 (Supplementary . More detailed N-glycan structural analysis was achieved by MS/MS analysis of selected molecular ions. This proved that the mono-fucosylated glycans can be both core or antenna fucosylated [fig_ref] Figure 2: Glycotope centric analysis of N-glycans from sample P3 [/fig_ref] , and that peaks with composition consistent with www.nature.com/scientificreports/ multiple LacNAc units are a mixture of structural isomers with varying numbers and lengths of antennae. Complex glycans with 2-4 LacNAc units were identified in the medium mass range and glycans with poly LacNAc units in the high mass range [fig_ref] Figure 1: MALDI-B who delivered at term in panel [/fig_ref]. Glycans above m/z 5000 included high numbers of LacNAc units exhibiting diverse modifications by Fuc and NeuAc . The MS/MS analysis reveals that structures with extended antennae are often favoured over branched structures [fig_ref] Figure 2: Glycotope centric analysis of N-glycans from sample P3 [/fig_ref]. These extended antennae were decorated by several fucose residues producing diverse poly Lewis antigens. LacdiNAc (GalNAcβ1-4GlcNAc) containing structures were identified at m/z 2285, 2326, 2500 and 2674, which were supported by MS/MS fragmentation analysis [fig_ref] Figure 2: Glycotope centric analysis of N-glycans from sample P3 [/fig_ref]. Interestingly, sample P5, from a donor with CST IV-B who delivered preterm at 31 + 3 weeks, showed a distinct pattern of glycan abundances compared with other samples. Its N-glycome was dominated by non-sialylated, and non-/ mono-fucosylated glycans [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref]. In addition, this sample had high levels of N-glycans with truncated antennae terminated by GlcNAc. A distinct N-glycan composition was also observed in CVF from non-pregnant donors [fig_ref] Figure 1: MALDI-B who delivered at term in panel [/fig_ref] , Supplementary [fig_ref] Figure 4: Fucosylation variations between non-pregnant and pregnant samples [/fig_ref]. Compared to pregnant women, spectra obtained from non-pregnant donors are dominated in the lower mass range by high mannose N-glycans (m/z 1579-2396, Man 5-9 GlcNAc 2 ), while the mid mass range is dominated by biantennary complex poly-fucosylated structures at m/z 2592, 2766 and 2940 (Gal 2 Man 3 GlcNAc 4 Fuc 3-5 ). The higher mass range is dominated by large polyLacNAc, heavily fucosylated glycans such as structures at m/z 3215, 3563, 3737, 4012, 4186, 4361, 4810 and 4984 (Gal 2-5 Man 3 GlcNAc 4-7 Fuc 3-9 ). Complex biantennary, triantennary and tetrantennary glycans capped by one to four sialic acids, were also detected, (eg., m/z 2605, 2966, 3211, 3776, 4124, 4473, 4761) but at lower abundance than exclusively fucosylated glycans. The complexity of CVF N-glycans with their multiple fucosylation and sialylation led us to apply additional complementary analytical strategies to facilitate deeper characterization of CVF N-glycan structures. Glyco-epitope centric N-glycan analysis using an Orbitrap mass spectrometer confirmed the presence of diverse glycotopes in a selected sample P3 [fig_ref] Figure 2: Glycotope centric analysis of N-glycans from sample P3 [/fig_ref]. MS 2 fragments of N-glycans detected LacdiNAc structures at m/z 505 and 679, terminal Lewis antigens at m/z 638 and 812, internal Lewis antigens at m/z 624, and Sialyl Lewis antigens at m/z 999. In addition, poly Lewis antigens were observed at m/z 1261 and m/z 1435. The MS 2 data also showed terminal GlcNAc or GalNAc at m/z 260, predicted to arise from bisecting glycans, glycans with truncated antennae, or LacdiNAc structures. The MS 2 peak at m/z 812 was selected for further fragmentation to obtain MS 3 data, which mainly detected the Lewis Y antigen. However, the MS 3 analysis of the MS 2 peak at m/z 638 revealed a mixture of Lewis X, Lewis A and the blood group H antigen, again highlighting the structural complexity of CVF N-glycans. Correlation of fucosylation and sialylation to pregnancy status. We next focused on quantitative analysis of the fucosylation and sialylation of the CVF N-glycans containing 2 or 3 LacNAc units [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref]. Glycans with 2 LacNAc units were shown to have up to 5 Fuc/glycan [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref] whereas glycans with 3 LacNAc units had up to 7 Fuc/glycan [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref]. In terms of sialylation, glycans with 2 LacNAc units had up to 2 sialic acids [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref] and glycans with 3 LacNAc units had up to 3 sialic acids [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref]. Variations of fucosylation and sialylation were observed among the samples. Quantitative analysis of Sample P5 showed extremely low levels of poly-fucosylation and sialylation for glycans with 2 or 3 LacNAc units [fig_ref] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc... [/fig_ref]. As shown in [fig_ref] Figure 4: Fucosylation variations between non-pregnant and pregnant samples [/fig_ref] , differential fucosylation was observed between non-pregnant and pregnant samples. For glycans with 2 LacNAc units, the pregnant samples had higher levels of non-fucosylated glycans and monofucosylated glycans. However, this trend started to reverse for bi-fucosylated glycans and was completely reversed for tri-and tetra-fucosylated glycans. Similar results were observed for glycans with 3 LacNAc units, except that the reversing point was at the tri-fucosylated glycans. The proportion of tetra-fucosylated glycans decreased to almost zero in the pregnant samples, but was retained at about 12% for glycans with 2 LacNAc units and 30% for glycans with 3 LacNAc units in the non-pregnant samples. An initial trend of fucosylation and sialylation changes were detected between preterm and term samples . CVF samples of women who delivered preterm showed higher levels of non-fucosylated glycans among glycans with 2 or 3 LacNAc units. The sialylation level among poly-fucosylated glycans tended to be lower for the preterm samples. ## Correlation of fucosylation and sialylation to microbial composition. community state type (CST) was used to characterise the vaginal microbial community status. Analysis of vaginal microbiota revealed that 3 of the pregnant samples had a vaginal bacterial community dominated by L. crispatus, consistent with Community State Type I (CST I) as described elsewhere, while 3 had a CST IV-B, characterised by G. vaginalis and A. vaginae dominance. Of the CST I samples two (P2, P4) were CST I-A (almost completely dominated by L. crispatus), and one (P1) was CST I-B (mostly dominated by L. crispatus but also containing low abundance of L. gasseri, L. jensenii and G. vaginalis). Of the samples that were classified as CST IV-B, there was a high relative abundance of G. vaginalis in two (P5 and P6), and a moderate relative abundance of A. vaginae and L. acidophilus in the other (P3) . The correlation between fucosylation and vaginal CST was investigated in relation to the number of Fuc residues per glycan . N-glycans with 2 LacNAc units and those with 3 LacNAc units showed the same trend: samples from CST IV-B donors had higher levels of non-fucosylated glycans and mono-fucosylated glycans, but lower levels of bi-fucosylated, tri-fucosylated and tetra-fucosylated glycans, compared with CST I-A/B donors. Detailed analysis of the correlation between sialylation and CST is shown in . Sialylation of glycans with different numbers of Fuc was analysed separately. The same trend was found regardless of the fucosylation status: CST IV-B samples consistently showed a lower level of sialylation compared to CST I-A/B samples. Our results suggest that the presence of a high relative abundance of Lactobacillus spp. is associated with a higher percentage of sialylation, bi-, tri-and tetra-fucosylated glycans, and lower levels of non-or mono-fucosylated glycans. www.nature.com/scientificreports/ Correlation of fucosylation and sialylation to pro-inflammatory cytokines. Strong correlation was found when fucosylation was mapped to levels of IL-1β and IL-18 (Figs. 8 and 9). The proportion of nonfucosylated glycans and the proportion of mono-fucosylated glycans to total glycans were positively correlated with cytokine concentration, while the proportion of poly-fucosylated glycans to total glycans and the proportion of highly-fucosylated glycans to poly-fucosylated glycans were negatively correlated with the cytokine concentrations. Similar correlations were observed for glycans with 3 LacNAc units [fig_ref] Figure 9: Correlation of fucosylation to IL-1 beta and IL-18 for glycans with 3... [/fig_ref]. No statistically significant correlations were seen with IL-8 and IL-6, however similar trends were observed (Supplementary Figs. S6 and S7). We also investigated levels of glycan sialylation and pro-inflammatory cytokines, however, only weak correlations were observed (Supplementary Figs. S1-S11). # Discussion Despite the importance of human CVF in shaping microbiota-host responses in the lower reproductive tract, to our knowledge, a detailed profiling of CVF N-glycans has not been reported. Our study characterises CVF as a glycan-rich environment with distinct and different N-glycosylation in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Our findings are based on a small cohort at present. This is due to a combination of factors including the collection device being more invasive than a simple swab and the labour intensive and high cost of sample processing. However, some initial trends have already been consistently observed. We also acknowledge that factors such as the stage of menstrual cycle or pregnancy, age, cervical length, blood group and ethnicity could influence the CVF glycosylation profile. We aim to study these factors with larger cohorts. The N-glycome of CVF is highly complex and abundant in paucimannose glycans and high mannose glycans in the low mass range. Complex glycans with 2-4 antennae in the medium mass range and glycans with poly LacNAc units in the high mass range are readily detectable. Antennae structures of detected N-glycans included terminal fucosylation of mono-fucosylated glycans, glycans with LacdiNAc structures, as well as sialylation and fucosylation patterns of extended poly LacNAc structures. Identification of a higher prevalence of glycan isomers carrying extended poly LacNAc chains compared to multi-antennary structures, suggests a biological role of the elongated antennae protruding from the mucin protein backbone [bib_ref] Loss of effector function of human cytolytic T lymphocytes is accompanied by..., Antonopoulos [/bib_ref] [bib_ref] Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis, Mkhikian [/bib_ref]. While extended poly-N-acetyllactosamine glycans can serve as spacers and carriers of additional glycotopes through their modification with fucose and sialic acid, poly LacNAc itself has been identified as a recognition motif of galectins [bib_ref] Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood..., Stowell [/bib_ref] , an important class of mammalian GBP involved in regulations of immune cell activities and microbial recognition as part of the innate immune system [bib_ref] Two distinct classes of carbohydrate-recognition domains in animal lectins, Drickamer [/bib_ref] [bib_ref] Roles of galectins in infection, Vasta [/bib_ref] [bib_ref] When galectins recognize glycans: from biochemistry to physiology and back again, Di Lella [/bib_ref]. N-Glycan structures carrying extended poly LacNAc chains have recently been reported as a characteristic feature of neutrophil granules 55 , which suggests these structures in CVF might partially be due to leukocyte infiltration. High mannose glycans can be recognized by DC-SIGN, the mannose receptor and other mannose binding GBP, which have been found to be involved in inflammation and infection [bib_ref] Emerging roles of protein mannosylation in inflammation and infection, Loke [/bib_ref]. Modifications of genital tract epithelia, mucus, and immune status occur throughout the menstrual cycle to accommodate conception and implantation [bib_ref] The cervicovaginal mucus barrier, Lacroix [/bib_ref]. At the time of conception, Sialyl Lewis X glycan epitopes on the human oocyte facilitates sperm-egg binding [bib_ref] Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona..., Pang [/bib_ref] , and glycans have also been implicated in the mediation of implantation [bib_ref] The expression and role of glycans at the feto-maternal interface in humans, Passaponti [/bib_ref] and immune homeostasis at the maternal-fetal interface [bib_ref] Sialic acid is a critical fetal defense against maternal complement attack, Abeln [/bib_ref]. Once implantation is successful, the cervicovaginal interface undergoes further modification, with the primary aim of forming a cervical barrier to infectious microbes to protect the developing fetus. Our study shows that this physiological switch to a pregnancy phenotype also involves marked changes to cervicovaginal glycan profiles. In both non-pregnant and pregnant women, N-glycans were found to be a mixture of paucimannose and high-mannose structures in the lower mass range, biantennary complex poly-fucosylated structures in the mid mass range, and large PolyLacNAc glycans the higher mass range. However, spectra of non-pregnant women CVF are clearly dominated by high mannose glycans in the lower mass range, which could be a sign of reduced glycan maturation and differentiation 7 or a different microbiota composition 25 compared to pregnant women. Oligosaccharides carrying terminal mannose residues are the main host receptors for bacteria such as E. coli. [bib_ref] Interaction of mannose-containing oligosaccharides with the fimbrial lectin of Escherichia coli, Firon [/bib_ref] A loss of high mannose residues on glycoproteins of the CVL from women with BV was observed by Wang et al. [bib_ref] Studying the effects of reproductive hormones and bacterial vaginosis on the glycome..., Wang [/bib_ref] , suggesting that native high mannose residues found on glycoproteins in fluids of the lower reproductive tract may act as natural inhibitors of pathogenic interactions, helping to prevent bacterial adhesion in women with normal microflora. We also demonstrated differences in glycan profiles between women who subsequently delivered preterm and women who delivered at term. The preterm samples showed higher levels of non-fucosylated glycans among glycans with 2 or 3 LacNAc units and lower sialylation among poly-fucosylated glycans. Desialylation of glycoproteins and glycolipids by sialidase leads to loss of or exposure of new glycan epitopes and interference with host immune recognition and binding of microbes [bib_ref] Multifarious roles of sialic acids in immunity, Varki [/bib_ref]. It is feasible that this could contribute to the mechanism of microbial driven preterm birth. In support of this, high sialidase concentrations have been associated with late miscarriage and preterm delivery [bib_ref] High sialidase levels increase preterm birth risk among women who are bacterial..., Cauci [/bib_ref] [bib_ref] Combination of vaginal pH with vaginal sialidase and prolidase activities for prediction..., Cauci [/bib_ref]. Further, we and others have reported an association between vaginal microbial composition and preterm birth risk [bib_ref] The pregnancy microbiome and preterm birth, Bayar [/bib_ref]. High diversity vaginal microbial composition (referred to as community state type IV, CST IV), or dominance of the niche by L. iners, is associated with higher rates of preterm birth, whereas L. crispatus (CST I) dominance is protective [bib_ref] Epidemiology and causes of preterm birth, Goldenberg [/bib_ref] [bib_ref] The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for..., Kindinger [/bib_ref] [bib_ref] Vaginal microbiome in early pregnancy and subsequent risk of spontaneous preterm birth:..., Tabatabaei [/bib_ref]. In this study N-glycans with 2 and 3 LacNAc units isolated from CVF of women with CST IV have lower levels of both poly and highly-fucosylated glycans and sialyated glycans compared to women with CST-I. Fucosylation and sialylation on LacNAc units can potentially produce different glycotopes, such as Lewis antigens, Sialyl Lewis antigens and poly Lewis antigens. Several studies have shown how glycans and their epitopes play a role in infective conditions such as HIV [bib_ref] Vaginal product formulation alters the innate antiviral activity and glycome of cervicovaginal..., Koppolu [/bib_ref] and Candida albicans [bib_ref] Cervical mucins carry alpha(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis, Domino [/bib_ref]. G. vaginalis is one of the main bacteria associated with bacterial vaginosis and CST IV, which is characterised by depletion of Lactobacillus species and polymicrobial overgrowth of anaerobes [bib_ref] Degradation, foraging, and depletion of mucus sialoglycans by the vagina-adapted Actinobacterium Gardnerella..., Lewis [/bib_ref] [bib_ref] Clue cells in bacterial vaginosis: immunofluorescent identification of the adherent gram-negative bacteria..., Cook [/bib_ref]. BV is also associated with increased vaginal concentrations of glycosidases such as sialidase, α-galactosidase, β-galactosidase and α-glucosidase [bib_ref] Large-scale characterisation of the pregnancy vaginal microbiome and sialidase activity in a..., Ng [/bib_ref] [bib_ref] Impact of bacterial vaginosis, as assessed by nugent criteria and hormonal status..., Moncla [/bib_ref] and decreased binding of lectins to both high mannose and α-2, 6 sialic acid. This observation suggests that bacteria sialidases and host sialoglycan receptors can be linkage specific, as also reported by other studies [bib_ref] Siglecs and their roles in the immune system, Crocker [/bib_ref] [bib_ref] Sialidases from gut bacteria: A mini-review, Juge [/bib_ref]. Findings from previous studies suggest that changes in glycosidases are accompanied by changes in glycosylation patterns in the vaginal fluid. On the other hand, a recent discovery found that Lactobacillus spp. Can create a protective micro-ecological environment through regulating the core fucosylation of vaginal epithelial cells [bib_ref] Lactobacillus spp. create a protective micro-ecological environment through regulating the core fucosylation..., Fan [/bib_ref]. Although glycosylation of the CVF could be influenced by hormone levels, it is thought that microbiota have a greater influence on CVF glycosylation than hormonal status [bib_ref] Studying the effects of reproductive hormones and bacterial vaginosis on the glycome..., Wang [/bib_ref]. Similarly to CVF, the human gastrointestinal (GI) mucosa consists of extensive glycoproteins overlaying epithelial cells, interspersed by immune cells, which form a protective physical barrier, and serve as first line of defence against microorganisms and harmful substances [bib_ref] How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to..., Josenhans [/bib_ref] [bib_ref] Roles and regulation of the mucus barrier in the gut, Cornick [/bib_ref]. Hydrolases, including exo-and endoglycosidases, are encoded in the genomes of mucin-degrading gut bacteria 73 allowing them to metabolize sugars [bib_ref] How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to..., Josenhans [/bib_ref] [bib_ref] Comparative genomic analysis of the human gut microbiome reveals a broad distribution..., Ravcheev [/bib_ref] , which are then exploited as nutrient source [bib_ref] Mucin glycan foraging in the human gut microbiome, Tailford [/bib_ref]. The mucins of the GI tract mucus layer also provide ligands for the attachment of bacteria 77 and may facilitate invasion; moreover, mucus-binding proteins such as adhesins and GBP have been described in many lactic acid bacilli [bib_ref] Introduction to the human gut microbiota, Thursby [/bib_ref]. The human gut microbiome and its role in both health and disease has been extensively studied and its involvement in human metabolism, nutrition, physiology, and immune function is now well established [bib_ref] Microbes, metabolites, and the gut-lung axis, Dang [/bib_ref] [bib_ref] Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation, Arpaia [/bib_ref] [bib_ref] Health and disease markers correlate with gut microbiome composition across thousands of..., Manor [/bib_ref] [bib_ref] Aspects of gut microbiota and immune system interactions in infectious diseases, immunopathology,..., Lazar [/bib_ref]. It is highly probable that similar interactions exist in the mucosa of the female lower reproductive tract, creating a tight interconnection between glycans, microorganisms colonising the female reproductive tract and cervicovaginal immune homeostasis. www.nature.com/scientificreports/ It is well established that local inflammation is a risk factor for preterm birth. However, only recently are we beginning to understand the relationship between inflammation, specific microbial communities, and preterm birth [bib_ref] Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix..., Osman [/bib_ref] [bib_ref] The role of inflammation and infection in preterm birth, Romero [/bib_ref] [bib_ref] Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and..., Pruski [/bib_ref] [bib_ref] The vaginal microbiome and preterm birth, Fettweis [/bib_ref]. Here we observed that non-fucosylated and monofucosylated glycans positively correlated with pro-inflammatory cytokines, whereas poly and high-fucosylated glycans are negatively correlated, highlighting the important role of N-glycosylation in maintaining immune homeostasis in the female reproductive tract. An abundance of paucimannose glycans was also detected in the low mass range of CVF, which could originate from infiltrating neutrophils, monocytes and macrophages [bib_ref] N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation..., Ugonotti [/bib_ref] [bib_ref] Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery, Venkatakrishnan [/bib_ref] [bib_ref] High-resolution longitudinal N-and O-glycoprofiling of human monocyte-to-macrophage transition, Hinneburg [/bib_ref] [bib_ref] Structural and functional diversity of neutrophil glycosylation in innate immunity and related..., Ugonotti [/bib_ref] [bib_ref] Human protein paucimannosylation: Cues from the eukaryotic kingdoms, Tjondro [/bib_ref]. These glycans could be derived from a recently described noncanonical truncation N-glycosylation pathway in human neutrophils [bib_ref] N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation..., Ugonotti [/bib_ref]. Paucimannose glycans are found on proteins in azurophilic granules of neutrophils, which can be selectively secreted upon pathogen stimulation, and their glycosylation status is involved in modulating multiple immune functions central to inflammation and infection [bib_ref] Paucimannose-rich N-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions, Loke [/bib_ref] [bib_ref] Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci-and phosphomannose glycans, Reiding [/bib_ref] [bib_ref] Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum, Thaysen-Andersen [/bib_ref]. The unusually high abundance of paucimannose structures found in CVF is potentially indicative of neutrophil invasion in the lower female reproductive tract, which could be used as a defence mechanism against invading pathogens. It is plausible that glycan-GBP interactions between pathogenic microbes and neutrophils leads to neutrophil degranulation, an abundance of paucimannoses, pro-inflammatory cytokine release and subsequent preterm labour. Another possible explanation for the paucimannose glycans is that they are the result of degradation by microbial glycohydrolytic enzymes [bib_ref] Large-scale characterisation of the pregnancy vaginal microbiome and sialidase activity in a..., Ng [/bib_ref] [bib_ref] Impact of bacterial vaginosis, as assessed by nugent criteria and hormonal status..., Moncla [/bib_ref]. It should be noted that CVF is also heavily O-glycosylated and we are undertaking similar analytic approaches to characterise these glycan structures. In summary, this study provides evidence that cervicovaginal glycans are associated with microbial composition and immune response in pregnancy where they may influence clinical outcome. Modulation of cervicovaginal glycans could thus represent a novel therapeutic strategy to prevent microbial driven preterm birth. www.nature.com/scientificreports/ ane, ammonium bicarbonate, EDTA, trypsin and Tris were from Merck (Poole, UK). PNGase F (cloned from Flavobacterium meningosepticum and expressed by E. coli), CHAPS and DTT were from Roche Applied Science (East Sussex, UK). 8 M guanidine hydrochloride (GuHCl) and Slide-A-Lyzer™ G2 Dialysis Cassettes, 3.5K MWCO were from Thermo scientific (Loughborough, UK). Glycomic sample processing was done following the protocol detailed previously [bib_ref] Glycomic profiling of cells and tissues by mass spectrometry: Fingerprinting and sequencing..., Jang-Lee [/bib_ref] [bib_ref] Mass spectrometric analysis of mutant mice, North [/bib_ref]. Briefly, CVF samples from aliquot one were sonicated in 25 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1% CHAPS, pH 7.4, dialysed in dialysis cassettes, reduced by DTT, carboxymethylated by IAA, and digested by trypsin. N-glycans were released by PNGase F and were permethylated. The permethylated glycans were cleaned by C18 cartridges and freeze dried before mass spectrometry analysis. Glycan profiling was done on AB Sciex 4800 MALDI-TOF/TOF mass spectrometer. The methylated glycans were dissolved in 10 ul methanol. One 1ul of sample was mixed with 1 ul of 10 mg/ml DABP matrix in 75% ACN. The mixture was spotted on a MALDI plate for MALDI-TOF-MS analysis. The data were analysed using Data Explorer™ version 4.6 from AB Sciex, Glycoworkbench 92 and MALDIquant [bib_ref] MALDIquant: A versatile R package for the analysis of mass spectrometry data, Gibb [/bib_ref]. The glycomic data were annotated based on monosaccharide composition derived from the molecular ion m/z value, knowledge of N-glycan biosynthetic pathways, the isotopic peak cluster patterns, the glycosylation patterns in the low and medium mass range, and MS/MS derived fragmentation. For glycans with overlapped isotopic peak clusters, the dominant one was annotated. The statistical analysis of the glycans was done using SQLite3 and R 3.6.3 on a Linux Ubuntu 20.04.4 LTS platform. The scripts are available on GitHub (Link: https:// github. com/ gw110/ CVF-glyco sylat ion. git). In order to have the least interference of overlapped isotopic peaks, Glycans with 2 or 3 LacNAc units were selected for quantitative analysis to assure high accuracy of data annotation and quantitation. The relative intensities of different glycan subgroups were calculated using SQLite3. The results were exported and visualized using ggplot2 in R 3.6.3. The correlation of glycan relative intensities with cytokines was done using the linear regression model. Pearson product-moment correlation coefficient and p value were calculated using the cor.test function in R. Box plot and two tailed Student's t test was used to compare glycosylation between sample groups. Glycans were further analysed following a glycotope-centric glycomic analysis based on a nanoLC-MS 2product dependent-MS 3 data acquisition workflow, previously established using the advanced Orbitrap Fusion Tribrid MS platform [bib_ref] Advancing a high throughput glycotope-centric glycomics workflow based on nanoLC-MS2-product dependent-MS3 analysis..., Hsiao [/bib_ref]. In essence, this takes advantage of the high acquisition speed, high mass accuracy and high sensitivity of the MS instrument to perform a comprehensive MS 2 mapping of the terminal glycotope based on preferred cleavage at the HexNAc of permethylated glycans. The respective intensity of each characteristic oxonium ion measured at 5 ppm mass accuracy or less can be summed from all glycan MS 2 spectral acquired throughout the LC-MS/MS run to provide a relative abundance index for the glycotope it represents. In cases when isomeric glycotopes need to be differentiated, as for the Lewis and H epitopes in this work, the MS 2 ion can be coupled with a further MS 3 event. The diagnostic MS 3 ions resulting from specifically eliminating the C3-substituent of the HexNAc + is sufficient to resolve the isomeric difference and their relative intensity can in turn provide a semi-quantitative estimation of the relative abundance of each isomeric constituent of that particular glycotope. Bacterial DNA extraction and metataxonomic profiling. Extraction of DNA from CVF samples and sequencing of 16S rRNA hyper variable regions was performed as previously described [bib_ref] Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and..., Pruski [/bib_ref]. Briefly, V1-V2 hyper variable regions of bacterial 16S rRNA genes were amplified using a mixed forward primer set (28f.-YM) consisting of the following primers mixed at a 4:1:1:1 ratio; 28F-Borrellia GAG TTT GAT CCT GGC TTA G; 28F-Chlorflex GAA TTT GAT CTT GGT TCA G; 28F-Bifido GGG TTC GAT TCT GGC TCA G; 28F GAG TTT GATCNTGG CTC AG. The reverse primer consisted of; 388R TGC TGC CTC CCG TAG GAG T 95 . Amplified products were then pooled equimolar and each pool was size selected in two rounds using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, Indiana) in a 0.7 ratio for both rounds. These were then quantified using the Quibit 2.0 Fluorometer (Life Technologies) and loaded on an Illumina MiSeq (Illumina, Inc. San Diego, California) 2 × 300 flow cell at 10 pM. All sequencing was performed at Research and Testing Laboratory (Lubbock, TX, USA). Trimming of primer sequences was performed using Cutadapt [bib_ref] cutPrimers: A new tool for accurate cutting of primers from reads of..., Kechin [/bib_ref] and QC performed using FastQC 97 . Resulting amplicon sequence variant (ASV) counts were calculated for each sample using the Qiime2 pipeline [bib_ref] Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2, Bolyen [/bib_ref]. DADA2 was used for denoising [bib_ref] DADA2: High-resolution sample inference from Illumina amplicon data, Callahan [/bib_ref] and taxonomic classification of sequences to species level was performed using the STIRRUPS reference database [bib_ref] Species-level classification of the vaginal microbiome, Fettweis [/bib_ref]. Samples were classified into vaginal community state types (CSTs) using the VAginaL community state typE Nearest CentroId classifier (VALENCIA). Using this standardised approach, CST 1 communities are characterised by Lactobacillus crispatus dominance and can be further subdivided into subtypes CST 1-A (almost complete dominance by L. crispatus) and CST 1-B (less L. crispatus, but still the majority). CST IV communities have a low relative abundance of Lactobacillus spp. and include subtypes CST IV-A, CST IV-B, and CST IV-C, depending on the relative abundances of G. vaginalis, A. vaginae, and BVAB1, respectively. Immune profiling of CVF samples. The stored supernatant from the CVF (aliquot two) was thawed on ice and used for Luminex® immunoassays to quantify cytokines by multiplexed bead-based immunoassays. The concentrations of 10 cytokines (IL-1β, IL-18, IL-6, IL-2, IL-4, IL-5, IL-10, IFN-γ, GM-CSF and TNF-α) were measured on a multiplex plate Human Premixed Multi-Analyte Kit (R&D Systems), following manufacturer's instructions. IL-8 was measured on a single-plex Human Premixed Analyte Kit (R&D Systems), following a 10-fold dilution using Calibrator Diluent RD6-52. All standards and samples were run in duplicate. Concentrations of IL-2, IL-5, IFN-γ, and GM-CSF were undetectable in all samples. IL-4, IL-10 and TNF-α were only detectable in a proportion of samples. [fig] Figure 1: MALDI-B who delivered at term in panel (c), a donor with CST IV-B who delivered preterm in panel (d) and a non-pregnant donor in panel (e). Donors in panel (a), (b) and (d) are blood group B, while donors in panels (c) and (e) are blood group O. The N-glycans from CVF were released by PNGase F and permethylated prior to MALDI-TOF and TOF-TOF profiling. Each spectrum is shown in a single panel, and all data are normalized to the most abundant component, which is designated as 100%. For clarity, a zoomed in panel is inserted for masses above 3500; colour coding has been used to distinguish families of glycans: paucimannose N-glycans are flagged as dark purple, peaks in green show high-mannose N-glycans, peaks in magenta are complex biantennary monofucosylated glycans, peaks in pink are sialylated complex N-glycans, peaks in orange are large PolyLacNAc structures decorated with multiple sialic acid and fucose residues, and in light blue are PolyLacNAc glycans decorated with 0-3 fucose residues. Peaks in light grey marked with X are known polyhexose contaminants. Structures of the colour coded peaks are represented in the corresponding coloured rectangle in panel (f). Main structures are depicted. Assignments are based on composition, tandem MS and knowledge of biosynthetic pathways. All molecular ions are [M + Na] + . Residues above a bracket have not had their location unequivocally defined. [/fig] [fig] Figure 2: Glycotope centric analysis of N-glycans from sample P3. All N-glycans were selected for CID fragmentation to produce MS2 ions. The summed intensity for each of the detected diagnostic MS2 ions normalized to their total was plotted to provide an overall assessment of the relative abundance of various glycotopes. The glycotopes represented by MS2 ions at m/z 638 and m/z 812 were further selected for additional stage of fragmentation (MS3) to identify their respective isomeric constituents, as shown in the insets. [/fig] [fig] Figure 3: Overall view of fucosylation and sialylation levels of N-glycans with 2 LacNAc units (a,b) and 3 LacNAc units (c,d). The glycans were subgrouped according to the number of Fuc or NeuAc they have. The summed intensity of glycans in a subgroup divided by the total glycan intensity was calculated as a relative intensity of the subgroup.Scientific Reports | (2022) 12:16948 | https://doi.org/10.1038/s41598-022-20608-7www.nature.com/scientificreports/ Glycans in the mid to high mass range of non-pregnant women show lower fucosylation and sialylation levels compared to pregnant women. This could be pointing again at a lower maturation and differentiation state of cervicovaginal proteins N-glycans and to a more quiescent immunological state which would allow easier transit of sperm and thus easier fertilisation. [/fig] [fig] Figure 4: Fucosylation variations between non-pregnant and pregnant samples. N-glycans with 2 LacNAc units (top panel) and 3 LacNAc units (bottom panel) were selected for analysis. The proportion of summed intensity of glycans with non-fucosylation, mono-fucosylation, bi-fucosylation, tri-fucosylation and tetra-fucosylation to total glycan intensity was calculated for data analysis. Scientific Reports | (2022) 12:16948 | https://doi.org/10.1038/s41598-022-20608-7 [/fig] [fig] Figure 5, Figure 6: Fucosylation and sialylation variations between preterm and term samples. N-glycans with 2 LacNAc units (top panel) and 3 LacNAc units (bottom panel) from pregnant samples were selected for analysis. Nonfucosylation % was calculated as the relative intensity of non-fucosylated glycans to the intensity of all glycans. Sialylation % was calculated as the relative intensity of sialylated glycans to the intensity of all poly-fucosylated glycans. recruitment and sampling. The study was conducted with approval of the NHS National Research Ethics Service (NRES) Committees London-Stanmore (REC 14/LO/0328), and in accordance with relevant guidelines, regulations, and the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. All patients and non-pregnant women provided written informed consent. Recruitment and sampling were performed at Queen Charlotte's and Chelsea Hospital, Imperial Col-Correlation of CST to fucosylation depended on the number of Fuc per glycan. N-glycans with 2 LacNAc units (top panel) and 3 LacNAc units (bottom panel)were selected for analysis. The proportion of summed intensity of glycans with non-fucosylation, mono-fucosylation, bi-fucosylation, tri-fucosylation and tetra-fucosylation to total glycan intensity was calculated. [/fig] [fig] Figure 7, Figure 8: CVF samples with CST IV-B had lower levels of sialylation than those with CST I, regardless of fucosylation. N-glycans with 2 LacNAc units (top panel) and 3 LacNAc units (bottom panel) were selected for analysis. The glycans were grouped based on the number of Fuc they have. In each group, the relative intensity of sialylated glycans to total glycan itensity was calculated as sialylation %. NHS Trust, London, UK. Non pregnant women were eligible if they were of reproductive age and aged 18 or over. Pregnant women at risk of preterm birth were eligible. Risk factors included having a short or open cervix, a previous preterm delivery or previous cervical treatment. Exclusion criteria included women under 18 years of age, those who had sexual intercourse within 72 h of sampling, vaginal bleeding in the preceding week, HIV or Hepatitis C positive status. Detailed maternal clinical metadata and birth outcome data was collected for all pregnant participants. CVF was sampled using the BBL™ CultureSwab™ MAXV liquid Amies swabs (Becton, Dickinson and Company, Oxford UK) for assessing microbial composition. CVF was then collected using a menstrual cup (Softdisc™, The Flex Company, USA) by placing it against the cervix for 20 min. After removal, material from both sides of the cup was retrieved by repeated pipetting of phosphate buffer saline (PBS) over each side leading to resuspension of material in a 1:5 weight: volume ratio within 30 min of collection. The suspension was distributed into separate aliquots to prevent unnecessary freeze thaw cycles. Aliquot one was reserved for glycomic profiling. Aliquot two was centrifuged (500×g, 10 min, 4 °C), and the supernatant was reserved for immune profiling. Samples were stored at − 80 °C until analysis.N-Glycan profiling of CVF samples. Methanol, acetonitrile, ammonia, chloroform, DMSO, propan-1-ol, sodium hydroxide, acetic acid were from Romil (Cambridge, UK). Idoacetic acid, sodium chloride, iodometh-Correlation of fucosylation to IL-1 beta and IL-18 for glycans with 2 LacNAc units. Pearson's product-moment correlation was used for statistical analysis. (a) Relative intensity of non-fucosylated glycans to the intensity of all glycans. (b) Relative intensity of mono-fucosylated glycans to the intensity of all glycans. (c) Relative intensity of poly-fucosylated glycans to the intensity of all glycans. (d) Relative intensity of highly fucosylated glycans to the intensity of poly-fucosylated glycans. Highly fucosylated glycans were defined as those with at least 3 Fuc per glycan. Poly-fucosylated glycans were defined as those with at least 2 Fuc per glycan. [/fig] [fig] Figure 9: Correlation of fucosylation to IL-1 beta and IL-18 for glycans with 3 LacNAc units. Pearson's product-moment correlation was used for statistical analysis. (a) Relative intensity of non-fucosylated glycans to the intensity of all glycans. (b) Relative intensity of mono-fucosylated glycans to the intensity of all glycans. (c) Relative intensity of poly-fucosylated glycans to the intensity of all glycans. (d) Relative intensity of highly fucosylated glycans to the intensity of poly-fucosylated glycans. Highly fucosylated glycans were defined as those with at least 4 Fuc per glycan. Poly-fucosylated glycans were defined as those with at least 2 Fuc per glycan. Scientific Reports | (2022) 12:16948 | https://doi.org/10.1038/s41598-022-20608-7 [/fig] [table] Table 1: Summary of CVF samples collected for glycomic analysis. [/table]

Associated characteristics and impact on recurrence and survival of free-floating tumor fragments in the lumen of fallopian tubes in Type I and Type II endometrial cancer A B S T R A C TObjective: This study sought to evaluate characteristics of cases of free-floating tumor fragments within the lumen of fallopian tubes ('floaters') on final pathology for Type I and Type II endometrial adenocarcinoma, including relationships with disease recurrence and mortality. Methods: A single institution experience of 1022 consecutive cases of uterine cancer presenting between 2005 and 2010 was retrospectively reviewed, with data extraction from electronic medical records. Associations of floaters with baseline characteristics were studied with logistic regression, and relationships with disease recurrence and survival were assessed with Cox proportional hazards models. Results: Among 816 included cases of Type I or Type II endometrial adenocarcinoma, floaters were identified on final pathology for 20 patients (2.5%). Patient characteristics of cases with floaters mirrored the overall sample. With adjustment, presence of floaters trended towards association with laparoscopic/robotic approach (OR = 3.84; 95%CI 0.98-15.1), and was significantly associated with lymphovascular invasion (OR = 9.65; 95%CI 2.35-39.6) and higher stage disease. Although floaters were associated with increased risk of recurrence in unadjusted analysis (HR = 3.22; 95%CI 1.41-7.37), after adjustment for disease type, stage, and patient comorbidities, no evidence for impact on disease recurrence or overall survival was found. Conclusions: The presence of floaters is rare. Floaters were generally associated with more extensive disease, but no evidence was found to show any independent prognostic impact on risk of recurrence or death. In agreement with prior research, this study found a trend towards association of floaters with laparoscopic/robotic approach, indicating the possibility of floaters sometimes being the result of trauma from uterine manipulator insertion. # Introduction Endometrial cancer is the most common gynecologic malignancy among women in the United States and accounts for 6% of malignancies in women worldwide. In the United States in 2017, 61,380 women will be diagnosed with endometrial cancer and 10,920 women will succumb to the disease [bib_ref] Cancer statistics, Siegel [/bib_ref]. Endometrial cancers exist in two forms, Type I and Type II [bib_ref] Two pathogenetic types of endometrial carcinoma, Bokhman [/bib_ref]. Type I disease (65-70% of all cases) includes International Federation of Gynecology and Obstetrics (FIGO) grade 1 and FIGO grade 2 endometrioid histology, and is often associated with unopposed estrogen exposure [bib_ref] Comparison of estrogen and progesterone receptor, Ki-67, and p53 immunoreactivity in uterine..., Lax [/bib_ref] [bib_ref] Should grade 3 endometrioid endometrial carcinoma be considered a type 2 cancer-a..., Voss [/bib_ref] [bib_ref] Uterine papillary serous carcinoma: patterns of metastatic spread, Goff [/bib_ref]. Type II disease includes FIGO grade 3 endometrioid, serous, or clear cell histology, and has a different genetic profile with development thought to be independent of estrogen exposure. Type II endometrial cancer is typically more aggressive than type I cancer and has a poorer prognosis [bib_ref] The more things change the more they stay the same, Mutch [/bib_ref] [bib_ref] Evaluation of unfavorable histologic subtypes in endometrial adenocarcinoma, Wilson [/bib_ref] [bib_ref] Hormonal interactions in endometrial cancer, Emons [/bib_ref] [bib_ref] Papillary adenocarcinoma of endometrium with psammoma bodies. Histology and fine structure, Hameed [/bib_ref] [bib_ref] Uterine papillary serous and clear cell carcinomas predict for poorer survival compared..., Hamilton [/bib_ref]. In 2009, updated staging recommendations were released by FIGO that made a series of small changes to the system, including eliminating a role for positive cytology from peritoneal washings in upstaging disease to stage IIIA [bib_ref] Revised FIGO staging for carcinoma of the endometrium, Creasman [/bib_ref] [bib_ref] Revised FIGO staging for carcinoma of the cervix, Pecorelli [/bib_ref]. Another pathologic finding that is sometimes noted on pathology reports is the presence of free floating fragments of tumor within the lumen of one or both fallopian tubes [bib_ref] FIGO staging of endometrial adenocarcinoma: a critical review and proposal, Zaino [/bib_ref]. A body of literature has accumulated assessing the role of positive cytology in prognosis, ultimately finding a relatively small impact in otherwise early stage disease, not warranting upstaging. On the other hand, research is lacking into the role of floaters in prognosis, as well as associations with other characteristics of the patient and case. The true incidence and etiology of floaters is unknown. In the only study of its kind, in 2013, DeLair et al. explored the difference in the incidence of floaters in laparoscopic (LH) versus robotic-assisted (RH) surgery for endometrial cancer [bib_ref] Tumoral displacement into fallopian tubes in patients undergoing robotically assisted hysterectomy for..., Delair [/bib_ref]. They found the incidence of floaters in LH to be 2.2% versus 11.7% in RH (P < 0.001). The majority of the patients with RH and tumor present in the tubes had Stage I disease (9/16, 56.2%) and Grade 1 tumors (9/16, 56.2%). Patients with floaters had a non-statistically significant higher body mass index. The authors postulated that floaters were a contaminant secondary to the placement of a uterine manipulator and also uterine manipulation, which they postulated was higher in RH [bib_ref] Tumoral displacement into fallopian tubes in patients undergoing robotically assisted hysterectomy for..., Delair [/bib_ref] [bib_ref] High incidence of positive peritoneal cytology in low-risk endometrial cancer treated by..., Sonoda [/bib_ref] [bib_ref] Does the use of a uterine manipulator with an intrauterine balloon in..., Lim [/bib_ref]. The objective of this study was to examine a series of cases with the finding of floaters on final pathology within a cohort of endometrial cancer patients in order to better understand the role of floaters in disease, including associations with patient, surgical, and pathologic characteristics, and assessment for impact on outcomes of time to progression and overall survival. # Methods ## Study design This study represents the experience of a single health system (Yale New Haven Health including Yale New Haven Hospital and the affiliated Bridgeport Hospital) with a new diagnosis of uterine cancer over a six year period. A retrospective cohort was formed from consecutive patients presenting to the health system's Division of Gynecologic Oncology with uterine cancer between January 1, 2005 and December 31, 2010. Diagnosis of pathologic subtype of uterine cancer was performed by trained gynecologic pathologists and all cases were reviewed at a multidisciplinary tumor board. This sample included both patients with preoperative biopsy showing uterine cancer followed by benign final pathology at the time of surgery, as well as patients with no sampling or benign preoperative pathology, and invasive cancer on final pathology of the surgical specimen. Study follow up was completed at the start of data extraction, defined as June 1st, 2016. ## Data extraction Institutional Review Board (IRB) approval was obtained before starting the study. Data was manually extracted from electronic health records. Extracted data included baseline patient characteristics, treatment, and outcomes (age, gravidity, parity, menopausal status, BMI, OCP use, HRT use, smoking status, medical and surgical history, adjuvant treatment, recurrence, death) from provider clinic and hospital notes, surgical characteristics (surgeon, approach, additional procedures) from operative notes, and pathological characteristics from preoperative, frozen, and final pathology notes. It should be noted that the presence of "floaters" on final pathology had no impact on the stage assigned to a patient as the International Federation of Gynecology and Obstetrics (FIGO) does not consider it in their staging system. The FIGO 2009 staging system was used for all patients [bib_ref] Revised FIGO staging for carcinoma of the endometrium, Creasman [/bib_ref]. Dates of death were confirmed with publically available obituary data. For patients lost to follow up, the date of last contact was used for the date of censure. For patients known to have died, but with no identifiable date of death, the date of last contact was approximated as the date of death. Data extraction was performed in parallel by four researchers (BA, JB, SG, MW, RP) in a standardized fashion using a single data entry form. ## Study sample The analysis in this study was performed on a subset of the above described cohort of patients with endometrial cancer, limited to patients with Type I (FIGO Grade 1 and 2 endometrioid adenocarcinoma) or Type II (FIGO Grade 3 endometrioid adenocarcinoma, clear cell carcinoma of the endometrium, uterine papillary serous carcinoma, and mixed endometrial carcinomas) disease. Patients with uterine sarcomas, carcinosarcomas, and squamous cell carcinomas were excluded. The study sample was further limited by excluding patients with missing data for variables critical for defining the type of disease (cellular histology on the final pathologic specimen), and for determining the outcome in time-to-event analysis (date of diagnosis and current disease status). ## Analyses The primary characteristics of interest in this study was the presence or absence of 'floaters.' For the purposes of this study, 'floaters' was defined as the presence of free floating tumor fragments identified within the fallopian tubes on sectioning and final pathological analysis of the specimen. Baseline characteristics of patients having pathology with and without the presence of floaters were compared using Student's t-test for continuous variables, and Pearson's χ 2 test for dichotomous and categorical variables. Descriptive statistics were used to describe the prevalence of floaters by cancer stage and nuclear grade at the time of surgery, as well as the relationship to positive peritoneal cytology. Of note, peritoneal cytology status was not originally included in the extracted data and was only assessed and available for cases with the presence of floaters. Associations of the presence of floaters with surgical and pathologic characteristics were investigated using bivariate and multivariate logistic regression. For primary analyses, time-to-event analysis was performed using the dates of diagnosis, progression, lost to follow up, and death. Bivariate and multivariate Cox proportional hazards models were created to assess the association of different patient and disease characteristics on the risk of progression and death. For all analyses, the date of diagnosis represented time zero and was defined as the date of tissue sampling showing endometrial cancer, whether disease was first identified on preoperative endometrial sampling or at the time of hysterectomy on frozen or final pathological analysis. For time to progression analysis, yielding hazard ratios for risk of progression, failure was defined by disease recurrence or progression, and patients were censored at date of lost to follow up or death without disease recurrence or progression. In addition to our primary exposure of floaters on final pathology, other risk factors for progression were considered, including cancer type and stage, and patient age and race. For overall survival analysis, yielding hazard ratios for risk of death, failure was defined by death from any cause, and patients were censored at date of lost to follow up. In addition to aforementioned risk factors for progression, a wider set of patient characteristics was included in multivariate models, as the risk of death from any cause is impacted by the overall health of the patient. We considered comorbidities including hypertension, diabetes, major cardiovascular disease (congestive heart failure, myocardial infarction, or stroke), psychiatric disease, history of additional primary cancer, and smoking history. Kaplan-Meier plots were constructed to visually compare time to progression and overall mortality with, and without the presence of floaters on final pathology, with and without adjustment. All analyses were performed using Stata 13.1 (Stata Corporation, College Station, TX). # Results ## Included sample Retrospective chart review was performed on 1022 consecutive patients diagnosed with uterine cancer presenting to our institution over a 6 year period from January 1st, 2005 to December 31st, 2010. The included cohort for this study was limited to a subset of 816 patients with Type 1 (n = 550) and Type 2 endometrial cancer (n = 266). Only four total patients were excluded for missing critical data. Within this sample, 20 patients (2.56%) were noted to have fragments of tumor cells noted within the fallopian tubes (floaters) on final pathology. ## Baseline patient characteristics Baseline patient characteristics for the cohort of patients, by Overall, the small group of 20 patient's found to have floaters on final pathology mirrored the overall population characteristics of the cohort. ## Description of cases with floaters The 20 cases in which floaters were identified on final pathology are described in more detail, by Type I vs Type II, nuclear grade, pelvic washing status, and surgery type, in [fig_ref] Table 2: Case distribution, by presence or absence of floaters, a for single institution... [/fig_ref]. Only 6 cases of floaters were identified among the 550 cases of Type 1 disease under review, 4 of which were found in patients with Stage I disease (stage IA n = 3; stage IB n = 1, implying that free floating fragments were found within the fallopian tubes without any coincident invasion of the adnexa. Despite these free floating fragments within the endometrial cavity, only 1 of the 6 (16.6%) cases with floaters in Type I disease also had positive pelvic washing cytology (stage IIIA due to presence of fallopian tube invasion). Floaters were present in a significantly greater proportion of Type II disease specimens (5.3% vs 1.1%, p < 0.001), a total of 14 of 266 cases. Unlike in Type 1 disease, 71.4% of cases with floaters in Type 2 disease were in patients with Stage III or IV disease, and 42.9% of cases with floaters also had positive pelvic washing cytology. Among the 20 cases with floaters, 15 were nuclear grade 3, while 5 were nuclear grade 2, and no cases were nuclear grade 1. In the analysis of surgery type, a trend towards a higher rate of floaters in cases done laparoscopically or robotically (OR = 3.84; 95%CI 0.98-15.1), in which a uterine manipulator would have been placed. ## Associations with surgical and pathologic characteristics The cases included in this cohort were generally treated according to the standard of care in practice at the time. The majority of patients underwent surgery that included staging with pelvic lymph node dissection (89.0%) and paraaortic lymph node dissection (71.2%), and, less commonly, additional procedures such as omentectomy (26.9%) or appendectomy (3.5%). The majority of patients underwent adjuvant treatment with vaginal brachytherapy (71.7%), while adjuvant carboplatin and paclitaxel chemotherapy was more commonly used for Type II cancers (65.1%) than Type I cancers (17.2%). Most patients that did not undergo adjuvant treatment had Type I disease (89.1%) with < 50% invasion (94.2%). Associations of the presence of floaters with surgical approach and other pathologic characteristics from the final specimen are presented in [fig_ref] Table 3: Associations of presence of floaters a with surgical approach and pathological characteristics... [/fig_ref]. In unadjusted bivariate logistic regression, the presence of floaters was positively associated with Type II disease, nuclear grade 3 (vs. grade 1 or 2), presence of lymphovascular invasion, and overall stage III or IV. As floaters were more common in Type II disease, and Type II disease is known to be associated with more extensive disease, it was necessary to use multivariate logistic regression to more accurately explore these associations. In adjusted analysis, the type of disease was found to be unrelated to the presence of floaters (OR = 1.77; 95%CI 0.21-14.6). The association of floaters with higher nuclear grade was similarly eliminated with adjustment. However, with multivariate adjustment, significant associations of floaters with positive lymphovascular invasion (OR = 9.65; 95%CI 2.35-39.6) and Stage III disease remained. Additionally, the extent of myometrial invasion was inversely related to the likelihood of observing floaters, with an OR of 0.08 for comparing no invasion to 100% invasion. Furthermore, a trend was observed towards an association of floaters with laparoscopic/robotic approach and uterine manipulator use (OR = 3.84; 95%CI 0.98-15.1; p = 0.054). ## Analysis of time to progression In unadjusted analysis, floaters were significantly associated with risk of progression/recurrence of disease (HR = 3.22, 95%CI 1.41-7.37), however this association was eliminated with adjustment for risk factors (HR = 1.10, 95%CI 0.46-2.60, [fig_ref] Table 4: Cox Proportional Hazard models for recurrence and death in a single institution... [/fig_ref]. In addition to cancer type (Type 2 vs Type 1) and stage (Stage III and IV vs Stage I), patient characteristics associated with increased risk of recurrence in adjusted models included age > 65 (HR = 2.22, 95%CI 1.23-4.00) and a trend towards increased risk in nonwhite race (HR = 1.62; 95%CI 0.99-2.66). As there is no biologic basis for patient medical comorbidities to impact disease recurrence, these variables were not found to be significantly associated with the outcome at hand, and were excluded from adjusted models for time to progression. Complete Cox regression data and Kaplan-Meier survival plots for unadjusted and adjusted analyses of time to progression can be seen in [fig_ref] Table 4: Cox Proportional Hazard models for recurrence and death in a single institution... [/fig_ref] and [fig_ref] Figure 1: Kaplan-Meier Survival Plots, adjusted and unadjusted, for [/fig_ref] ## Analysis of overall survival Floaters were not associated with decreased overall survival in unadjusted (HR = 1.84, 95%CI 0.86-3.91) or adjusted analysis. In the adjusted model, patients with Type 2 disease were at significantly increased risk of death . Increasing disease stage and patient age were also tightly associated with risk of death. History of smoking and of major cardiovascular disease (congestive heart failure, myocardial infarction, or stroke) at the time of diagnosis were significantly associated with risk of death (HR = 1.79, p = 0.001; HR = 1.84, p = 0.025, respectively). Interestingly, non-white race remained a near significant predictor of increased risk of death, even in the adjusted model (HR = 1.46; 95%CI 0.98-2.18; p = 0.066). Complete Cox regression data and Kaplan-Meier survival plots for unadjusted and adjusted analyses of overall survival can be seen in [fig_ref] Table 4: Cox Proportional Hazard models for recurrence and death in a single institution... [/fig_ref] and [fig_ref] Figure 1: Kaplan-Meier Survival Plots, adjusted and unadjusted, for [/fig_ref] # Discussion The true incidence and etiology of floaters is unknown and there is scant evidence about their relationship with recurrence and survival. Furthermore, the presence of floaters does not play a role in the FIGO Staging System for endometrial cancer [bib_ref] Revised FIGO staging for carcinoma of the cervix, Pecorelli [/bib_ref]. Therefore, when this pathologic finding is encountered, there exists a diagnostic and therapeutic conundrum: In early stage cancers with floaters, should patients be treated similar to a Stage IIIA with adnexal involvement or should they be treated as a Stage I with floaters being a non-significant incidental finding? We report an overall incidence of floaters of 2.5%, which is within range of the only published incidence rate. Delair et al. reported a range from 2 to 11%; 2% when laparoscopic hysterectomy was performed and 11% when Robotic-assisted laparoscopic hysterectomy was performed [bib_ref] Tumoral displacement into fallopian tubes in patients undergoing robotically assisted hysterectomy for..., Delair [/bib_ref]. Our data showed that floaters were more common in Type II disease specimens when compared to Type I specimens (5.3% vs 1.1%, p < 0.001). In addition, it was found that in early stage disease, floaters were more common in Type II cancers (2.45% vs 0.83%; p = 0.107). When looking at both risk of recurrence and death, however, the presence of floaters did not appear to play a significant or causative role. Thus, it is more likely that in Type II cancer, where stage at presentation was often more advanced, it is the extent of disease and overall aggressiveness which are responsible for the high risk of recurrence and death, and likely not the presence of floaters within the fallopian tube lumen. Given that positive washings in the setting of floaters is three times more likely in Type II versus Type I cancers, it could further be assumed that it is the late stage of presentation in Type II cases which causes positive washings versus the floaters themselves. Based on these results, there should be no change in management practices based on the presence of floaters in Type II cancers. In early stage disease, there was no increased risk of recurrence or death when floaters were present. It stands to reason that the presence of floaters is somewhat analagous to the presence of positive peritoneal cytology, in that it is a pathologic finding without major prognostic impact in the setting of otherwise, low risk disease [bib_ref] FIGO staging of endometrial adenocarcinoma: a critical review and proposal, Zaino [/bib_ref]. Therefore, based on the current available data, for both Type I and Type II cancers, we do not recommend the presence of floaters be considered as a factor in determining whether or not a patient needs adjuvant therapy. We also find evidence that, controlling for cancer stage and other pathologic findings, floaters were more likely to be found in cases performed laparoscopically or robotically as compared to abdominal hysterectomy (HR = 3.85, p = 0.05 for all stages, HR = 7.34; p = 0.05 for stage I or II). This finding indicates the possibility that floaters could sometimes be a result of tissue dislodged by intra-operative use of a uterine manipulator, a theory that was also proposed by Uterine manipulators have been previously postulated to cause higher rates of positive peritoneal cytology and "psuedo" lymphovascular space invasion by disruption of tissue [bib_ref] High incidence of positive peritoneal cytology in low-risk endometrial cancer treated by..., Sonoda [/bib_ref] [bib_ref] Vascular pseudo invasion in laparoscopic hysterectomy specimens: a diagnostic pitfall, Logani [/bib_ref]. However, both of these associations, as well as any impact on outcomes from uterine manipulator use have been discounted in a number of more recent studies [bib_ref] Lymphovascular space invasion in robotic surgery for endometrial cancer, Hopkins [/bib_ref] [bib_ref] Effects of uterine manipulation on surgical outcomes in laparoscopic management of endometrial..., Lee [/bib_ref] [bib_ref] Does the type of surgery for early-stage endometrial cancer affect the rate..., Momeni [/bib_ref] [bib_ref] Does a uterine manipulator affect cervical cancer pathology or identification of lymphovascular..., Rakowski [/bib_ref] [bib_ref] Relationship between minimally invasive hysterectomy, pelvic cytology, and lymph vascular space invasion:..., Zhang [/bib_ref]. The major limitation to this study is that it was retrospective in nature and limited to a single institution. As this study was conducted retrospectively, no prospective power analysis was performed for the question at hand. We recognize that the small number of cases limited our power to detect a small difference in rare outcomes, in particular for death and thereby overall survival. With that being said, there is a scarcity of data on the role of floaters as a pathologic finding, their association with other pathologic findings, and their impact on patient outcomes. Our findings are the first of their kind and further pooled data would be useful. [fig] Figure 1: Kaplan-Meier Survival Plots, adjusted and unadjusted, for (A) Time to Progression and (B) Overall Survival 1 , by presence or absence of free floating tumor fragments within the fallopian tubes ('floaters'), in Type I and Type II endometrial cancer, single institution cohort presenting 2005-2010. [/fig] [table] Table 1: Demographic characteristics of single institution cohort of patients with type I and type II endometrial adenocarcinoma, by presence or absence of floaters. a nificance; for dichotomous and categorical variables, percent with characteristic shown, Pearson's χ 2 test for significance. Floaters were defined as evidence of free-floating tumor fragments within the fallopian tubes on final pathologic specimen analysis. b Includes history of congestive heart failure, myocardial infarction, and stroke. c Includes diagnoses of depression, anxiety, bipolar, and schizophrenia. [/table] [table] Table 2: Case distribution, by presence or absence of floaters, a for single institution cohort of patients with type I and type II endometrial adenocarcinoma, 2005-2010. Floaters were defined as evidence of free-floating tumor fragments within the fallopian tubes on final pathologic specimen analysis. b Pelvic washing status data only recorded for cases with presence of floaters. [/table] [table] Table 3: Associations of presence of floaters a with surgical approach and pathological characteristics in a single institution cohort of patients with type I and type II endometrial ade- [/table] [table] Table 4: Cox Proportional Hazard models for recurrence and death in a single institution cohort of patients with type I and type II endometrial adenocarcinoma,2005-2010. [/table]

Top-down resolution of visual ambiguity – knowledge from the future or footprints from the past? Current theories about visual perception assume that our perceptual system weights the a priori incomplete, noisy and ambiguous sensory information with previous, memorized perceptual experiences in order to construct stable and reliable percepts. These theories are supported by numerous experimental findings. Theories about precognition have an opposite point of view. They assume that information from the future can have influence on perception, thoughts, and behavior. Several experimental studies provide evidence for precognition effects, other studies found no such effects. One problem may be that the vast majority of precognition paradigms did not systematically control for potential effects from the perceptual history. In the present study, we presented ambiguous Necker cube stimuli and disambiguated cube variants and systematically tested in two separate experiments whether perception of a currently observed ambiguous Necker cube stimulus can be influenced by a disambiguated cube variant, presented in the immediate perceptual past (perceptual history effects) and/or in the immediate perceptual future (precognition effects). We found perceptual history effects, which partly depended on the length of the perceptual history trace but were independent of the perceptual future. Results from some individual participants suggest on the first glance a precognition pattern, but results from our second experiment make a perceptual history explanation more probable. On the group level, no precognition effects were statistically indicated. The perceptual history effects found in the present study are in confirmation with related studies from the literature. The precognition analysis revealed some interesting individual patterns, which however did not allow for general conclusions. Overall, the present study demonstrates that any future experiment about sensory or extrasensory perception urgently needs to control for potential perceptual history effects and that temporal aspects of stimulus presentation are of high relevance.PLOS ONEPLOS ONE | https://doi.org/10.1371/journal.pone. # Introduction Our everyday experience suggests that we perceive the world as it is. However, due to the limited capacities of our senses the available information is a priori remarkably incomplete, noisy, and to varying degrees ambiguous. The perceptual system has to construct-as fast as possiblestable and reliable perceptual interpretations out of this limited information. One strategy to overcome this perceptual inference problem, as already described by Helmholtz and others (for a nice historic overview see, is to take temporal aspects of perception-on different time scales-into account. Our perceptual environment changes only slightly from one moment to the next. We can thus exploit perceived regularities from our immediate perceptual history to make predictions about the immediate perceptual future. But also longer-term memories can influence how we perceive the world. In the recent years, predictive coding theories have successfully modeled the influence of the perceptual history for normal visual perception and altered perception in psychiatric disorders. Different lines of research demonstrate the influence of the perceptual history from different time scales on the current percept. Studies reported positive effects (i.e. current stimuli are likely to be perceived in the same way as previous ones), like positive priming, positive hysteresisor serial dependence. In contrast, other studies reported negative effects (e.g. current stimuli are likely to be perceived as opposite of the previous one), like adaptation or negative hysteresis. Further examples are motion aftereffects, contrast aftereffects, or repetition suppression. Moreover, perceptual history effects can be found at different time scales and different complexity levels along the perceptual processing chain, from low-level visual functions, like contrast or motion perception, up to the processing of emotional content of faces and even beyond. Moreover, the perceptual history not only influences our current percepts, but our current percepts also add to our perceptual memories and change them. We thus never process the same sensory input in the same way [e.g.]. ## Long-term perceptual history effects One convincing example of how our perceptual memories contribute to our current percept is 3D perception and in particular, perception of the ambiguous Necker cube: During our first steps of visual processing, images of three-dimensional objects are projected on twodimensional retinae [e.g.. We thus only have direct access to two out of three dimensions. Our perceptual system needs to reconstruct the missing third dimension out of secondary information, like occlusion, stereopsis, movement parallax, etc. [e.g . The Necker cube (see the Necker lattice, i.e. a construction of 3x3 Necker cubes in is a 2D drawing of a 3D cube grid that demonstrates the limits of such perceptual 3D reconstruction. The 2D image on our retinae induced by the Necker cube drawing is equally compatible with two 90˚angle cubes with opposite spatial orientations. As a consequence, perception of the Necker cube is unstable and reverses between a "front-side to the bottom-right" perspective ("B"-perspective, and a "front-side to the top left" perspective ("T"-perspective, . Moreover, most observers' percepts are biased towards a B-perspective . One post-hoc explanation of this bias is close to Helmholtz's (and others) perceptual inference approach and concurrently demonstrates influences from long-term perceptual memory: During our lifetime we much more often look down on objects in our environment than up to them. This makes the B-perspective of the Necker cube more probable than a T-perspective interpretation. In the following, we will call this the long-term perceptual history effect. Even more confirmation for long-term memory effects comes from the fact that the Necker cube image on our retinae is in principle compatible with almost infinitively many geometric objects [for a nice demonstration of this see and the common view is that 90˚interpretations are simply more probable because of the many 90˚objects in our environment. ## Immediate perceptual history effects The Necker cube example can also nicely demonstrate how the immediate perceptual history can influence our current percept. Having observed a disambiguated cube variant can have priming (identical percept) or adaptation effects (opposite percept) for the subsequent perception of the Necker cube, depending on how long the preceding disambiguated variant had been observed [e.g . Furthermore, it has been shown that with a discontinuous presentation mode (Necker cube presentations are interrupted by short blank screen gaps) the dynamics of perceptual reversals [i.e. its "dwell time", e.g. 30] strongly depend on the duration of the intermediate gap. ## Precognition effects Precognition is the proposed ability to perceive or sense events or more general information at the present moment, although this information will only be generated in the future. A large body of phenomenal reports about precognition experiences, together with a number of empirical studies and meta-analyses thereof, indicate the existence of precognition phenomena and have even evoked hypotheses about the relevance of precognition during the evolution of human cognition. On the other hand, phenomenal reports cannot be confirmed statistically and replications of many of the empirical studies failed to reproduce the effect, taking the existence of precognition into question. One critical factor for precognition experiments is randomization. The stimulus material needs to be presented in a randomized order to prevent any regular temporal pattern of stimulus presentation that may allow predicting the future stimuli and thus confound the study results [e.g. 51]. A second aspect of an appropriate randomization is making sure that all used stimuli are presented about equally often over the whole experiment in order to prevent possible quantitative confounds in the results. However, presenting different types of stimuli in a precognition experiment, using an appropriate randomization procedure and concurrently making sure that each stimulus occurs with about the same frequency, may not be enough control for precognition experiments. Another potentially confounding but so far hardly recognized factor in previous precognition studies is the influence of the perceptual history. As discussed above, it has been shown that what we see at the current moment is influenced by what we saw a millisecond, a second, a minute (or even longer) before. And even with a randomization procedure it is possible that certain stimuli have in some way stronger and/or longer lasting memory effects on the perceptual system than other stimuli. It is thus mandatory for any precognition analysis to control for perceptual history effects. Ambiguous figures, like the Necker cube, are regarded as interesting stimuli to study precognition phenomena, because observing an ambiguous figure evokes unstable perceptual states. The idea is that observers in unstable perceptual states or more generally unstable mental states are more sensitive for the proposed information from the future than observers in stable perceptual and/or mental states. Some years ago, Biermanstudied precognition effects, using the ambiguous Necker cube and two apparently disambiguated cube variants, corresponding to the two most probable Necker cube percepts. His participants observed a continuous presentation of the Necker cube and were instructed to press one key when perceiving the B-perspective (Response 1) and another key when perception changed to the T-perspective (Response 2). Immediately after Response 2, the computer program replaced the Necker cube with a random selection of one of the two disambiguated cube variants. Bierman compared the duration of the B-perspective during observation of the ambiguous Necker cube (i.e. the time between Response 1 and Response 2) as a function of the identity of the subsequent disambiguated variant, presented after Response 2. The basic assumption underlying this study was that the two disambiguated cube variants retrocausally or precognitively influence the duration of the B-perspective perception of the preceding Necker cube in different directions. Bierman executed three separate experiments in this way, a pilot (N = 5 participants) and two follow-up experiments with N = 26 and N = 122. According to his description, the pilot experiment served as hypothesis generating, and its results indicated a retrocausal adaptation effect. In detail the duration of the B-perspective percept was shorter when a disambiguated T-perspective cube followed, compared to a subsequent disambiguated B-perspective cube variant. In Experiment 2, he found a weak confirmation of this result, in Experiment 3 only a tendency. The study described above, contained a number of obscurities. It was unclear, why Bierman restricted his analyses only to the duration of B-perspective Necker cube percepts, how and at exactly what time (before or after execution of the experiment) the criteria for outliers had been defined (particularly in the pilot experiment) and why he integrated the data from the hypothesis-generating pilot experiment into the confirmatory final analysis, to name only some. Moreover, and particularly relevant for the present study, Bierman interpreted his results as evidence for perceptual precognition without controlling for perceptual history effects. In the present study, we also presented Necker cube stimuli and disambiguated stimulus variants, but used a slightly different experimental paradigm and a different analysis approach than Bierman (2011), as will be motivated below. Overall, we executed two experiments, where we systematically investigated both potential influences from the perceptual future but also potential influences from the perceptual past. In summary, in our Experiment 1, we found strong evidence for perceptual history effects, some indication for precognition-like effects in individual participants but no significant precognition effects on the group level. In the subsequent Experiment 2, we again found perceptual history effects and some participants with precognition-like effect indications but no precognition effects on the group level. # Methods-experiment 1 Participants 13 participants (7 women, 6 men) took part in Experiment 1. The median age was 24 with a range between 21 and 34 years. All participants had normal or corrected-to-normal visual acuityand gave their written consent to participate in this study. The study was performed in accordance with the ethical standards laid down in the Declaration of Helsinkiand was approved by the local ethics board (Ethik-Kommission der Albert-Ludwigs-Universität Freiburg). ## Stimuli We used a so-called Necker lattice, which is a variant of the famous Necker cubeconsisting of 3x3 Necker cubes . This Necker lattice is most often perceived in two different and mutual exclusive 3D configurations, either with the front side pointing to the bottom right (B-perspective) or with the front side pointing to the top left (T-perspective). We also used two disambiguated lattice variants, corresponding to these two perceptual interpretations of the Necker lattice . These disambiguated lattices were transparent like the Necker lattices but contained depth cues, based on a drawing model incorporating shading, central projection, and aerial perspective (OpenGL lighting model;. All lattices had light grey edges on a dark background and were presented with a visual angle of 7.5˚x 7.5˚. The Necker lattice had a uniform luminance of 40 cd/m 2 . Due to the depth cues of the disambiguated lattice, the edges had inhomogeneous luminance with darker edges of the apparent back layer than of the apparent front layer. We chose the edge luminance of those lattices in a way that the average luminance across the eight outer lattice corners was at 40 cd/m 2 . The stimuli were presented with the open software PsychoPyon an Apple Mac mini computer. ## Paradigm Participants had a distance of 114 cm from a Philips GD 402 monochrome CRT screen (refresh rate = 85 Hz, screen resolution = 800 x 600 pixel) and were instructed to fixate a cross in the middle of the screen. Experiment 1 consisted of four conditions. Within each condition, the lattice stimuli were presented in pairs forming a so-called observation sequence ("OS") in the following way (see also : The first stimulus S1 was presented for 800 ms, followed by an inter-stimulus interval ("ISI") with a grey screen for 400 ms, which was followed by stimulus S2 for 800 ms and finally by another grey screen (inter-observation-sequence interval, "IOSI") for 1000 ms. Within each OS, participants had two tasks. In Task 1 they indicated their percept of the presented lattice stimulus S1 per key press (different keys for B-perspective and T-perspective). In Task 2 they compared their percept of stimulus S2 with their previous percept of S1 and indicated a changed percept by one key and perceptual stability (i.e. identical percepts of S1 and S2) by a second key. In the conditions with the ambiguous lattice stimuli, perceptual reversals occur endogenously within the perceptual system of the observer. According to the literature, such endogenous reversals take place with a probability of 0.3 on average (e.g.. The disambiguated lattices were alternated pseudo-randomly to simulate the spontaneous perceptual reversals of the ambiguous variants. We adopted the reversal probability from ambiguous variants for the exogenous reversal rate of the disambiguated Necker lattice variants. Each condition was subdivided in 3 experimental blocks of about 9 minutes duration and each experimental block consisted of 180 observation sequences from the respective condition. We varied the ambiguity levels of S1 and S2 between experimental conditions but kept them constant within experimental conditions (and thus within experimental blocks). This resulted in four experimental conditions as defined in . In total, each participant thus performed 12 experimental blocks within three successive time periods with breaks of about 10 minutes between periods. The order of the experimental blocks was pseudo-randomized. One experimental session lasted for about 3 hours with roughly 1.5 hours measurement time. The study described here was part of a larger research project and included EEG recordings. Separate and non-overlapping aspects of this study have already been published elsewhere. In the present analysis, we only focused on the behavioral data from the conditions AD Lattice stimuli were presented in pairs, the first lattice stimulus S1 was followed by the second lattice stimulus S2. S1 was presented for 800 ms and participants indicated with different keys whether they perceived it in the B-or the T-perspective. After a grey screen inter-stimulus interval (ISI) of 400 ms lattice S2 was presented for 800 ms. Participants compared their percept of S2 with their memorized percept of S1 and indicated by key press either perceptual stability (same perceived perspectives of S1 and S2) or perceptual reversal (change between B-and T-perspective). S2 was followed by an inter-observation interval (IOSI) of 1000 ms. In the present example case, both S1 and S2 were disambiguated lattice versions. In our study, the ambiguity levels of S1 and S2 stayed constant within but varied between experimental conditions. A variant of this figure has already been presented in a recent publication from our lab [see and DA, in which ambiguous Necker lattices alternate with disambiguated lattice stimuli. The here analyzed aspects of the study data are novel and had not been part of the above mentioned publication. # Data analysis Perceptual history effects. When focusing on Condition AD to study the influence of the immediate perceptual history on the current percept of an ambiguous Necker lattice, we sorted the observation sequences (OS) into eight separate groups of stimulus/percept sequences: BBB-Sequences (AD Condition): - The disambiguated lattice S2 from the previous OS had a B-perspective - The currently observed ambiguous lattice S1 from the current OS is perceived in the Bperspective . Within one OS two lattice stimuli S1 and S2 were presented in succession and participants had to execute respective tasks, as explained in In Condition AD, stimulus 1 (S1) was always an ambiguous lattice ("A") and S2 a disambiguated lattice variant ("D"). In Condition DA, S1 was the disambiguated lattice and S2 the ambiguous lattice variant. Lattice ambiguity levels stayed constant within but differed between experimental blocks/conditions. A variant of this figure has already been presented in a recent publication from our lab [see . https://doi.org/10.1371/journal.pone.0258667.g003 . Experimental condition. ## Conditions Ambiguity level of S1 and S2 Condition 1: AA S1 and S2 ambiguous ## Plos one Knowledge from the future or footprints from the past? - The disambiguated lattice S2 from the current OS will have a B-perspective BBT-Sequences: - The disambiguated lattice S2 from the previous OS had a B-perspective - The currently observed ambiguous lattice S1 from the current OS is perceived in the Bperspective - The disambiguated lattice S2 from the current OS will have a T-perspective The logic of this grouping is visualized inAccording to this rule the other six groups were BTB, BTT, TBB, TBT, TTB and TTT. The number of occurrences of either of those groups will be labeled with an "n"-prefix (e.g. nBTB = number of BTB group occurrences). The average number of analyzed sequences across participants is reported in. A similar logic applies when focusing on Condition 3 (DA), again with eight separate groups of sequences: BBB-Sequences (DA Condition): - The disambiguated lattice S1 from the current OS had a B-perspective - The currently observed ambiguous lattice S2 from the current OS is perceived in the Bperspective Grouping of analysis sequences. The logic behind the grouping underlying the data analysis, exemplified for the history effect analysis. We compared the percentage of group occurrences of percepts of the ambiguous stimulus (S1 current OS, green frame) given that the subsequently presented lattice variants will be identical but the preceding lattice variants differed. For the precognition effect analysis we varied the ambiguity level of the subsequent stimulus but kept it for the preceding stimuli constant.: P-perspective; T: T-perspective. https://doi.org/10.1371/journal.pone.0258667.g004 ## Plos one Knowledge from the future or footprints from the past? - The disambiguated lattice S1 from the subsequent OS will have a B-perspective BBT-Sequences: - The disambiguated lattice S1 from the current OS had a B-perspective - The currently observed ambiguous lattice S2 from the current OS is perceived in the Bperspective - The disambiguated lattice S1 from the subsequent OS will have a T-perspective According to this rule the other six groups were BTB, BTT, TBB, TBT, TTB and TTT. The number of occurrences of those groups will be labeled with an "n"-prefix (e.g. nBTB = number of BTB group occurrences). The average number of analyzed sequences across participants is reported in. Please keep in mind that in the AD condition the temporal distance between onset of the ambiguous lattice and the preceding disambiguated lattice variant was 1000 ms whereas in the DA condition it was only 400 ms and thus 600 ms shorter (see. Accordingly, the temporal distance between onset of the ambiguous lattice and the subsequent disambiguated lattice variant in the AD condition was 400 ms whereas in the DA condition it was 1000 ms and thus 600 ms longer. Further, the tasks differed between stimuli S1 and S2 (see , which results in another difference between the AD and DA data in terms of the relation between stimulus ambiguity level and task. In condition AD, participants had to perform a comparison task in response to disambiguated variants (S2) and an identification task in response to ambiguous variants (S1). In condition DA, however, participants had to perform an identification task in response to disambiguated variants (S1) and a comparison task in response to ambiguous variants (S2). In order to study perceptual history effects and precognition effects, we first calculated probabilities of B-percepts of the ambiguous Necker lattice as a function of the identity of the preceding and subsequent disambiguated lattice variants in the following way (the bold letters indicates the perceptual interpretation of the ambiguous Necker lattice): [formula] P BBB ð Þ ¼ nBBB nBBB þ nBTBð1ÞP BBT ð Þ ¼ nBBT nBBT þ nBTTð2Þ [/formula] ## Plos one Knowledge from the future or footprints from the past? [formula] P TBB ð Þ ¼ nTBB nTBB þ nTTB ð3Þ P TBT ð Þ ¼ nTBT nTBT þ nTTTð4Þ [/formula] We restricted our analysis to B-percepts of the ambiguous Necker lattice for the following reason: Focusing on Eq (1) it becomes immediately clear that P(BBB) + P(BTB) = 1. The same principle applies to Eqs 2-4. The probabilities of T-percepts of the Necker lattice as a function of preceding and subsequent disambiguated Necker lattice are thus completely dependent on the probabilities of the B-percepts. For the statistical analyses of perceptual history and precognition effects, we calculated an ANOVA with the factors CONDITION, (immediate) HISTORY and (immediate) FUTURE, each with two steps. The ANOVA thus compares P(BBB) with P(BBT) and correspondingly P (TBB) with P(TBT) to test for precognition effects, while controlling for the immediate perceptual history. The ANOVA further compares P(BBB) with P(TBB) and P(BBT) with P(TBT) to test for history effects, while controlling for possible precognition effects. Given that the Necker lattice is physically completely ambiguous, we can postulate equal probabilities of p = 0.5 to perceive it in the B-and T-perspective if no history effects (neither short-term nor long-term) and no precognition effects are present (null hypothesis). Systematic deviations from the 0.5 level would be evidence in favor of an a priori perceptual bias for the Necker cube (i.e. a long-term perceptual history effect). The ANOVAs thus further tested for this with the factor BIAS. # Results-experiment 1 A graphical demonstration of the different perceptual probabilities on both the level of participants and grand means is presented inThe contrasts mentioned above for both the perceptual history and precognition analyses (differences between the respective probability values) are graphically presented in Figs 7 and 8. In these difference graphs, any systematic deviation of the differences of probability values from zero would indicate effects in the respective directions. Participants perceived the ambiguous Necker lattice overall more often in the B-perspective than in the T-perspective, independent of the identities of the disambiguated preceding and the subsequent lattice variants and of the experimental conditions (p = 2.9 � 10 −09 , F(1,96) = 42.84, η 2 = 0.22 for the factor BIAS).this by the fact that the majority of icons are above the (grey dashed) horizontal 0.5-line. Further, in the condition AD the probability to perceive the Necker lattice in the B-perspective was significantly larger, when the preceding stimulus was the disambiguated lattice variant with the T-perspective, than when it was in the B-perspective. This can also be observed as a tendency in the DA condition, but the effect is much weaker (p = 0.003, F(1,96) = 9.27, η 2 = 0.02 for the interaction between the factors HIS-TORY and BIAS). This can be seen inwhere more of the icons in the TBB and TBT sequences are above the 0.5-line than in the BBB and BBT sequences and where this pattern is more prominent in the AD condition (left) than in the DA condition (right). ## Plos one We found no statistically significant precognition effect, no further difference between the experimental conditions and no further interaction. # Discussion-experiment 1 In Experiment 1, we presented ambiguous Necker lattices and disambiguated lattice variants in alternation and investigated whether the perceptual interpretations of the ambiguous Necker lattice can be influenced by the identity of the preceding (history effects) and/or of the subsequent (precognition effects) disambiguated lattice variants. ## History effects We found strong evidence for long-term perceptual history effects. Participants perceived the ambiguous Necker lattices in general more often in the B-perspective than in the T-perspective, independent of the experimental condition and also independent of the identities of the ## Plos one Knowledge from the future or footprints from the past? preceding and subsequent disambiguated lattice variants. This is a confirmation of reports about an a priori bias in favor of the B-perspective, when viewing an ambiguous Necker cube stimulus. The current (post-hoc) explanation of this effect is that we look much more often down on objects in our environment than up on them. This recurring perceptual experience over lifetime may thus serve as a long-term perceptual memory bias in favor of an a priori from-above perspective (i.e. the B-perspective). The probability of B-perspective percepts increased even more, when the preceding stimulus was a disambiguated lattice in the T-perspective. However, this short-term perceptual history effect was only present in the Condition AD, but not in the Condition DAmore grey triangular icons are above the 0.5-line on the left than on the right, see also difference values in. In the following, we discuss, how these apparently inconsistent results between conditions may be explained in the context of priming and adaptation. Several studies with ambiguous figures reported both positive (priming) and negative (adaptation) effects of the immediate perceptual history on the current percept [8,10,31,65- 72]. A recent study even indicated erroneous percepts of a currently observed stimulus, which could be traced back to the immediate perceptual history. The basic idea of priming in the context of ambiguous figures is, that the disambiguated stimulus pre-activates the related neuronal representation. The pre-activated representation then dominates perception of the subsequent ambiguous stimulus. The basic idea underlying adaptation is that perceiving the disambiguated stimulus variant for a longer time period reduces the responsiveness and/or stability of the underlying neural representation. At the time point when the ambiguous stimulus variant replaces the disambiguated stimulus, the not-yet adapted neural representation of the alternative stimulus interpretation dominates the adapted one and the observer perceives the ambiguous stimulus in this interpretation. After a previously dominant neural representation has become non-dominant, it starts to recover from adaptation with a stimulus-specific and most probably also participant-specific time constant. A number of studies are particularly interesting for the present findings, starting with work by Orbach et al., where the authors observed that recovery from adaptation starts immediately after the stimulus is turned off. However, if the next stimulus occurs before recovery has finished, the novel adaptation adds to the remaining level at stimulus onset-and so on (like a saw-tooth, however with an overall increase of adaptation level, until a maximum is reached). In a follow-up study, the same group found that, presenting a disambiguated version of the Necker cube, changes the perceptual probabilities of the two interpretations of a subsequently presented ambiguous Necker cube. In another relevant study, Long et al.worked with two separate ambiguous figures, namely with the Necker cube and with overlapping squares. In separate conditions they presented disambiguated variants of these stimuli for varying durations, ranging from 0 to 160 seconds. The disambiguated stimuli were followed by the ambiguous variants and participants indicated their percept of the latter. For short presentation durations of the disambiguated stimulus variants up to about 20 seconds, Long et al. found priming effects, i.e. participants perceived the ambiguous stimulus in the same way as the preceding disambiguated variant. For longer presentation times of the disambiguated stimulus variants, starting at about 80 seconds, they reported adaptation effects, i.e. participants perceived the ambiguous stimulus in opposite orientation as the preceding disambiguated variant. The authors termed this opposite pattern of perception of an ambiguous stimulus as a function of the duration of a previously observed disambiguated stimulus variant as "reversebias effect". In yet another study, Toppino et al. found that the adaptation effect becomes weaker as a function of the duration of a gap between the disambiguated and the ambiguous stimulus. The authors recently replicated their findings and provided an elaborated overview of the topic in the introduction of their publication. The literature above indicates that priming and adaptation are two independent processes that work on different time scales. Further, both neural representations (the B-perspective and the T-perspective) can be adapted independently. Moreover, the impacts of both priming and adaptation start to decay immediately after the conditioning stimulus (in our case the disambiguated lattice) is turned off (and in our case replaced by a grey screen in the ISI and IOSI). In addition, if the next conditioning stimulus occurs before the adaptation state of the perceptual system hasn't fully recovered, the new adaptation adds upon the remaining adaptation state. Given this background, one explanation for the heterogeneous results for the immediate history effects in the Conditions AD and DA may be the following: We assume that neither the ISI nor the IOSI are enough time for full recovery of adaptation states. As a consequence, due to the repetitive presentation of the disambiguated stimuli, adaptation of both perceptual representations may cumulatively reach a relatively high level of adaptation. In both conditions priming may also take place, however priming works on a much shorter time scale than adaptation. In Condition AD, the grey screen gap between the preceding disambiguated lattice (i.e. the IOSI, seeand the ambiguous lattice is 600 ms longer than in the DA Condition (i.e. the ISI, see. The compensatory power of the priming effect (against adaptation) may thus be weaker-due to the stronger decay of priming effects-resulting in a stronger adaptation effect in the AD condition than in the DA condition. ## Precognition We found no significant precognition effect on the group level. However, some interesting individual patterns of precognition-like effects are indicated in Two participants showed a difference between the compared probability values larger than 0.1 (indicated by green and red squares in . Particularly, one participant (the red square, surrounded by a black circle) showed a consistent deviation of the difference value from zero larger than 1.5 standard deviations and with identical signs in both experimental conditions the BBB minus BBT contrasts in AD and DA; the grey dashed and dotted lines indicate 1.5 and 2 standard deviations, respectively). The finding of relatively strong group-level history effects for the perception of the ambiguous Necker lattice but no group-level precognition effects, raises questions about the role of the perceptual history for potential precognition effects and whether precognition effects can be identified in an experimental setting without an influential immediate perceptual history as in this Experiment 1. We focused on this question in our Experiment 2. # Methods-experiment 2 ## Participants Twenty-one participants (14 women, 7 men) took part in Experiment 2. The median age was 24, ranging from 19 to 31. All participants had normal or corrected-to-normal visual acuityand gave their written consent to participate in this study. The study was performed in accordance with the ethical standards laid down in the Declaration of Helsinkiand was approved by the local ethics board. ## Stimuli We used the same stimuli as in Experiment 1. ## Paradigm The paradigm of Experiment 2 was very similar to that of Experiment 1 with the following exceptions: 1. Experiment 2 was executed with a new sample of participants. 2. Experiment 2 contained only Conditions AA and AD. We analyzed the Condition AD. 3. Each experimental block contained only three observation sequences. This reduced the length of an experimental block from about 9 minutes (Experiment 1) to about 9 seconds (Experiment 2). Concurrently, we drastically increased the number of experimental blocks per condition by factor 40 from 3 blocks (Exp. 1) to 120 blocks (Exp. 2). The reason for this change will be explained in the following.. At the beginning of each experimental block, we announced the experimental condition to which this block belonged with abstract symbols (see. 5. Experiment 2 was interlaced with a second unrelated experiment. This additional experiment also contained experimental blocks of about 9 seconds each, where we presented happy or sad smiley faces. The blocks from Experiment 2 and from the additional experiment alternated with each other (for a schematic overview see. The basic idea of this interlacing was to eliminate within-experiment visual short-term memories from one block of one experiment to the next of the same experiment. As a result, each first OS in a block (labeled as OS1 inshould be free of an immediate experiment-specific perceptual history. OS1 should thus serve to study precognition effects without any influence from the immediate perceptual history. The two subsequent pairs (OS2 and OS3 inmay show effects of accumulating experiment-specific entries into short-term memory. In Experiment 2, the nomenclature of the various sequences (BBB, BBT, etc.) within OS2 and OS3 are identical to those of Experiment 1. For OS1, however, no directly preceding ). OS1 was preceded by a symbolic announcement of the upcoming experimental condition (in blue rectangles). Experiment 2 consisted of two experimental conditions. Each stimulus pair in Condition AD consisted of an ambiguous lattice (S1) followed by a disambiguated lattice (S2). The stimulus pairs in Condition AA contained only ambiguous lattices. Like in Experiment 1, participants indicated the perceived 3D orientation of lattice S1 (Task 1). After presentation of S2 they compared the perceived 3D orientation of S2 with the memorized percept from S1 and indicated either reversed percepts or perceptual stability by key press (see ## Plos one Knowledge from the future or footprints from the past? disambiguated stimulus exists. Therefore, the sequences in OS1 are sorted and labeled in the following way: BB: - The currently observed ambiguous lattice S1 from the current OS is perceived in the Bperspective. - The disambiguated lattice S2 from the current OS will have a B-perspective. BT: - The currently observed ambiguous lattice S1 from the current OS is perceived in the Bperspective. - The disambiguated lattice S2 from the current OS will have a T-perspective. According to this rule the other two groups were TB and TT. # Data analysis We calculated a separate precognition analysis for OS1, comparing the probability of perceiving the ambiguous Necker lattice in the B-perspective as function of a subsequent disambiguated lattice also in the B-perspective versus a subsequent disambiguated lattice in the Tperspective. To do so, we performed classical repeated-measures t-tests and for confirmation purposes additional Wilcoxon tests. For the OS2 and OS3 we calculated a repeated-measures ANOVA with the factors (immediate) HISTORY, (immediate) FUTURE and OBSERVATION SEQUENCE, each of these factors containing two factor steps. As in the analysis from Experiment 1, the ANOVA further tested the individual probabilities against p = 0.5 (factor BIAS). # Results-experiment 2 A graphical demonstration of the different perceptual probabilities on both the level of participants and grand means is presented indifference values subtracting the specific probability values from each history and precognition contrasts. In these graphs, any systematic deviation of the differences of probability values from zero would indicate effects in the respective directions.provides the averages of the numbers of sequences per participant that entered the analyses. Overall, both the ANOVA results anda strong perceptual long-term history effect: The majority of icons in OS2 and OS3 are above the dashed horizontal 0.5 probability line. This means that the participants tend to perceive the Necker lattice more often in the Bperspective than in the T-perspective, independent of the identity of the preceding and the subsequent disambiguated lattice stimulus, as is also indicated by the ANOVA results (p = 2 � 10 −16 , F(1,160) = 243.92, η 2 = 0.6). This effect was even stronger if the preceding disambiguated lattice had been presented in the B-perspective (p = 0.019, F(1,20) = 6.5, η 2 = 0.24 for the factor HISTORY), compared to disambiguated predecessor lattices in the T-perspective. The ANOVA indicated no effects for the factor OBSERVATION SEQUENCE and no interaction between HISTORY and OBSERVATION SEQUENCE. The ANOVA further indicated no effects for the factors HISTORY and OBSERVATION SEQUENCE and no related interactions. Interestingly, the separate analysis of OS1 indicated neither a history effect nor a precognition effect. Like in Experiment 1, despite no significant precognition effect on the group level (factor FUTURE), we found some interesting precognition-like patterns on the level of individual participants . Moreover, closer inspection of some interesting variability patterns with smaller within-group variability in OS1 compared to OS2 and OS3. We thus performed exploratory post-hoc Barlett-tests between the data set from OS1 and the data sets from OS2 and OS3. We also performed a Barlett-test comparing the data sets from OS2 and OS3 with each other. The results are presented inand indicate significantly smaller variance in the data set from OS1 compared to all other data sets. Further, the variability of the data sets from OS2 and OS3 do not differ between each other. ## Plos one Knowledge from the future or footprints from the past? # General discussion In Experiment 1, we found statistical evidence for large long-term perceptual history effects (the a priori bias) and smaller short-term perceptual history effects (influence of the immediate perceptual history). No statistically significant precognition effect was indicated, but some interesting precognition-like patterns on the level of individual participants could be observed. In Experiment 2, we modified the paradigm from Experiment 1 to test for potential precognition effects in a situation without a stimulus-specific perceptual history (no immediately preceding stimulus-specific OS) and with a slowly accumulating perceptual history (one preceding OS and two preceding OS). We found again strong long-term perceptual history effects and weaker short-term perceptual history effects for OS2 and OS3, i.e. in the case of one and two preceding OS. Interestingly, the observed short-term perceptual history effects from Experiment 2 had an opposite sign (priming) compared to the short-term perceptual history effects from Experiment 1 (adaptation) (compare Figs 7 and 11). Again, we found no statistically significant precognition effects in OS2 and OS3 of Experiment 2, but some interesting patterns on the level of individual participants. In this Experiment 2, we particularly focused on OS1, the only OS without a perceptual short-term history of potentially influential disambiguated lattice variants. Also this OS1 revealed no significant precognition effect, regarding perceptual probability values. However, in an exploratory additional analysis we found smaller within-group variability for the data set of difference values from OS1 compared to the data sets from OS2 and OS3. ## Potential explanations of the pattern of perceptual history effects At first glance, the opposite pattern of short-term perceptual history effect findings from Experiments 1 and 2 seem to be contradictory and puzzling and suggest type-1-errors rather than substantial effects. However, these results can be easily integrated into the existing literature. Already in the Discussion section of Experiment 1 we introduced literature findings about priming and adaptation effects during observation of ambiguous figures. Xx no return hereThe priming effects found in OS2 from our Experiment 2 (perceiving the ambiguous Necker lattice more often in the B-perspective if the preceding disambiguated variant had been presented in this B-perspective), may result from a kind of "beneficial" pre-activation of the the whole Experiment 1 lasted for about 1.5 hours, the data sets contained more trials with long (> 80 seconds) than with short perceptual histories (< 20 seconds). Keep in mind that the perceptual adaptation theories postulate that each neural representation (here the T-and the B-perspectives) can be adapted independently (e.g.. We thus assume that in Experiment 1 both neural representations became adapted to a certain and probably comparably large degree over a certain number of presentations of the respective stimuli. In the highly adapted states, the immediate disambiguated lattice antecessor may then tip a given adaptation balance in the respective direction. This postulated explanation can unfortunately not be tested with the present data sets, because we do not have enough trials to also take into account the perceptual histories at earlier time points t (-2) , t etc. Such an extended analysis would increase the number of data sets that have to be separated from each other. This, in turn, would reduce the number of trials per data set dramatically, making reliable statistics impossible. Further, it was found that not only the preceding stimulus identity, but also knowledge about the occurrence probabilities of previously presented stimuli influence the current perceptual interpretation. Current models of perception, like Bayesian probability, predictive codingor circular inference modelspostulate that past perceptual experiences are used to make predictions of upcoming sensory information and highlight the influence of this integration on current perceptual processes [for electrophysiological correlates see 64]. These theories suggest a highly complex and hierarchical network of predictive influences from the perceptual past on the perceptual presence and a full experimental control of all such aspects is a real challenge. ## Discussion of the heterogeneous precognition-like patterns One critical point in precognition research and also more general in research about extrasensory perception or psychokinesis experiences ("PSI": the causes underlying extrasensory perception and psychokinesis experiences cannot be explained by known physical or biological mechanismsis the recurring pattern that positive study findings cannot be reliably replicated. One attempt to explain these replication problems refers to the possibility that only some particularly gifted persons show large PSI effects, while the majority of participants do not [e.g.. Theoretically assuming that this is the case and additionally assuming that such gifted persons are a priori rare, any study that randomly collects participants will be convicted to produce either non-significant or only weakly significant results on the group level. Taking this option into account, we identified potentially gifted "outlier participants" with maximal deviations of the calculated difference values from zero (above 1.5 and 2 standard deviations, "SD") in each of the analyzed data sets and checked whether they systematically show extreme values also in the other analyzed data set. In Experiment 1, we identified one outlier participant (red square in , who in both Conditions AD and DA in the "BBB minus BBT" showed differences of probability values with the same sign and both above 1.5 SD (0.08 in Condition AD and about 0.12 in Condition DA). A similar pattern was found in Experiment 2 for the observation sequences OS2 and OS3. Two participants in this experiment showed consistent deviations of their differential probability values of about 0.22 or larger (red star and dark green square in across two separate contrasts. Again, both were above the 1.5 SD line. While for one participant (red star in this deviation was observed in the same contrast (BBB-BBT) over observation sequences OS2 and OS3, the contrast type changed from BBB-BBT (OS2) to TBB-TBT (OS3) for the other participant (dark-green square in . Even if we assume the correctness of the null hypothesis of no precognition effects, we should expect some variability of the data from individual participants among the expected zero mean . Yet we should not expect the repetitive pattern of individual participants as described above. However, the main focus of our Experiment 2 was on OS1, where we tried to reduce the potential influence from the perceptual history as a potentially confounding factor for the precognition analysis as much as possible. It turned out that the three remarkable outlier participants across OS2 and OS3 were unremarkable in the OS1 where the influences of perceptual history were minimized. This, in turn, indicates that the perceptual history is the most probable causal factor for the pattern these special participants show in OS2 and OS3, even though it will be difficult to explain how this influence may unfold in these cases [but see 82 for a speculative alternative explanation]. another interesting and related pattern: The variability of probability difference values was much smaller in OS1 (no immediate perceptual lattice history) compared to OS2 and OS3 (accumulating perceptual lattice history), where the variability was similar but clearly larger than in OS1. A post-hoc exploratory analysis indicated statistical significance of these observations (see. This indicates that the tendency for larger deviations from zero concerning precognition effects seems to be somehow dependent on the existence or non-existence of a stimulus-specific immediate perceptual history. We regard the observations on the individual level interesting and worth mentioning, although they are not statistically supported and thus do not allow for strong conclusions. We regard the variability effects in Experiment 2 as also highly interesting. The effects are visible in the precognition contrasts, but the given paradigm clearly indicates that the perceptual history plays a role, as already discussed above. Again, the present data do not allow for strong conclusions, but they invite to have a closer look on the variability parameter in future studies. The literature contains vivid and highly controversial discussions about the question ofwhether it is at all theoretically possible that effects like precognition exist, or whether it is worth or even ethically justifiable to execute related studies [e.g.. The aim of the present article is not to contribute to such-in our view-dispensable discussions. Rather, we aim to emphasize the importance of the perceptual history for how we perceive the world at the present moment and how we will perceive it in a future moment. Future precognition studies should thus also take into account that past percepts leave their "footprints" in our perceptual system and some footprints can be deeper than others. The latter can nicely be demonstrated with the a priori perceptual bias of the Necker cube in favor of the B-perspective, what we labeled as the long-term perceptual history effect and what we already described in the introduction. The proposed preference of our perceptual system for the B-perspective most probably reflects the majority of our everyday experiences and can thus be regarded as a "very deep footprint from the past". In the present paradigm, it is of course not difficult to comprehend the direct influence from past percepts of a disambiguated lattice figure on the perception of a highly similar but ambiguous lattice variant. In other precognition paradigms, such as some of those used in the experiments of the seminal Bem paper, the potential role of the perceptual history is not as directly comprehensible as in the present study-which does not necessarily rule it out. Particularly, given that it is even unclear so far, which factors of past perceptual experiences contribute to the perception of a present sensory information, it will be extremely hard, if not impossible, to exclude all possible influences from the perceptual past on a given precognition effect. In a perfect experiment, one would have to know the history of the participant's exposure to similar stimuli, the predictive models formed within the participant's brain and how the sensory information presented within the experiment is represented in the participant. Only in the case of full access to all underlying processes within the participant's brain, one would be able to doubtlessly disentangle potential influences from the past and potential influences from the future. An additional complication may result from the potential individual differences concerning the amount of impact of past perceptual experiences. In conclusion, the present study is an example for a central feature of our perceptual system: We prefer to perceive what we already know, and what we currently perceive can strongly influence what we see next. Future studies about perception, whether they regard sensory or extrasensory perception, need to take this into account.

Characteristics and outcomes of primary pleural angiosarcoma Primary pleural angiosarcoma (PPA) is an extremely rare malignancy for which there is no consensus on treatment. The clinical course of PPA is usually quickly fatal, regardless of the treatment used.We summarized and evaluated a relatively large population of published PPA cases to assess prognostic factors, diagnostic approaches, treatment methods and clinical outcomes. Using the CNKI, Embase, and PubMed databases, literature published in English and Chinese from 1988 through 2020 was searched using the terms "primary pleural angiosarcoma," "pleural angiosarcoma," and "pleuropulmonary angiosarcoma."A total of 43 patients with PPA were identified in retrospective case series and case reports. The median age at diagnosis was 64 years (range 24-87 years), and the median overall survival was 4 months (range 0.1-180 months). Approximately 80% of patients died from PPA within 10 months of diagnosis, and the 2-year survival rate was approximately 4.4%. In univariate analyses, the presence of pleural effusion and hemothorax were significant predictors of decreased survival, with hazard ratios (HRs) of 2.7 (P = .04) and 3.3 (P = .006), respectively. Sixteen patients received no therapy, and their prognosis was worse than patients who did receive therapy (P = .019). Radiation therapy improved survival more than no radiation therapy (P = .007). Patients appeared to derive clinical benefit from chemotherapy (P = .048). However, tumor resection did not seem to provide a survival benefit (P = .051). In multivariate analysis, tumor resection, and radiation were independent, statistically significant, positive predictors of better survival, with HRs of 0.3 (P = .017) and 0.1 (P = .006), respectively. The presence of hemothorax was an independent predictor of worse prognosis (P = .006).Primary angiosarcoma of the pleura is a rare, poorly understood malignancy with a poor prognosis; hence, the clinical spectrum of PPA is not completely defined. By multivariate analysis, this retrospective study showed a survival benefit of tumor resection or radiation therapy, and the presence of hemothorax was a significant prognostic factor for poor outcomes.Abbreviations: CI = confidence interval, HR = hazard ratio, PPA = primary pleural angiosarcoma. # Introduction Angiosarcoma is a rare malignant tumor of endothelial origin that accounts for only 1% to 2% of all soft tissue sarcomas. [bib_ref] Predictive value of grade for metastasis development in the main histologic types..., Coindre [/bib_ref] Tumors can occur in any part of the body, but the most common primary sites are cutaneous lesions (approximately 60% of cases), particularly in the head and neck; although, they can also present within the soft tissues, visceral organs, bone, and retroperitoneum. [bib_ref] Clinicopathological features and prognostic factors in angiosarcoma: a retrospective analysis of 200..., Wang [/bib_ref] [bib_ref] Pathologic angiogenesis of malignant vascular sarcomas: implications for treatment, Khan [/bib_ref] There are major differences in the characteristics, behaviors, and prognosis of angiosarcoma among primary sites. [bib_ref] Clinicopathological features and prognostic factors in angiosarcoma: a retrospective analysis of 200..., Wang [/bib_ref] Primary pleural angiosarcoma (PPA), a highly malignant disease, is an extremely rare malignancy, and the literature is limited to case reports. [bib_ref] Primary pleural angiosarcoma: case report and literature review, Sedhai [/bib_ref] [bib_ref] Primary pleural angiosarcoma as a mimicker of mesothelioma: a case report *..., Kao [/bib_ref] [bib_ref] Malignant epithelioid vascular tumors of the pleura: report of a series and..., Zhang [/bib_ref] [bib_ref] Primary angiosarcomas of the chest wall and pleura, Alexiou [/bib_ref] Delayed diagnosis and the rarity of these tumors contribute to difficulties in determining the best treatment and prognostic factors. With no consensus as to the most effective treatments, current treatment methods are guided by angiosarcoma studies and prior PPA cases. The treatments for PPA include surgical excision, radiotherapy and chemotherapy. [bib_ref] Primary pleural angiosarcoma: case report and literature review, Sedhai [/bib_ref] [bib_ref] Primary pleural angiosarcoma as a mimicker of mesothelioma: a case report *..., Kao [/bib_ref] [bib_ref] Malignant epithelioid vascular tumors of the pleura: report of a series and..., Zhang [/bib_ref] [bib_ref] Primary angiosarcomas of the chest wall and pleura, Alexiou [/bib_ref] Nevertheless, the clinical course is usually aggressive and often becomes rapidly fatal, regardless of the treatment used. [bib_ref] Malignant epithelioid vascular tumors of the pleura: report of a series and..., Zhang [/bib_ref] [bib_ref] Primary angiosarcomas of the chest wall and pleura, Alexiou [/bib_ref] [bib_ref] Primary de novo angiosarcoma of the pleura, Lorentziadis [/bib_ref] Approximately 75% of patients die from PPA within 7 months, and all die within 24 months. [bib_ref] Primary pleural angiosarcoma as a mimicker of mesothelioma: a case report *..., Kao [/bib_ref] PPA remains difficult to manage clinically due to the paucity of cases. For this study, we retrospectively summarized and evaluated 43 patients with PPA. To our knowledge, the study in this review represents the largest PPA cohort to date that identifies the clinical profiles, treatment outcomes, and prognostic factors associated with PPA. To achieve the study goals, the demographics and tumor characteristics of PPA patients at the time of diagnosis were summarized and evaluated in comparison with survival. # Methods ## Data collection We summarized and evaluated a relatively large population of recently published PPA cases to assess prognostic factors, diagnostic approaches, treatment methods, and clinical outcomes. Using the CNKI, Embase, and PubMed databases, literature published in English and Chinese from 1988 through 2020 was searched using the terms "primary pleural angiosarcoma," "pleural angiosarcoma," and "pleuropulmonary angiosarcoma." Cases were carefully reviewed, and published data were entered into a spreadsheet for inspection and analysis. Approval of the medical ethics committee was not required for this review because the case information was publicly available. # Statistical analysis Survival was defined as the interval between date of PPA diagnosis and date of death or last follow-up. Survival was evaluated by Kaplan-Meier plots and Cox log-rank test. The Cox proportional hazards regression model was used to perform multivariate analyses and identify independent prognostic factors. P values <.05 were considered to indicate significant differences. The statistical software packages R (http://www.Rproject.org, The R Foundation) and Empower Stats (http://www. empowerstats.com, X&Y Solutions, Inc., Boston, MA) were used to analyze all data. # Results ## Patient characteristics and clinical presentation Data from 43 PPA cases between 1988 and 2020 were included in the review. The median age at diagnosis was 64 years (range 24-87 years). The baseline characteristics of the patients are shown in [fig_ref] Table 1: Baseline characteristics of patients [/fig_ref]. There was a male predominance (65% of cases). The ethnic distribution of these patients was 44% White, 5% Black, 49% Asian, and 2% other. Tuberculosis exposure was confirmed in 21% of patients. Tobacco smoking was reported in 28% and prior radiation therapy was reported in 9% of cases. Chronic expanding hematoma of the chest was considered a specific type of chronic empyema and was reported in 1 case. Only 1 patient (2%) was asymptomatic, and the most common presenting signs and symptoms were typical of pleural effusion, which occurred in 32 patients (74%). Symptoms were not restricted to dyspnea and included chest pain, hemoptysis, cough, fever, weight loss, fatigue and anorexia. Histopathological subtype data were provided for 43 patients and included 24 (56%) epithelioid angiosarcomas and 19 (44%) classical angiosarcomas. Fourty two cases had chest CT scan and/or MR image results, and positron emission computed tomography (PET-CT) data were present in 11 cases; in these cases, a chest mass was noted in 44% and pleural invasion was noted in 70% of cases, and invasion was circumferential in 26% and local in 44% of cases. Seventy four percent of patients had pleural effusion, but only 2% and 7% had pneumothorax and calcification, respectively. Nine percent of the patients were found to have bilateral multiple nodules, and the detection rates of single nodules or unilateral multiple nodules were 16% and 27%, respectively. A total of 47% of the patients were anemic, and 19% did not have anemia; 10 cases had hemoglobin values, and the median hemoglobin level was 71.5 g/ L. Eight (19%) patients underwent blood transfusion. Metastasis occurred in 40%, and the sites of metastasis included but were not limited to the contralateral lung, liver, brain, and bone. ## Diagnostic approach In the present study, the median time until diagnosis (reported in 31 cases) was 1.4 months. Biopsy or tumor resection by thoracotomy were the most common diagnostic methods for PPA (n = 14, 33%) [fig_ref] Table 2: Diagnostic methods, treatments, and clinical outcomes [/fig_ref]. Diagnosis was made by thoracoscopy in 12 (28%) patients. Postmortem diagnosis was made in 4 (9%) cases. Sixteen cases underwent percutaneous lung/pleural biopsy; although, tumor tissue was successfully obtained in only 7 cases. Thoracentesis and drainage were performed in 25 patients; among them, the pleural effusion in 24 contained blood. Pleural cytology in 16 patients and bronchoscopy in 10 patients were performed but failed to obtain diagnostic material. The pathology of 6 cases was determined through metastasis. ## Treatment and clinical outcomes Treatment data are shown in [fig_ref] Table 2: Diagnostic methods, treatments, and clinical outcomes [/fig_ref]. Sixteen patients never received any antitumor therapy, and their prognosis was worse than that of the treatment group (HR 2.6; 95% confidence interval (CI, 1.2-5.7; P = .019) [fig_ref] Figure 1: Kaplan-Meier curves for median overall survival [/fig_ref] and [fig_ref] Table 3: Univariate and multivariate analysis of predictors affecting survival [/fig_ref]. Diagnosis was verified by postmortem investigations in 4 cases, and 3 cases without prognostic data were excluded from the prognostic outcome analysis. Radiation treatment was used in 7 patients (17%), and survival was better than that in patients who received no radiation treatment (P = .007) [fig_ref] Figure 1: Kaplan-Meier curves for median overall survival [/fig_ref]. Twenty nine percent of patients underwent tumor resection. However, surgery did not seem to provide a survival benefit (P = .051) [fig_ref] Figure 1: Kaplan-Meier curves for median overall survival [/fig_ref]. Pleurectomy also failed to provide a survival benefit (HR 0.4; 95% CI, 0.1-1.3; P = .123) [fig_ref] Figure 1: Kaplan-Meier curves for median overall survival [/fig_ref]. Four patients were treated with targeted drugs: bevacizumab (2 cases), pazopanib (1 case), and sorafenib (1 case) with or without chemotherapy. Approximately 80% of patients died from PPA within 10 months of diagnosis, and the 2-year survival rate was approximately 4.4% [fig_ref] Figure 2: Survival times of 36 cases of pleural angiosarcoma [/fig_ref]. Univariate analysis was used to describe clinical characteristics by age, sex, tuberculosis, and radiation exposure, tobacco use, race, histologic subtype, pleural effusion, hemothorax, anemia, metastasis, and treatment (Table 3). The presence of pleural effusion and hemothorax were significant predictors of decreased survival, with HRs of 2.7 (P = .04) and 3.3 (P = .006), respectively [fig_ref] Figure 1: Kaplan-Meier curves for median overall survival [/fig_ref]. In multivariate analysis, a Cox regression model was used to analyze the risk factors using the following possible prognostic factors: age, pleural effusion, hemothorax, metastasis, systemic chemotherapy, tumor resection, radiation, no therapy, and single-organ involvement in the chest. Thirty six cases (84%) had complete outcome data and were included in the analysis. Tumor resection and radiation were independent statistically significant positive predictors of better survival, with HRs of 0.3 (P = .017) and 0.1 (P = .006), respectively [fig_ref] Table 3: Univariate and multivariate analysis of predictors affecting survival [/fig_ref]. The presence of hemothorax demonstrated a trend toward worse outcomes (HR 3.4; 95% CI, 1.43-8.25; P = .006). # Discussion PPA is an extremely rare malignancy. We performed a systematic literature search in CNKI, Embase and PubMed between 1988 and 2020 and retrospectively summarized 43 published cases with PPA to outline clinical features and to analyze outcomes. Given the rarity of PPA, its pathogenesis and etiology remain unclear and are still the object of much speculation. However, several exposure-related risk factors are known for this disease, including prior radiotherapy and a history of tuberculosis. [bib_ref] Malignant epithelioid vascular tumors of the pleura: report of a series and..., Zhang [/bib_ref] [bib_ref] Angiosarcoma of serous membranes, Mccaughey [/bib_ref] [bib_ref] Angiosarcoma developing from chronic pyothorax, Aozasa [/bib_ref] [bib_ref] Angiosarcoma in Japan. A review of 99 cases, Naka [/bib_ref] Likewise, tuberculosis exposure, radiation exposure and chronic expanding hematoma of the chest were reported in 14 (32%) cases in this study but did not correlate with outcome. Chronic inflammation and persistent physical stimulation are purported to result in PPA. [bib_ref] Pleural epithelioid angiosarcoma with lymphatic differentiation arisen after radiometabolic therapy for thyroid..., Cabibi [/bib_ref] [bib_ref] Angiosarcoma of the pleura, Pramesh [/bib_ref] [bib_ref] A case of pyothorax-associated pleural angiosarcoma diagnosed by autopsy, Saitou [/bib_ref] The final diagnosis of PPA was based upon a histological study of the tumor samples. In the present series, the histologic subtype was epithelioid in 56% and classical in 44% of cases. This is inconsistent with the findings of previous reports that show that epithelioid variants account for 75% of PPA. [bib_ref] Primary de novo angiosarcoma of the pleura, Lorentziadis [/bib_ref] [bib_ref] Primary pleural angiosarcoma in a 63-yearold gentleman, Abu-Zaid [/bib_ref] Univariate analysis of histologic subtype did not differ significantly with respect to overall survival in this case series. This is also incompatible with the results of a previous study that showed that histologic subtype correlated directly with malignant potential in patients with PPA. [bib_ref] Primary pulmonary epithelioid angiosarcoma presenting as a solitary pulmonary nodule on image, Yang [/bib_ref] Potential explanations include poor overall survival and a relatively small number of cases in this retrospective study. Intriguingly, metastases of PPA are not associated with a worse outcome, unlike other tumor types. [bib_ref] Survival and prognosis with osteosarcoma: outcomes in more than 2000 patients in..., Smeland [/bib_ref] [bib_ref] Multiple treatment lines and prognosis in metastatic colorectal cancer patients, Carlomagno [/bib_ref] This difference may be explained by the fact that patients often die of recurring hemothorax and/or local progression within a short time. [bib_ref] Primary de novo angiosarcoma of the pleura, Lorentziadis [/bib_ref] [bib_ref] Bilateral and unilateral spontaneous massive hemothorax as a presenting manifestation of rare..., Varsano [/bib_ref] [bib_ref] Primary epitheloid angiosarcoma of the pleura: an exceptional tumor location, Azzakhmam [/bib_ref] [bib_ref] Primary pleural epithelioid angiosarcoma. A case report and review of the literature, Dainese [/bib_ref] The clinical symptoms of PPA are not specific and are commonly represented by chest pain, dyspnea, hemoptysis, pleural effusion, and recurring hemothorax. [bib_ref] Primary angiosarcomas of the chest wall and pleura, Alexiou [/bib_ref] [bib_ref] Primary pleural angiosarcoma in a 63-yearold gentleman, Abu-Zaid [/bib_ref] [bib_ref] Two cases of epithelioid angiosarcoma involving the thyroid and a brief review..., Goh [/bib_ref] [bib_ref] Malignant vascular tumours of the pleura in "asbestos" workers and endothelial differentiation..., Attanoos [/bib_ref] Radiology provides multiparametric morphological and important help for the diagnosis of PPA, but the radiological signs are also not very specific. PET-CT was used in 11 cases to estimate the stage and primary site. The diagnosis of PPA is strongly dependent on pathological findings. The median time until diagnosis was 1.4 months. Biopsy or tumor resection performed by thoracotomy or by video-assisted thoracoscopic surgery were by far the most frequently applied and helpful diagnostic tool for PPA, and they were applied in 26 (61%) cases. [bib_ref] Epithelioid angiosarcoma: a rare cause of pericarditis and pleural effusion, Durani [/bib_ref] [bib_ref] Pleural myiasis associated with pleural angiosarcoma, Patel [/bib_ref] Percutaneous lung/ pleural biopsy was also supported by the present series. [bib_ref] Primary pleural angiosarcoma in a 63-yearold gentleman, Abu-Zaid [/bib_ref] [bib_ref] Primary epithelioid angiosarcoma of the pleura: a case report and review of..., Zhang [/bib_ref] [bib_ref] Significant response to gemcitabine monotherapy in primary pleural epithelioid angiosarcoma, Tsubouchi [/bib_ref] Pleural fluid cytology yields a diagnosis in one-fourth of cases of pleural mesothelioma. [bib_ref] Thoracoscopy in pleural malignant mesothelioma: a prospective study of 188 consecutive patients...., Boutin [/bib_ref] Unlike for pleural mesothelioma, pleural cytology had negative findings in all cases of PPA. [bib_ref] Epithelioid angiosarcoma: a rare cause of pericarditis and pleural effusion, Durani [/bib_ref] Treatment modalities for PPA include surgery, radiotherapy and/or chemotherapy, and surgery followed by adjuvant radiotherapy is considered the optimal current treatment modality. One patient with a large solitary angiosarcoma was successfully treated with surgery and postoperative radiotherapy, remaining well and asymptomatic after 15 years of follow-up. [bib_ref] Primary angiosarcomas of the chest wall and pleura, Alexiou [/bib_ref] Vascular embolization can be helpful in supporting hemostasis. [bib_ref] Primary de novo angiosarcoma of the pleura, Lorentziadis [/bib_ref] [bib_ref] Diffuse primary angiosarcoma of the pleura: a case report and review of..., Kurtz [/bib_ref] There are no consensus criteria for chemotherapy regimens for PPA; 16 (38%) patients received chemotherapy with or without targeted agents. Chemotherapy drugs included gemcitabine, docetaxel, paclitaxel, cisplatin, ifosfamide, doxorubicin, and/or albumin-bound paclitaxel. [bib_ref] Angiosarcoma of the thoracic wall responded well to nanoparticle albumin-bound paclitaxel: a..., Hara [/bib_ref] Chemotherapy was associated with survival benefit in univariate analysis, which discordant results occurred in multivariate analysis. The role of chemotherapy may be limited, but radiotherapy can be effective in PPA. In addition, targeted medicines and immunotherapy have recently been studied as promising treatments for angiosarco- mas. [bib_ref] Cutaneous angiosarcoma: the possibility of new treatment options especially for patients with..., Fujisawa [/bib_ref] [bib_ref] Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven..., Florou [/bib_ref] [bib_ref] Histology-and non-histology-driven therapy for treatment of soft tissue sarcomas, Casali [/bib_ref] [bib_ref] Sorafenib for patients with advanced angiosarcoma: a phase II Trial from the..., Ray-Coquard [/bib_ref] Tyrosine kinase inhibitors (TKIs) have been implemented in targeted therapy of angiosarcomas, especially sorafenib and pazopanib. [bib_ref] Cutaneous angiosarcoma: the possibility of new treatment options especially for patients with..., Fujisawa [/bib_ref] [bib_ref] Histology-and non-histology-driven therapy for treatment of soft tissue sarcomas, Casali [/bib_ref] [bib_ref] Sorafenib for patients with advanced angiosarcoma: a phase II Trial from the..., Ray-Coquard [/bib_ref] In the present series, 4 patients were treated with targeted drugs: bevacizumab, pazopanib, and sorafenib. [bib_ref] Morphoproteomic study of primary pleural angiosarcoma of lymphangioendothelial lineage: a case report, Quesada [/bib_ref] [bib_ref] Case of primary pleural angiosarcoma with malignant seeding along the pleural tap..., Yamaguchi [/bib_ref] Recently, a study described PD-L1 detection in 16/24 (66%) angiosarcoma samples. [bib_ref] Programmed death ligand 1 (PD-L1) expression in primary angiosarcoma, Botti [/bib_ref] One previous study reported a case of angiosarcoma of the nose treated with pembrolizumab, in which the patient received pembrolizumab for 13 cycles, which resulted in marked shrinkage of his liver disease and no new facial lesions. [bib_ref] Angiosarcoma treated successfully with anti-PD-1 therapy -a case report, Sindhu [/bib_ref] Current treatment options for PPA are limited and poorly studied, making it necessary to consider treatment with tyrosine kinase inhibitors and checkpoint blockade therapies when appropriate. However, more studies are needed to establish the efficacy and safety of agents targeting the VEGF/VEGFR signaling pathway and immune checkpoint pathway in patients with PPA. Although we represent the largest study to date on published PPA cases, our findings and conclusions should be interpreted with caution because of the relatively small number of cases and retrospective design of the study. The value of combined modality treatment was not assessed in this review due to few patients receiving combination therapy, which limits our ability for further analysis. Additionally, our characterization analysis of hemoglobin in patients with PPA was insufficient due to incomplete, limited data and a lack of corresponding information. Despite these limitations, we believe our analysis provides helpful information to physicians caring for individuals with PPA in clinical practice because the rarity of PPA makes it impossible to conduct prospective controlled trials or prospective cohort studies. # Conclusions In summary, PPA remains an exceedingly rare and poorly defined malignancy. We report the first study of PPA that identified potentially useful clinical correlates for outcome. Specifically, tumor resection and radiation may be beneficial. The presence of hemothorax demonstrated a trend toward worse outcomes. Our findings provide important insight into this extremely rare and aggressive malignancy, and further study is needed to validate our findings and identify more accurate prognostic indicators of PPA. # Author contributions [fig] Figure 1: Kaplan-Meier curves for median overall survival: (A) No therapy vs therapy. Median survival time: 1.2 minutes vs 5.2 minutes, HR = 2.6, P = .019; (B) Radiation vs no radiation. Median survival time: 18 minutes vs 3.0 minutes, HR = 0.2, P = .007; (C) Tumor resection vs no surgery. Median survival time: 8.0 minutes vs 3.0 minutes, HR = 0.4, i = .051; (D) Chemotherapy vs no chemotherapy. Median survival time: 7.8 minutes vs 1.4 minutes, HR = 0.5, P = .048; (E) Pleural rffusion vs no pleural effusion. Median survival time: 4.0 minutes vs 7.0 minutes, HR = 2.7, P = .040; (F) hemothorax vs no hemothorax. Median survival time: 1.4 minutes vs 6.0 minutes, HR = 3.3, P = .006. [/fig] [fig] Figure 2: Survival times of 36 cases of pleural angiosarcoma. Approximately 80% of patients died within 10 months of diagnosis. [/fig] [table] Table 1: Baseline characteristics of patients. [/table] [table] Table 2: Diagnostic methods, treatments, and clinical outcomes. [/table] [table] Table 3: Univariate and multivariate analysis of predictors affecting survival. * * P < .05 was considered significant. HR = hazard ratio, CI = confidence interval. [/table]

Recurrent Stroke in Minor Ischemic Stroke or Transient Ischemic Attack With Metabolic Syndrome and/or Diabetes Mellitus on behalf of the CHANCE Investigators* Background--We aimed to determine the risk conferred by metabolic syndrome (METS) and diabetes mellitus (DM) to recurrent stroke in patients with minor ischemic stroke or transient ischemic attack from the CHANCE (Clopidogrel in High-risk patients with Acute Non-disabling Cerebrovascular Events) trial.Methods and Results--In total, 3044 patients were included. Patients were stratified into 4 groups: neither, METS only, DM only, or both. METS was defined using the Chinese Diabetes Society (CDS) and International Diabetes Foundation (IDF) definitions. The primary outcome was new stroke (including ischemic and hemorrhagic) at 90 days. A multivariable Cox regression model was used to assess the relationship of METS and DM status to the risk of recurrent stroke adjusted for potential covariates. Using the CDS criteria of METS, 53.2%, 17.2%, 19.8%, and 9.8% of patients were diagnosed as neither, METS only, DM only, and both, respectively. After 90 days of follow-up, there were 299 new strokes (293 ischemic, 6 hemorrhagic). Patients with DM only (16.1% versus 6.8%; adjusted hazard ratio 2.50, 95% CI 1.89-3.39) and both (17.1% versus 6.8%; adjusted hazard ratio 2.76, 95% CI 1.98-3.86) had significantly increased rates of recurrent stroke. No interaction effect of antiplatelet therapy by different METS or DM status for the risk of recurrent stroke (P=0.82 for interaction in the fully adjusted model of CDS) was observed. Using the METS (IDF) criteria demonstrated similar results.Conclusions--Concurrent METS and DM was associated with an increased risk of recurrent stroke in patients with minor stroke and transient ischemic attack. ( J Am Heart Assoc. 2017;6:e005446. DM indicates diabetes mellitus; HR, hazard ratio; METS, metabolic syndrome. *Model 1: adjusted for age and sex. † Model 2: adjusted for age, sex, history of ischemic stroke, transient ischemic attack, myocardial infarction, angina, congestive heart failure, known atrial fibrillation or flutter, valvular heart disease, smoking status, index event and National Institutes of Health Stroke Scale on admission, and time to randomization. T he metabolic syndrome (METS) refers to a cluster of highly interrelated metabolic risk factors. [bib_ref] Impact of metabolic syndrome on prognosis of symptomatic intracranial atherostenosis, Ovbiagele [/bib_ref] Regardless of the details of several criteria by different organizations for its diagnosis, [bib_ref] American Heart Association; National Heart, Lung, and Blood Institute. Diagnosis and management..., Grundy [/bib_ref] [bib_ref] Diagnosis and management of the metabolic syndrome: a Chinese Diabetes Society Scientific..., Xiang [/bib_ref] it is generally accepted that the prevalence of METS in diverse racial populations is increasing (between 10% and 84%). [bib_ref] Prevalence of the metabolic syndrome among US adults: findings from the third..., Ford [/bib_ref] [bib_ref] Metabolic syndrome: a closer look at the growing epidemic and its associated..., O&apos;neill [/bib_ref] Previous prospective studies showed that the presence of METS identifies persons at an elevated risk for ischemic stroke or transient ischemic attack (TIA). [bib_ref] Metabolic syndrome clusters and the risk of incident stroke: the Atherosclerosis Risk..., Rodriguez-Colon [/bib_ref] [bib_ref] Influence of metabolic syndrome and general obesity on the risk of ischemic..., Chen [/bib_ref] There are limited data, however, on the relationship between METS and the risk of stroke recurrence. Previous studies found that METS may not be predictive for stroke recurrence in patients with general ischemic stroke, [bib_ref] On behalf of the investigators for the survey on Abnormal Glucose Regulation..., Mi [/bib_ref] [bib_ref] Risk of stroke and cardiovascular events after ischemic stroke or transient ischemic..., Callahan [/bib_ref] whereas another study demonstrated that METS was associated with higher risk of stroke recurrence in patients with ischemic stroke. [bib_ref] Metabolic syndrome and three of its components as risk factors for recurrent..., Liou [/bib_ref] The relationship between METS and recurrence of stroke after a stroke or TIA remains controversial. METS was defined for use in persons without diabetes mellitus (DM), but the definition has developed in recent decades to include those with DM. [bib_ref] Metabolic syndrome compared with type 2 diabetes mellitus as a risk factor..., Najarian [/bib_ref] [bib_ref] Impact of the metabolic syndrome on macrovascular and microvascular outcomes in type..., Cull [/bib_ref] Given a synergistic relationship among these components, the collective entity of METS provides better stroke risk estimates. Nevertheless, this integration of each component of METS made it difficult to understand the effect of DM or other components on stroke recurrence compared with the role of METS as an independent risk factor. Minor ischemic stroke and TIA account for %65% of all acute ischemic cerebrovascular events [bib_ref] Transient ischemic attack and minor stroke are the most common manifestations of..., Von Weitzel-Mudersbach [/bib_ref] and lead to a risk of 10% to 15% stroke occurrence within 90 days. [bib_ref] Risk of stroke early after transient ischaemic attack: a systematic review and..., Giles [/bib_ref] Factors associated with a high risk of recurrence in patients with TIA or minor stroke were different from those of general stroke [bib_ref] Factors associated with a high risk of recurrence in patients with transient..., Ois [/bib_ref] ; however, data from previous studies were derived from trials or cohorts in which patients were recruited weeks or months after their initial event and underestimated early recurrence, especially for minor stroke or TIA. [bib_ref] On behalf of the investigators for the survey on Abnormal Glucose Regulation..., Mi [/bib_ref] [bib_ref] Risk of stroke and cardiovascular events after ischemic stroke or transient ischemic..., Callahan [/bib_ref] [bib_ref] Recurrent vascular events in lacunar stroke patients with metabolic syndrome and/or diabetes, Zhu [/bib_ref] Consequently, the risks of recurrent stroke caused by METS and DM in patients with a minor stroke or TIA should be further examined. We compared the risk of recurrent stroke in patients with different METS and DM status and minor stroke or TIA from the CHANCE (Clopidogrel in High-risk patients with Acute Non-disabling Cerebrovascular Events) trial. Our hypothesis was that METS and DM were associated with an increased risk of recurrent stroke after a minor stroke or TIA. # Methods ## Study patients The CHANCE trial was a randomized, double-blind, controlled trial that enrolled 5170 patients within 24 hours after onset of minor stroke (National Institutes of Health Stroke Scale [NIHSS] ≤3) or high-risk TIA (ABCD 2 ≥4) from 114 clinical centers in China. [bib_ref] Clopidogrel with aspirin in acute minor stroke or transient ischemic attack, Wang [/bib_ref] [bib_ref] Clopidogrel with aspirin in acute minor stroke or transient ischemic attack (CHANCE)..., Wang [/bib_ref] [bib_ref] Rationale and design of a randomized, double-blind trial comparing the effects of..., Wang [/bib_ref] In total, 73 (64%) of 114 participating hospitals voluntarily participated in the serum biomarker substudy in the CHANCE trial. The triglyceride and highdensity lipoprotein levels, which are the significant items for the diagnosis of METS, were analyzed in the laboratory using collected serum. A total of 3044 patients in these 73 centers with available triglyceride and high-density lipoprotein levels were included in this analysis. ## Standard protocol approvals, registrations, and patient consents The CHANCE trial is registered at ClinicalTrials.gov (identifier NCT00979589). The protocol and data collection of the CHANCE trial were approved by the ethics committee of Beijing Tiantan Hospital and all other study centers. All participants or his or her representatives provided written informed consent before being entered into the study. ## Measurements Baseline demographics and clinical characteristics, including age, sex, medical history of ischemic stroke, TIA, myocardial infarction, angina, congestive heart failure, known atrial fibrillation or flutter, valvular heart disease, hypertension, DM, hypercholesterolemia, and baseline NIHSS and ABCD 2 scores were collected through face-to-face interviews by neurologists from clinical centers. Body mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared (kg/m 2 ). Blood pressure was measured in the left arm of supine patients using a mercury or electronic sphygmomanometer. Venous blood was drawn from fasting patients 24AE12 hours after randomization in 3044 patients of these 73 centers. [bib_ref] Glycated albumin predicts the effect of dual and single antiplatelet therapy on..., Li [/bib_ref] Plasma glucose after overnight fasting was then analyzed. The serum specimens were collected and shipped on ice by overnight courier from each participating hospital to Beijing Tiantan Hospital (China), where all data analyses were performed. The triglyceride and high-density lipoprotein levels were analyzed in the laboratory using collected serum by testing personnel blinded to clinical data in Beijing Tiantan Hospital. Data were analyzed with a Roche Modular P800 system. DM was defined as a fasting glucose level ≥7.0 mmol/L (126 mg/dL), self-reported history of DM, or receiving treatment for DM. METS was defined using the definitions of the Chinese Diabetes Society (CDS), [bib_ref] Diagnosis and management of the metabolic syndrome: a Chinese Diabetes Society Scientific..., Xiang [/bib_ref] which is the only official recommendation for the diagnosis of METS in the Chinese population, and International Diabetes Foundation (IDF). [bib_ref] American Heart Association; National Heart, Lung, and Blood Institute. Diagnosis and management..., Grundy [/bib_ref] We excluded patients with DM or fasting plasma glucose ≥7.0 mmol/L in both definitions of METS. Patients in our study were stratified into 4 groups: neither METS or DM, METS only, DM only, or both. METS (CDS) was determined by the presence of ≥3 of the following metabolic risk factors: overweight or obesity (BMI ≥25); fasting plasma glucose of 6.1 to 6.9 mmol/L (110-125 mg/dL), 2-hour plasma glucose ≥7.8 mmol/L (140 mg/dL), or a history of DM with antidiabetic medication; elevated blood pressure (≥140/≥90 mm Hg) or a history of hypertension with antihypertensive medication; and dyslipidemia, which includes increased triglyceride levels (≥1.7 mmol/L [150 mg/dL]) or reduced high-density lipoprotein levels (<0.9 mmol/L [35 mg/dL] in men and <0.9 mmol/L [39 mg/dL] in women). [bib_ref] Diagnosis and management of the metabolic syndrome: a Chinese Diabetes Society Scientific..., Xiang [/bib_ref] METS (IDF) was defined by abdominal visceral obesity (increased waist circumference , ≥90 cm in men and ≥80 cm in women for an Asian population) plus any ≥2 of the following factors: triglyceride level (≥150 mg/dL) or history of hyperlipemia with antihyperlipemia medication; reduced highdensity lipoprotein level (<40 mg/dL in men and <50 mg/dL in women); elevated blood pressure (≥130/≥85 mm Hg) or a history of hypertension with antihypertensive medication; fasting plasma glucose of 100 to 125 mg/dL. 2 BMI ≥25 was used as a proxy for abdominal obesity because WC data are not available in the CHANCE study. METS (IDF) was used to perform sensitivity analysis. ## Efficacy outcomes The primary efficacy outcome was a new stroke (ischemic or hemorrhagic) within 90 days. [bib_ref] Clopidogrel with aspirin in acute minor stroke or transient ischemic attack, Wang [/bib_ref] Recurrent stroke was defined by the presence of a sudden new symptomatic neurological deficit on a background of stability or improvement after the presenting event. [bib_ref] Early use of Existing Preventive Strategies for Stroke (EXPRESS) study. Effect of..., Rothwell [/bib_ref] Secondary efficacy outcome contained composite events (ischemic stroke, hemorrhagic stroke, myocardial infarction, or vascular death). [bib_ref] Clopidogrel with aspirin in acute minor stroke or transient ischemic attack, Wang [/bib_ref] All events were evaluated and confirmed by a central adjudication committee that was blinded to the study group assignments. # Statistical analysis Continuous variables of baseline characteristics were presented as medians with interquartile ranges and categorical variables as proportions. Baseline variables between patients included in and excluded from this analysis were compared with the Wilcoxon rank sum test for continuous variables and the v 2 test for categorical variables. Baseline variables among different METS and DM status (both, METS only, DM only, or neither) were compared with the Kruskal-Wallis test for continuous variables and the v 2 test for categorical variables. The interaction effect of METS and DM status with antiplatelet therapy group assignment was examined using METS/DM status by treatment group assignment in multivariable Cox models. We further assessed the relationship between METS and DM status and outcomes of minor stroke or TIA using multivariable Cox regression models with the neither group as reference. Adjusted hazards ratios (HRs) and their 95% CIs were calculated. All potential confounding variables were adjusted. The proportional hazards assumption for the Cox models was examined by adding a time-dependent covariate with interaction of METS/DM status and a logarithmic function of survival time in the model. In sensitivity analyses, the relationship between METS/DM status and patient outcomes was assessed by propensity score methods. The generalized propensity score for each METS/DM category was estimated using a nonparsimonious multivariable multinomial logistic regression model. All baseline variables were included to calculate the generalized propensity score. Then, HRs with their CIs were estimated by Cox regression models with adjustment of propensity score or weighting of inverse probability of METS/DM category. [bib_ref] Generalized propensity score for estimating the average treatment effect of multiple treatments, Feng [/bib_ref] We also performed a similar analysis using METS (IDF) criteria in a sensitivity analysis. A 2-sided P<0.05 was considered to be statistically significant. All analyses were performed with SAS software version 9.4 (SAS Institute Inc). # Results ## Baseline characteristics Among 5170 patients, a total of 3044 patients (59%) with minor stroke or TIA were included from these 73 centers, and 2126 patients (41%) were excluded because of missing data of triglyceride and high-density lipoprotein levels. The patients included in and excluded from the study were well balanced, except for a slightly higher proportion of DM and TIA and less severity in symptom presentation in excluded patients [fig_ref] Table 1: Baseline Characteristics of the Patients Included in and Excluded From This Analysis [/fig_ref]. For the included patients, the baseline characteristics in the clopidogrel-aspirin and aspirin-alone groups were well balanced [fig_ref] Table 2: Baseline Characteristics of the Patients Included in the Analysis by Treatment Group [/fig_ref]. The baseline characteristics of 3044 included patients are listed in [fig_ref] Table 3: Baseline Characteristics of Patients According to METS [/fig_ref]. Of the 3044 participants, the average age was 62.2 years, and 1017 (33.4%) were female. Using the CDS definition of METS, 53.2%, 17.2%, 19.8%, and 9.8% of the [fig_ref] Table 3: Baseline Characteristics of Patients According to METS [/fig_ref]. As shown in [fig_ref] Table 5: Distributions of Metabolic Factors in Patients With or Without METS [/fig_ref] , hypertension was the most prevalent metabolic component of METS (CDS), detected in 99.1% of the nondiabetic patients, followed by dyslipidemia (89.9%) and obesity (88.2%), whereas elevated fasting glucose was relatively uncommon (33.1%). were also associated with increased risk of recurrent stroke (FigureB). METS only did not show an increased risk of recurrent stroke in both definitions (adjusted . In sensitivity analyses, we observed similar results using the propensity score method [fig_ref] Table 8: Sensitivity Analysis of Hazard Ratios Estimated by Propensity Score Method [/fig_ref]. Similar results were observed in the secondary outcomes of composite events and ischemic stroke in both criteria. # Discussion In this post hoc analysis of the CHANCE study, patients with DM only or concurrent METS and DM had higher recurrent stroke risk than those with neither condition; however, METS only was not associated with stroke recurrence in patients with minor stroke or TIA. There was no difference in the effect of antiplatelet treatment in reducing these events in patients with or without METS or DM. As we hypothesized, DM only and concurrent METS and DM were significant risk factors of recurrent stroke in patients with minor stroke or TIA in present study. Different from what we expected, METS showed only a trend of increased risk of recurrent stroke and did not reach statistical significance. Even with medical intervention, minor stroke and TIA still led to a high risk of recurrence that could raise the disability rate. 14,15 METS was frequently found in patients with minor Patients with METS only did not have significantly higher risk of any recurrent stroke than those with neither condition. This finding might be associated with the fact that the definition of METS in our study did not include patients with DM. A previous study showed that METS likely played a crucial role in the development of recurrent ischemic stroke in patients with ischemic stroke or TIA. [bib_ref] Metabolic syndrome and three of its components as risk factors for recurrent..., Liou [/bib_ref] The definition of METS in this study included DM; however, results of substudies of the SPARCL (Secondary Analysis of the Stroke Prevention by Aggressive Reduction in Cholesterol Levels) trial and the SPS3 (Secondary Prevention of Small Subcortical Strokes) study demonstrated that patients with METS only were not at an increased risk of recurrent stroke. [bib_ref] Risk of stroke and cardiovascular events after ischemic stroke or transient ischemic..., Callahan [/bib_ref] [bib_ref] Recurrent vascular events in lacunar stroke patients with metabolic syndrome and/or diabetes, Zhu [/bib_ref] Unlike previous studies, the definition of METS in these 2 studies did not include DM, similar to our present study. Consequently, the definition of METS including DM or not might be a factor that influenced the relationship between METS and risk of stroke recurrence. Several organizations formulated different criteria for METS diagnosis, but all showed 4 main categories of metabolic abnormalities: atherogenic dyslipidemia, increased blood pressure, abnormal glucose regulation, and obesity. [bib_ref] American Heart Association; National Heart, Lung, and Blood Institute. Diagnosis and management..., Grundy [/bib_ref] [bib_ref] Diagnosis and management of the metabolic syndrome: a Chinese Diabetes Society Scientific..., Xiang [/bib_ref] Among these factors, abnormal glucose regulation was an established risk factors of recurrent stroke in patients with stroke or TIA. [bib_ref] Prediabetes in patients with stroke or transient ischemic attack: prevalence, risk and..., Fonville [/bib_ref] The physiology underlying the elevated risk of recurrent ischemic stroke in diabetic METS may be that it was a recognized risk factor of intracranial atherosclerosis. [bib_ref] Metabolic syndrome is more associated with intracranial atherosclerosis than extracranial atherosclerosis, Park [/bib_ref] [bib_ref] Association of the metabolic syndrome with intracranial atherosclerotic stroke, Bang [/bib_ref] Previous studies also reported that diabetic METS was associated with recurrent ischemic stroke in patients with large-vessel infarction or lacunar stroke, 10,17 which we were not able to examine in our study. Patients enrolled in the CHANCE trial primarily were minor stroke patients with stroke subtypes of large-artery atherosclerosis and smallvessel occlusion. [bib_ref] Clopidogrel with aspirin in acute minor stroke or transient ischemic attack, Wang [/bib_ref] [bib_ref] Rationale and design of a randomized, double-blind trial comparing the effects of..., Wang [/bib_ref] Therefore, patients with concurrent METS and DM had higher risk of recurrent stroke than those with nondiabetic METS (METS only) and neither in our study. In this post hoc analysis, we applied BMI rather than WC for definition of abdominal fat based on a previous study that showed WC and BMI can both be used in the prediction of abdominal visceral obesity for Chinese adults, 1 and the BMI cutoff for abdominal obesity was ≥25. [bib_ref] Prediction of abdominal visceral obesity from body mass index, waist circumference and..., Jia [/bib_ref] [bib_ref] Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1:..., Alberti [/bib_ref] [bib_ref] Epidemiological study on obesity and its comorbidities in urban Chinese older than..., Jia [/bib_ref] Even so, this still might be a bit inaccurate for the diagnosis of METS (IDF). Nevertheless, it is unlikely that using BMI as a proxy would alter the results because similar patients were diagnosed with METS regardless of whether BMI or WC was used. [bib_ref] American College of Endocrinology position statement on the insulin resistance syndrome, Einhorn [/bib_ref] Large epidemiologic studies have shown a high correlation between BMI and WC. [bib_ref] Trends in waist circumference among U.S. adults, Ford [/bib_ref] Furthermore, the association between obesity and insulin resistance was similar regardless of whether WC or BMI was used for obesity diagnosis. [bib_ref] Insulin resistance and hypersecretion in obesity. European Group for the Study of..., Ferrannini [/bib_ref] Using METS with the IDF definition in a sensitivity analysis in our study, the results also demonstrated similar results. Our study has some limitations. First, data on 2-hour plasma glucose, which is an item for assessment of METS (CDS), were not available in the CHANCE trial; therefore, we may have missed some patients who could be diagnosed as having METS. Second, only Chinese patients were enrolled in our study; further evaluation of METS in other races might be required. Third, the characteristics of minor stroke and TIA patients enrolled in this study were different from those of a typical minor stroke or TIA sample from populationbased cohorts. This study enrolled only minor stroke patients with noncardiogenic embolism and high-risk TIA patients (ABCD 2 scores ≥4), which may have resulted in high events rates. Furthermore, large-scale population-based cohorts assessing the association of METS and recurrent stroke are needed to confirm this finding. Finally, duration of DM was not recorded in the CHANCE trial. The duration of DM might be associated with the prognosis of patients with stroke. [bib_ref] Duration of diabetes and risk of ischemic stroke: the Northern Manhattan Study, Banerjee [/bib_ref] # Conclusions The results from our study showed that patients with DM only or concurrent METS and DM were associated with an elevated risk of stroke among patients with minor ischemic stroke and TIA. Nondiabetic METS (METS only) was not observed to be associated with stroke recurrence in patients with minor stroke or TIA. [table] Table 1: Baseline Characteristics of the Patients Included in and Excluded From This Analysis [/table] [table] Table 2: Baseline Characteristics of the Patients Included in the Analysis by Treatment Group [/table] [table] Table 3: Baseline Characteristics of Patients According to METS (Chinese Diabetes Society) Status BMI indicates body mass index; DM, diabetes mellitus; IQR, interquartile range; METS, metabolic syndrome; NIHSS, National Institutes of Health Stroke Scale; TIA, transient ischemic attack. *ABCD 2 stroke risk scores range from 0 to 7, with higher scores indicating higher risk. Data are provided only for the group of 817 patients for whom TIA was the qualifying event for inclusion in the trial. Using the CDS criteria of METS, patients with DM only (16.1% versus 6.8%; adjusted HR 2.50, 95% CI 1.89-3.39) and both (17.1% versus 6.8%; adjusted HR 2.76, 95% CI 1.98-3.86) were associated with increased risk of recurrent stroke (FigureA). Using the IDF criteria of METS, patients with DM only (15.7% versus 6.5%; adjusted HR 2.53, 95% CI 1.89-3.37) and both (17.7% versus 6.5%; adjusted HR 3.08, 95% CI 2. [/table] [table] Table 4: Baseline Characteristics of Patients According to METS (International Diabetes Foundation) Status [/table] [table] Table 5: Distributions of Metabolic Factors in Patients With or Without METS (Chinese Diabetes Society) [/table] [table] Table 6: Risk of Stroke at 3 Months for Clopidogrel-Aspirin Combined Therapy Comparing With Aspirin Alone by METS (Chinese Diabetes Society) StatusModel 2: adjusted for age, sex, history of ischemic stroke, transient ischemic attack, myocardial infarction, angina, congestive heart failure, known atrial fibrillation or flutter, valvular heart disease, smoking status, index event and National Institutes of Health Stroke Scale on admission, and time to randomization. [/table] [table] Table 7: Risk [/table] [table] Table 8: Sensitivity Analysis of Hazard Ratios Estimated by Propensity Score Method [/table]

Return to Play Following Shoulder Stabilization: A Systematic Review and Meta-analysis Background: Anterior shoulder instability can be a disabling condition for the young athlete; however, the best surgical treatment remains controversial. Traditionally, anterior shoulder instability was treated with open stabilization. More recently, arthroscopic repair of the Bankart injury with suture anchor fixation has become an accepted technique.Hypothesis: No systematic reviews have compared the rate of return to play following arthroscopic Bankart repair with suture anchor fixation with the Bristow-Latarjet procedure and open stabilization. We hypothesized that the rate of return to play will be similar regardless of surgical technique.Study Design: Systematic review; Level of evidence, 4.Methods:We performed a systematic review and meta-analysis focused on return to play following shoulder stabilization. Inclusion criteria included studies in English that reported on rate of return to play and clinical outcomes following primary arthroscopic Bankart repair with suture anchors, the Latarjet procedure, or open stabilization. Statistical analyses included Student t tests and analyses of variance.Results: Sixteen papers reporting on 1036 patients were included. A total of 545 patients underwent arthroscopic Bankart repair with suture anchors, 353 with the Latarjet procedure, and 138 with open repair. No significant difference was found in patient demographic data among the studies. Patients returned to sport at the same level of play (preinjury level) more consistently following arthroscopic Bankart repair (71%) or the Latarjet procedure (73%) than open stabilization (66%) (P < .05). Return to play at any level and postoperative Rowe scores were not significantly different among studies. Recurrent dislocation was significantly less following the Latarjet procedure (3.5%) than after arthroscopic Bankart repair (6.6%) or open stabilization (6.7%) (P < .05).Conclusion: This systematic review demonstrates a greater rate of return to play at the preinjury level following arthroscopic Bankart repair and the Latarjet procedure than open stabilization. Despite this difference, >65% of all treated athletes returned to sport at their preinjury levels, with other outcome measures being similar among the treatment groups. Therefore, arthroscopic Bankart repair, the Latarjet procedure, and open stabilization remain good surgical options in the treatment of the athlete with anterior shoulder instability. Anterior shoulder instability can be a disabling condition for the young athlete. Treatment of the athlete with shoulder instability continues to evolve and remains a topic of debate in the literature. The ultimate goal of many of these patients, especially younger ones, is to return to the activities and sports in which they were participating prior to injury. Unidirectional anterior instability is most commonly caused by sequelae of traumatic anterior shoulder dislocation, which can tear the anterior labrum and cause injury to the anterior capsule and/or glenoid. [bib_ref] Mechanisms of glenohumeral joint stability, Lippitt [/bib_ref] Anterior shoulder instability, particularly in older individuals, can oftentimes be satisfactorily treated nonoperatively through physical therapy and activity modification. [bib_ref] History of surgical intervention of anterior shoulder instability, Levy [/bib_ref] However, for cases of recurrent dislocations among younger patients, for patients participating in collision/contact sports, or for those patients otherwise at risk for ongoing instability, surgery is typically indicated to prevent continued instability. [bib_ref] Anterior shoulder stabilization by Bristow-Latarjet procedure in athletes: return-to-sport and functional outcomes..., Beranger [/bib_ref] [bib_ref] History of surgical intervention of anterior shoulder instability, Levy [/bib_ref] [bib_ref] Shoulder sport-specific impairments after arthroscopic Bankart repair: a prospective longitudinal assessment, Stein [/bib_ref] Although open stabilization has been regarded as the gold standard in the surgical management of anterior shoulder instability, arthroscopic repair of the Bankart injury with suture anchor fixation (arthroscopic Bankart procedure) has become an accepted and primary treatment option. [bib_ref] History of surgical intervention of anterior shoulder instability, Levy [/bib_ref] Owens et al [bib_ref] Surgical trends in Bankart repair: an analysis of data from the American..., Owens [/bib_ref] highlighted the increasing utilization of arthroscopic techniques in the treatment of shoulder instability, with a nearly 20% increase in arthroscopic procedures over a 5-year period. Open stabilization involves direct open repair of the injury to the capsule. Another popular procedure, the Bristow-Latarjet procedure, involves transfer of the coracoid process to the anterior rim of the glenoid to act as a bone block preventing anterior translation and subsequent dislocation of the humeral head from the glenoid. [bib_ref] Anterior shoulder stabilization by Bristow-Latarjet procedure in athletes: return-to-sport and functional outcomes..., Beranger [/bib_ref] [bib_ref] Arthroscopic Latarjet procedure, Lafosse [/bib_ref] This improves the inherent stability of the glenohumeral joint because of the increased excursion required to dislocate, and it provides an anteroinferior soft tissue sling.The arthroscopic Bankart repair improves stability by directly tightening the anterior capsule as well as repairing the anterior labrum into a socalled anteroinferior bumper. [bib_ref] Functional outcome and the structural integrity of arthroscopic Bankart repair: a prospective..., Zhu [/bib_ref] In the literature to date, there are data suggesting that arthroscopic and open techniques, including open capsular repair and reconstruction and the Latarjet procedure for addressing anterior shoulder instability, are safe and effective, with few complications and low rates of recurrent instability regardless of the technique. [bib_ref] Arthroscopic Bankart repair versus open Bristow-Latarjet for shoulder instability: a matched-pair multicenter..., Blonna [/bib_ref] [bib_ref] Arthroscopic versus open stabilization for anterior shoulder subluxations, Owens [/bib_ref] The focus of the current literature comparing these techniques, however, has largely been to investigate any difference in rates of redislocation or need for revision surgery. To our knowledge, no systematic reviews have examined the rate of return to play following arthroscopic Bankart repair with suture anchor fixation as compared with open stabilization and the Latarjet procedure. Given the many smaller series evaluating these techniques, this study is ideal for a systematic review to provide additional clinical information to surgeons and patients. It was our aim to describe rates of return to sport in the arthroscopic and open variants of surgical correction of anterior shoulder instability. We describe return to the same and lower level of play, as well as time to return to play. Our hypothesis was that there would be no difference in rates of return to play among these 3 common surgical techniques. # Methods A systematic review and meta-analysis of the literature were performed following the PRISMA guidelines (Preferred Reporting Items for Systematic Reviews and Metaanalyses). [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] A protocol was established stating the goal of the review as well as the search strategies. ## Search strategy An electronic search of the literature was performed in MEDLINE via PubMed, Scopus, Embase, and the Cochrane Library of Systematic Reviews. Searches included the keywords "return to play/sport," "shoulder stabilization," "Bankart repair," "Latarjet," and "open stabilization." The final search was performed on November 1, 2016. The references were checked for each article, and a manual search of related articles was performed. ## Selection criteria Inclusion criteria were studies in the English language (levels of evidence 1-4) that evaluated return to sport and clinical outcomes after treatment of traumatic shoulder instability. Surgical treatments included primary arthroscopic capsule-labral repair (the arthroscopic Bankart procedure) with suture anchors, primary Latarjet procedure, and primary open stabilization (open capsular repair or the open Bankart procedure). Exclusion criteria were reviews of the literature, expert opinions, nonclinical studies, case reports, and clinical studies that did not assess return to play. Associated lesions (superior labral anterior-posterior lesions, rotator cuff tears) were not exclusion criteria. Two authors selected the abstracts and then analyzed them separately. When the abstract seemed pertinent, the article was analyzed. ## Data extraction and synthesis Data extraction was performed according to a standardized form developed prior to data search. It included (1) characteristics of the study (design, year, number of patients, and level of evidence), (2) characteristics of the study participants (age, sex, level of sport, sports played, dominant shoulder), (3) characteristics of pathology (number of times dislocated, dislocation during sport), (4) treatment technique (arthroscopic Bankart repair with suture anchors, Latarjet procedure, or open stabilization), and (5) clinical outcome measures (rate of return to sport at preinjury level or lower, time to return to play, Rowe scores). Data were analyzed with PSPP (GNU Project) statistical data software. The main judgment criterion was whether a study participant was able to return to sport following surgery. Subgroup analysis was also performed assessing whether the participant was able to return to preinjury level of sport, as well as time to return to sport. Redislocation rate following surgery was analyzed. Rowe score was also analyzed as a self-reported clinical outcome measure. Bivariate data were analyzed with a Student t test, while 1-way analysis of variance (ANOVA) was used to analyze differences among the means of the 3 repair techniques. P ¼ .05 was considered statistically significant in all tests. # Results Initial search, including studies reporting on outcomes following surgical stabilization, yielded 1756 studies [fig_ref] Figure 1: Systematic review flowchart [/fig_ref]. Sixty-three of these records reported on return to play following surgical stabilization. After removal of duplicate studies, 47 studies remained for abstract review. An additional 26 studies were found to be nonpertinent and were thus removed following abstract review. The remaining 21 studies underwent full study review. Five studies were removed, leaving 16 papers remaining for inclusion in quantitative and qualitative synthesis [fig_ref] TABLE 1 Studies: Meeting Inclusion Criteria for Meta-analysis a [/fig_ref]. Sixteen papers were reviewed, including 1036 patients. Nine studies were identified that described return to sport after arthroscopic shoulder stabilization. These studies included a total of 545 patients (438 male, 72 female, 35 not reported). Mean age at surgery was 27.6 years. Length of follow-up was highly variable, ranging from 32 to 82 months. A majority of the patients (58%) underwent arthroscopic Bankart repair in the lateral decubitus position. Six studies were identified that described return to sport after the Latarjet procedure. These studies included a total of 353 patients (329 male, 24 female). Mean age at surgery was 26.5 years. Three studies were identified that described return to sport after open shoulder stabilization procedure. These studies included a total of 138 patients (113 male, 25 female) and 150 shoulders. Mean age at time of surgery was 24 years. Length of follow-up was again variable among the studies, with a range from 6 months to 28 years. No significant difference was found among the demographics of these groups. A 1-way ANOVA was conducted to compare the returnto-sport rate at the preinjury level among patients who underwent Latarjet stabilization, open repair, and arthroscopic suture anchor stabilization. Patients returned to their desired sport at the same level of play (preinjury level) more consistently following arthroscopic Bankart repair with suture anchors (71%) or the Latarjet procedure (73%) than those treated with open stabilization (66%) (P < .05). Arthroscopic and Latarjet procedures were found to be associated with a higher rate of return to play at the same level when compared with open stabilization (P < .05) [fig_ref] TABLE 3: Rate of Return to Sport a [/fig_ref]. However, return to sport at any level was not significantly different among the groups. The time to return to sport was longer following arthroscopic Bankart repair with suture anchors (8 months # Discussion This systematic review and meta-analysis demonstrated no statistically significant difference in rate of return to play following arthroscopic Bankart repair, Latarjet procedure, and open stabilization. No differences were found among the groups when analysis included patients returning to all levels of sport, including lower level than that prior to injury. Rowe scores were not statistically significant different among the 3 treatment options analyzed. The Latarjet procedure demonstrated a lower overall redislocation rate in comparison with arthroscopic Bankart repair utilizing suture anchors and open stabilization. As surgical stabilization is becoming a more commonly performed procedure for the young active patient with a history of traumatic glenohumeral dislocation, [bib_ref] Surgical trends in Bankart repair: an analysis of data from the American..., Owens [/bib_ref] the rate of return to play is an increasingly important outcome measure. Arthroscopic Bankart repair has become the treatment of choice for many surgeons in the setting of minimal glenoid bone loss. [bib_ref] Arthroscopic Bankart repair in a high demand patient population, Bacilla [/bib_ref] [bib_ref] Long-term outcomes after Bankart shoulder stabilization, Harris [/bib_ref] [bib_ref] Long-term results with the Bankart and Bristow-Latarjet procedures: recurrent shoulder instability and..., Hovelius [/bib_ref] [bib_ref] Arthroscopic versus open stabilization for anterior shoulder subluxations, Owens [/bib_ref] [bib_ref] Mid-term results of arthroscopic Bankart repair: a review of 31 cases, Tordjman [/bib_ref] Despite this fact, the current literature demonstrates wide variation with regard to the comparison of arthroscopic stabilization with open stabilization. [bib_ref] The open Latarjet procedure is more reliable in terms of shoulder stability..., Bessiere [/bib_ref] [bib_ref] Coracoid bone block versus arthroscopic Bankart repair: a comparative paired study with..., Bessiere [/bib_ref] [bib_ref] Arthroscopic Bankart repair versus open Bristow-Latarjet for shoulder instability: a matched-pair multicenter..., Blonna [/bib_ref] [bib_ref] Arthroscopic versus open Bankart repair for traumatic anterior shoulder instability, Cole [/bib_ref] Few studies have compared the Latarjet procedure with arthroscopic Bankart repair with suture anchors, and no studies to date have analyzed the rates of return to play among the 3 most common stabilization procedures, possibly owing to the fact that there are different indications for each procedure for many surgeons. Arthroscopic Bankart repair has demonstrated an excellent rate of return to sport to the preinjury athletic level. 14,31-33 Blonna and colleagues, 5 for example, Several outcome measures are typically reported in studies of anterior stabilization surgery, including Rowe score, 12-Item Short Form Survey scores, and American Shoulder and Elbow Surgeons scores. Our systematic review demonstrated wide variation in outcome scores reported. Most athletes define surgical success as ability to return to sport at the preinjury level, with partial success being defined as return to some level of sport. [bib_ref] Arthroscopic Bankart repair versus open Bristow-Latarjet for shoulder instability: a matched-pair multicenter..., Blonna [/bib_ref] [bib_ref] Validity and reliability of the SPORTS score for shoulder instability, Blonna [/bib_ref] [bib_ref] Shoulder sport-specific impairments after arthroscopic Bankart repair: a prospective longitudinal assessment, Stein [/bib_ref] This holds especially true at the higher levels of competition. For these reasons, we focused our study on the rate of return to play as well as the redislocation rate. Although some scores have been validated (eg, Subjective Shoulder Value-Sport), [bib_ref] Validity and reliability of the SPORTS score for shoulder instability, Blonna [/bib_ref] the variation in outcome measures makes it difficult to compare among multiple studies. Redislocation rates were similar among the 3 procedures assessed in our study, with the Latarjet procedure slightly outperforming arthroscopic suture anchor and open stabilization. This is consistent with most of the currently published literature, 20,23 although wide variation in postoperative redislocation rates exists. Tordjman and colleagues 32 reported a redislocation rate of 16% following arthroscopic stabilization, while other studies reported much lower incidence of recurrent instability. [bib_ref] Arthroscopic Bankart shoulder stabilization in athletes: return to sports and functional outcomes, Gerometta [/bib_ref] [bib_ref] Arthroscopic suture anchor fixation of bony Bankart lesions: clinical outcome, magnetic resonance..., Plath [/bib_ref] Despite these similar dislocation rates, the rate of return to play at the preinjury level was lower among those patients who underwent open stabilization. This suggests that other factors, such as shoulder stiffness and loss of motion or strength with open stabilization, may play a role in return to sport, especially at the collegiate or professional level. These factors should continue to be examined in the future. Given its excellent rate of return to play, especially at a high level of competition with limited complications, arthroscopic Bankart procedure should be considered an option for athletes with recurrent shoulder instability. The Latarjet procedure and open stabilization remain options for the modern-day athlete. Limitations of this study include the inherent limitations of a systematic review and meta-analysis, including multiple and inconsistent outcome measures reported in the individual studies. Furthermore, the type of sport involved was inconsistently reported in the included studies. The major strength of this study is the large number of patients included, especially for arthroscopic Bankart repair utilizing only suture anchor fixation. Also, this is the first study to date to compare rates of return to play among the 3 most prevalent stabilization procedures. Our outcomes are consistent with published data but elucidate the rate of return to play as well as time to return with current techniques. # Conclusion This systematic review demonstrated no overall difference in the rate of return to play, as well as patient-reported outcomes, following arthroscopic Bankart repair, the Latarjet procedure, and open stabilization. Despite this difference, >65% of all treated athletes returned to sport at their preinjury levels, with other outcome measures being similar among the treatment groups. Therefore, arthroscopic Bankart repair, the Latarjet procedure, and open stabilization remain good surgical options in the treatment of the athlete with anterior shoulder instability. [fig] Figure 1: Systematic review flowchart. [/fig] [table] TABLE 1 Studies: Meeting Inclusion Criteria for Meta-analysis a [/table] [table] TABLE 3: Rate of Return to Sport a [/table]

Reduced Surface Expression of Epithelial E-Cadherin Evoked by Interferon-Gamma Is Fyn Kinase-Dependent Interferon gamma (IFNc) is an important regulatory cytokine that can exert a pro-inflammatory effect in the gut, where it has been shown to increase epithelial permeability via disruption of the tight junctions. Here we investigated the potential for IFNc to regulate the adherens junction protein E-cadherin, an important mediator of normal epithelial tissue function, using the model T84 human colonic epithelial cell line. IFNc (10 ng/ml) stimulated increased internalization of E-cadherin as assessed by immunofluorescence microscopy; internalization was reversed when cells were treated with PP1 (125 nM), a Src kinase-selective inhibitor. Immunoprecipitation studies demonstrated loss of E-cadherin from membrane fractions following IFNc treatment and a corresponding increase in cytosolic E-cadherin and its binding partners, p120-catenin and betacatenin: effects that were Src-kinase dependent. E-cadherin and p120-catenin phosphorylation was increased by IFNc treatment and siRNA studies showed this was dependent upon the Src-kinase isoform Fyn. E-cadherin ubiquitinylation and subsequent proteasomal degradation stimulated by IFNc was found to be dependent upon Fyn and the E-cadherinselective ubiquitin ligase, Hakai. Use of Fyn and Hakai siRNA inhibited the internalization of E-cadherin as shown by immunoblotting and confocal fluorescence microscopy. Finally, IFNc treatment resulted in a more fragile T84 cell monolayer with increased cell detachment in response to physical stress, which was prevented by PP1 and siRNA targeting Fyn or Hakai. Collectively, these results demonstrate a Fyn kinase-dependent mechanism through which IFNc regulates E-cadherin stability and suggest a novel mechanism of disruption of epithelial cell contact, which could contribute to perturbed epithelial barrier function. # Introduction The integrity of the intestinal epithelial monolayer constitutes an important regulated barrier that controls access of the gut microflora, an abundant population of commensal and potentially pathogenic microbes, to the mucosa and thus is a key modulator of immune-mediated inflammatory activity within the intestinal submucosa [bib_ref] Autophagy, microbial sensing, endoplasmic reticulum stress, and epithelial function in inflammatory bowel..., Kaser [/bib_ref]. Furthermore, increases in enteric epithelial permeability often parallel the onset of inflammatory disease and also potentially colorectal cancer [bib_ref] Microenvironmental regulation of stem cells in intestinal homeostasis and cancer, Medema [/bib_ref]. As the association between microbial-driven inflammation and cancer becomes more apparent, a greater awareness of mechanisms of altered gut epithelial function during inflammatory responses may lead to additional or improved treatment strategies. Interferon-gamma (IFNc) is a key inflammatory cytokine primarily secreted by T cells and natural killer (NK) cells that has a well-described role during intestinal inflammation [bib_ref] Interferon-c is causatively involved in experimental inflammatory bowel disease in mice, Ito [/bib_ref] [bib_ref] Differential regulation of interleukin 17 and interferon-c production in inflammatory bowel disease, Rovedatti [/bib_ref]. IFNc stimulates increased intestinal epithelial permeability by reducing tight junction stability (paracellular permeability) [bib_ref] Interferon-c directly affects barrier function of cultured intestinal epithelial monolayers, Madara [/bib_ref] , and our work, and that of others, has shown that IFNc signalling through phosphatidylinositol 3-kinase (PI3K) and the Src-kinase family member, Fyn, promotes increased epithelial uptake of commensal bacteria and macromolecules in in vitro model systems [bib_ref] Interferon-c induces translocation of commensal Escherichia coli across gut epithelial cells via..., Clark [/bib_ref] [bib_ref] Phosphatidylinositol 39-kinase is a critical mediator of interferon-c-induced increases in enteric epithelial..., Mckay [/bib_ref] [bib_ref] Interferon-c-induced increases in intestinal epithelial macromolecular permeability requires the src kinase fyn, Smyth [/bib_ref]. Thus, IFNc may serve to exacerbate inflammatory responses via its effects on the epithelial barrier. Src kinase activity has been widely-associated with epithelial dysfunction. Src kinases have been linked to epithelial to mesenchymal transition (EMT) in response to growth factors or oxidative stress [bib_ref] The SRC-induced mesenchymal state in late-stage colon cancer cells, Avizienyte [/bib_ref] [bib_ref] Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition, Avizienyte [/bib_ref]. c-Src-mediated epithelial cell scattering has been suggested as an important step during acquisition of a transformed phenotype [bib_ref] Overexpression of pp60c-src elicits invasive behavior in rat colon epithelial cells, Pories [/bib_ref] , and the Src-family Fyn kinase has recently been identified as a potential catalyst for the development of prostate cancer [bib_ref] Fyn is over-expressed in human prostate cancer, Posadas [/bib_ref] [bib_ref] Fyn: a novel molecular target in cancer, Saito [/bib_ref]. However, extensive studies of Fyn kinase participation in the intestinal epithelial response to IFNc have not been conducted. A key mediator of the stability of the intestinal epithelium is the adherens junction. E-cadherin is a critical intercellular junctional protein that is maintained at the cell surface by interactions with p120-catenin, beta-catenin and additional proteins mediating adhesion to the actin cytoskeleton [bib_ref] Adherens junctions: from molecules to morphogenesis, Harris [/bib_ref]. Loss of E-cadherin function is associated with development of chronic inflammatory diseases including Crohn's disease [bib_ref] Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is..., Muise [/bib_ref]. E-cadherin is also considered a tumour suppressor, as loss of E-cadherin expression or activity is highly correlative to the onset of epithelial-derived cancers [bib_ref] Prognostic significance of the wnt signalling pathway molecules APC, beta-catenin and Ecadherin..., Lugli [/bib_ref]. Fyn kinase has been reported to induce E-cadherin internalization following epithelial cell exposure to acidic pH or growth factors in vitro [bib_ref] Fyn mediates transforming growth factor-b 1 -induced down-regulation of E-cadherin in human..., Kim [/bib_ref] [bib_ref] An acidic extracellular pH disrupts adherens junctions in HepG2 cells by src..., Chen [/bib_ref] , and p120-catenin, whose key function is to bind and stabilize E-cadherin, is known to be a Fyn kinase substrate [bib_ref] Identification of src phosphorylation sites in the catenin p120ctn, Mariner [/bib_ref]. Thus, we sought to determine whether IFNc-stimulated Fyn kinase activity affected epithelial adherens junction stability, specifically the expression and location of Ecadherin. Here we show that IFNc stimulates increased internalization of E-cadherin in the human colonic T84 epithelial cell line. Ecadherin internalization was reduced by treatment with the Src inhibitor PP1 and siRNA targeting Fyn, and by siRNA targeting Hakai, which has been characterized as a Src-dependent, Ecadherin-specific ubiquitin ligase. Blockade of Fyn or Hakai reduced the fragility of cell-cell adhesion in IFNc-treated plasticgrown T84 cell monolayers. The data illustrate that an adherens junction-destabilizing pathway involving Fyn kinase and Hakai can be activated by IFNc in T84 epithelia, and we speculate that this could be relevant to epithelial cell-cell adhesion and communication, and potentially enteric inflammatory disease. # Materials and methods ## Reagents and antibodies Cell culture supplements and pharmacologic inhibitors, were purchased from Sigma-Aldrich (Oakville, Ontario, Canada) unless otherwise indicated. The Src inhibitor PP1 was purchased from Biomol (Enzo Life Sciences, Plymouth Meeting, PA, USA). Recombinant human IFNc was from Ebioscience Inc. (San Diego, CA, USA). Mouse anti-E-cadherin and mouse anti-betacatenin antibodies were purchased from BD Transduction Labs (Mississauga, ON, Canada). Mouse anti-p120 catenin and mouse anti-phosphotyrosine (clone 4G10) antibodies were purchased from Upstate/Millipore (Billerica, MA, USA). Rabbit antioccludin was purchased from Zymed/Invitrogen (Carlsbad, CA). Rabbit anti-zonula occludens (ZO-1) antibody was purchased from Invitrogen. Goat anti-actin antibody, rabbit anti-CBLL/ Hakai, mouse anti-Fyn antibody and HRP-conjugated secondary antibodies were from Santa Cruz Biotech (Santa Cruz, CA, USA). AlexaFluor goat anti-mouse 488 and goat anti-rabbit 594 fluorescent secondary antibodies were purchased from Molecular Probes/Invitrogen (Carlsbad, CA, USA). ## Cell culture The immortalized human colon-derived T84 epithelial cell line (ATCC, Manassas, VA, USA) was cultured at 37uC/5% CO 2 in 1:1 Dulbecco's modified Eagle's Medium/Ham's F-12 medium supplemented with 2% (vol./vol.) penicillin-streptomycin, 1.5% HEPES, 5% NaHCO 3 , 1% L-glutamine, 1% sodium pyruvate (all from Invitrogen, Burlington, ON, Canada) and 10% fetal bovine serum (PAA Laboratories, VWR International, Edmonton, AB, Canada). All cytokine stimulations with IFNc were conducted using 10 ng/ml recombinant cytokine (equivalent to 250 biological units of activity/ml cell culture medium). Transient Transfection of T84 Cells with Small Interfering (si) RNA siRNAs targeting Fyn and Hakai were created using the Stealth siRNA oligomer design platform (Invitrogen). Target oligomer sequences used in this study are as follows: Fyn 59-GAGCGACAGCTATTGTCCTTTGGAA. Hakai 59 -CAACATGTGCCACATGAGCA CTATA. The control siRNA sequence used was 59-GAGACATCGTTACTGTTCGGAA. Transfections were performed as previously described [bib_ref] Interferon-c-induced increases in intestinal epithelial macromolecular permeability requires the src kinase fyn, Smyth [/bib_ref]. Briefly, 20 pM of siRNA in Lipofectamine 2000/Opti-MEM (500 mg/ml) (Invitrogen) was added to suspension cultures of T84 cells (1610 6 / ml) in antibiotic-free FBS-containing culture medium. The cells were then either seeded onto filter supports or 12-well culture dishes and following an overnight incubation, adherent cells were washed and transferred to antibiotic-containing culture medium. ## Immunoprecipitation and immunoblotting One million T84 cells were seeded onto 12 mm 2 permeable filter supports (Greiner Bio-One) in 12-well plates and cultured until confluent as assessed by phase-contrast microscopy and electrical confluence (transepithelial resistance (TER)$1000 V.mm 2 as measured by voltmeter and paired electrodes (Millipore) was accepted as an electrically confluent monolayer). As indicated, cell lysates were prepared following two washes with ice-cold phosphate-buffered saline (PBS). For low-salt, detergent-free lysate generation, cells were scraped into hypotonic Buffer A as described in [bib_ref] A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers..., Andrews [/bib_ref] supplemented with protease and phosphatase inhibitors (CompleteH protease inhibitor cocktail (Roche/Mannheim), 1 mM sodium orthovanadate, 1 mM sodium fluoride). Lysates were incubated with gentle agitation at 4uC for 30 min, centrifuged at 10,0006g and supernatants were collected and stored at -80uC. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA). For isolation of membrane or cytoskeletal components, T84 monolayers were lysed in 1% sodium dodecyl sulphate (SDS)/PBS supplemented with protease inhibitors as above. Isolation of equivalent quantities of cell material was assured by adjustment to Triton X-100 protein lysate concentrations of identically plated cell cultures. For immunoprecipitation experiments, monolayers were extracted using radioactive immunoprecipitation assay (RIPA) buffer (100 mM NaCl, 24 mM Tris-Cl, 1% (vol./vol.) NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with CompleteH protease inhibitor cocktail, sodium orthovanadate and sodium fluoride). Four hundred mg of clarified cell lysates were incubated in 2 mg/ml anti-E-cadherin antibody overnight at 4uC with gentle agitation. Immune complexes were isolated by incubation with EZ-link protein A-agarose beads (Sigma) at 4uC for 90 mins, followed by two washes in RIPA buffer and one wash with PBS. Immune complexes were eluted with 2X Laemmli buffer and set aside for immunoblotting. Protein lysate immunoblotting was performed by addition of 20 mg of lysates to Laemmli buffer, which were subsequently boiled and resolved on 8% SDS-PAGE. Separated proteins were blotted to Immobilon nitrocellulose membranes (Millipore), and blots were blocked at room temperature for 1 h in 5% non-fat milk/wash buffer (0.15% Tween-20/ Tris-buffered saline (TBS/T)). Primary antibodies (see Results) were incubated in 1% bovine serum albumin/TBS/T (for phosphoprotein analysis) or 5% non-fat milk/TBS/T (total proteins) overnight at 4uC with gentle rocking. Blots were washed three times in TBS/T and species-appropriate, HRP-conjugated secondary antibodies were applied with gentle rocking for 1 h at room temperature. Blots were washed, subjected to chemiluminescence (Western LightningH PLUS, PerkinElmer, Waltham, MA, USA) and subsequently exposed to Kodak XB-1 film (Eastman Kodak, Rochester, NY, USA). Densitometric quantification of phospho-E-cadherin and ubiquitinylated-E-cadherin chemiluminescence was performed by analysis of 16-bit JPEG blot images with Image J (version 1. [bib_ref] Src family kinases as potential therapeutic targets for malignancies and immunological disorders, Benati [/bib_ref] , NIH open access software, W. Rasband). Measurement was conducted on three replicate experimental immunoblots. Levels of phospho-Ecadherin or ubiquitinylated-E-cadherin were normalized to total Ecadherin immunoprecipitated per sample, and measurements presented as relative ratios compared to non-stimulated control (scored as a value of '1'). As controls, input lysate levels of E-cadherin were assessed and actin levels from unbound, or flow-through lysate, were also verified. ## Immunofluorescence microscopy T84 cells were plated (3610 5 cells/ml) onto glass coverslips in 12 well culture plates and grown to 70% confluent, at which time IFNc6PP1 (125 nM) was added for 48 h. Coverslips were washed three times in 4uC PBS, fixed in 4% paraformaldehyde (PFA), washed three times in PBS and then blocked with 10% goat serum for 1 h at room temperature. Monolayers were then incubated for 24 h at 4uC with mouse anti-E-cadherin (1:350) or anti-ZO-1 (1:100) antibody in PBS containing 10% goat serum and 0.1% Tween. Following three washes with 1X PBS, monolayers were incubated for 1 h at room temperature with AlexaFluor 488 goat anti-mouse secondary antibody (1:500). DAPI (1:500) was added for 1 min followed by 2 washes with 1X PBS, and coverslips were mounted onto slides with Fluorosave (Calbiochem), allowed to dry and stored in dark at 4uC. For visualization, slides were analyzed using an Olympus 4100BX epifluorescence microscope (Olympus) using the 40X objective lens: regions of monolayer were randomly selected based on DAPI-identification of nuclei and then specific immunofluorescence observed and images captured of that area. Confocal laser scanning microscopy. For some experiments, T84 cells were transfected with control, Fyn or Hakai siRNA and seeded onto 6 mm 2 filter supports at 1610 5 cells/ml. Following 72 h, cells were either left untreated or stimulated with IFNc for 48 h, and then washed and fixed as above. Subsequently, cells were stained with anti-E-cadherin antibody or anti-occludin antibody (1:350) then with appropriate AlexaFluor secondary antibodies (1:500), mounted and images captured on an Olympus FV1000 confocal scanning fluorescent microscope (40X objective). Images were collected and analyzed using FV10-ASW2.1 imaging software (Olympus). As described above, cell viability and localization were verified by nuclear DAPI staining. Determination of plane depth for analysis of occludin and E-cadherin was . The Src inhibitor PP1 blocks IFNc -stimulated E-cadherin internalization but does not affect the focal loss of zonula occludens-1 (ZO-1). T84 cells were grown on glass coverslips and stimulated with IFNc (10 ng/ml, 48 h)6the Src inhibitor PP1 (125 nM). Left panels: immunofluorescence microscopy of epithelial monolayers probed with a C-terminal-specific anti-E-cadherin antibody showed IFNc caused increased E-cadherin internalization (*), which was inhibited by PP1 co-treatment, whereas in contrast (right panels) the focal discontinuities in the distribution of ZO-1 induced by IFNc (arrows) was unaffected by PP1 co-treatment. Images collected are representative of two independent experiments each using replicate epithelial cell monolayers per treatment, the junctional pattern with E-cadherin reflects the more diffuse adherens junction which is in contrast to the more localized tight junction and associated plaque proteins (i.e. ZO-1). Scale bar = 10 mm. doi:10.1371/journal.pone.0038441.g001 carried out using occludin-immunoreactivity as the indicator of the apical aspect of the epithelial layer. For each captured image Ecadherin was imaged 0.2 mm deeper than the z-plane slice selected for occludin localization. ## Cell detachment assays T84 cells were seeded on 12 mm 2 permeable filter supports as described previously and grown to confluence and indicated by TER$1000 V.cm 2 . Where indicated, cells were transfected with control, Fyn or Hakai targeted siRNA. Control siRNAtransfected cells were either left untreated or treated with IFNc6125 nM PP1. Fyn and Hakai siRNA-treated T84 cells were also stimulated with IFNc. Following 48 h cytokine treatment, TER was measured, the cells washed twice with 1 ml of 1X PBS and incubated for 15 minutes at 37uC in 500 ml serum-free PBS. Subsequently, epithelial monolayers were rinsed gently by pipeting with PBS (ten passes per monolayer) and detached cells were collected into 1.5 ml Eppendorf tubes, placed on ice, and centrifuged for 2 min at 30006g. Cell number was assessed in two ways: (1) cells were re-suspended in 100 ml PBS and were deposited onto slides (50 ml/preparation) by Cytospin, stained with Cresyl violet, and then counted at 206 magnification on a bright field inverted microscope. Counts were made for each filter-grown epithelial monolayer, with a minimum of triplicate filters used per treatment condition; (2) cell number was approximated by quantification of total protein from the lysis of collected detached cells (using 100 ml RIPA buffer/filter collected) using the Bradford assay. Protein was measured according to a standard concentration curve using bovine serum albumin and results were plotted graphically as mg of detached cell protein isolated per filter; cell detachment was plotted as a percentage of detachment relative to non-stimulated control (which was assigned a percentage value of 100). # Statistical analysis Quantitative data are presented as mean6standard error of the mean (SEM), with n values given as the number of epithelial observations from replicate experiments. Single group comparisons were performed using Student's t test and multiple group statistical analysis was by a one-way analysis of variance (ANOVA) followed by pair-wise post-hoc statistics. For cell detachment studies, sample sizes were: non-stimulated (control), n = 5; IFNc, n = 6; IFNc+PP1, n = 5; IFNc+Fyn siRNA, n = 5; IFNc+Hakai, n = 4. In all analyses a p,0.05 was accepted as a level of statistically significant difference. # Results ## E-cadherin internalization is increased following ifnc treatment in a src-dependent manner Previous research indicates that IFNc is a potent modulator of intestinal tight junction form; however, relatively little attention has focused on the role of IFNc in the regulation of the adherens junction. IFNc has been reported to reduce surface expression of E-cadherin in intestinal epithelial cells [bib_ref] Proinflammatory cytokines disrupt epithelial barrier function by apoptosisindependent mechanisms, Bruewer [/bib_ref] , but the intracellular mechanism(s) responsible for this was not determined. Given our data identifying a key role for the Src kinase Fyn in IFNc-evoked increases in epithelial barrier function [bib_ref] Interferon-c-induced increases in intestinal epithelial macromolecular permeability requires the src kinase fyn, Smyth [/bib_ref] , we sought to determine if Src activity was required for IFNc-evoked changes in E-cadherin expression and localization in T84 epithelia. Initial studies used epifluorescence microscopy to obtain a view of the global impact of IFNc treatment on E-cadherin expression. Forty-eight hours after IFNc treatment (10 ng/ml), there was a marked accumulation of E-cadherin in cytosolic punctate structures and loss from . Increased solubilization of E-cadherin, p120-catenin and beta-catenin by IFNc is Src-dependent. A) Representative immunoblot showing differential solubility of E-cadherin, but not the peri-junctional area, that was reduced by co-treatment with the pan-Src inhibitor PP1 . As a comparison with tight junction structure, a 48 h treatment with IFNc resulted in the expected focal discontinuities in ZO-1 [bib_ref] Proinflammatory cytokines disrupt epithelial barrier function by apoptosisindependent mechanisms, Bruewer [/bib_ref] , that was unaffected by PP1 co-treatment . ## Ifnc treatment stimulates increased adherens junction protein accumulation in a soluble cytoplasmic isolate E-cadherin typically localizes to a highly insoluble cellular fraction enriched with cytoskeletal components. We hypothesized that IFNc stimulation would provoke a release of E-cadherin and interacting proteins from the cell membrane to the cytosol, where it would be extractable under less stringent isolation conditions. Lysis of equivalent-density cell cultures in either detergent-free, hypotonic conditions (termed low-salt buffer) or 1% SDS buffer (a highly disruptive, anionic detergent buffer) indicated that the majority of E-cadherin remained insoluble in non-stimulated T84 cells . Conversely, equivalent amounts of the tight junction protein occludin were identified by immunoblotting of extracts retrieved by the low-salt and SDS buffers. Analysis of T84 cell extracts obtained with the low-salt buffer revealed increased Ecadherin after 6 h of IFNc treatment, indicative of movement out of the membrane and into the cytosol . Similarly, coimmunoprecipitation experiments employing a monoclonal antibody raised against a C-terminal E-cadherin epitope revealed increased amounts of detectable p120-catenin and beta-catenin, two binding partners of the C-terminal domain of E-cadherin, in the low-salt extract: suggestive of the E-cadherin/p120 catenin/ beta-catenin disengagement from the cell membrane. No detectable E-cadherin, p120 catenin or beta-catenin could be recovered from control, non-immune IgG immunoprecipitated samples (data not shown). Inhibition of Src kinases with PP1 (125 nM, a concentration that targets Fyn) reduced the solubility of all three proteins following IFNc stimulation. In contrast to low-salt extracted conditions, immunoprecipitation of high-salt lysis bufferextracted E-cadherin showed a minor and insignificant increase in protein levels of both E-cadherin and p120-catenin following IFNc treatment . ## Ifnc-stimulated tyrosine phosphorylation of e-cadherin and p120 catenin is fyn-dependent We subsequently focused on identifying potential mechanisms responsible for the IFNc-evoked increase in E-cadherin solubility (i.e. dissociation from the membrane). Studies of v-Src transformed cell lines suggest that increased tyrosine phosphorylation of Ecadherin results in its dissociation from the cell surface; while p120-catenin phosphorylation has been shown to affect its ability to stabilize membrane-bound E-cadherin [bib_ref] The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at..., Fukumoto [/bib_ref]. Anticipating that an increase in phosphorylation of total E-cadherin would reflect the increased amounts of solubilised E-cadherin , epithelial cell lysis was conducted using RIPA buffer followed by SDS-PAGE and immunoblotting. IFNc treatment evoked a gen-eral increase in tyrosine phosphorylation in T84 cells [fig_ref] Figure 3: IFNc-stimulated phosphorylation of E-cadherin and p120-catenin is reduced by PP1 and Fyn... [/fig_ref]. Following 6 h of IFNc stimulation, there was increased tyrosine phosphorylation of E-cadherin, as demonstrated by immunoblotting of immunoprecipitated E-cadherin with the anti-phosphotyrosine antibody 4G10. Consistent with our observation of altered E-cadherin solubility, the Src inhibitor PP1 treatment strongly inhibited IFNc-stimulated E-cadherin phosphorylation [fig_ref] Figure 3: IFNc-stimulated phosphorylation of E-cadherin and p120-catenin is reduced by PP1 and Fyn... [/fig_ref]. We had previously reported that use of PP1 at low concentrations could exclude potential off-target inhibition of tyrosine phosphorylation by receptor tyrosine kinases such as EGFR or by inhibition of Janus kinases, and we showed that Fyn was a major Src family member stimulated by IFNc in T84 cells [bib_ref] Interferon-c-induced increases in intestinal epithelial macromolecular permeability requires the src kinase fyn, Smyth [/bib_ref]. Therefore, the effects of Fyn siRNA upon E-cadherin phosphorylation were examined. Consistent with the data from PP1, immunoprecipitates of Ecadherin demonstrated reduced tyrosine phosphorylation following IFNc treatment in the presence of Fyn siRNA as compared to control siRNA-treated lysates [fig_ref] Figure 3: IFNc-stimulated phosphorylation of E-cadherin and p120-catenin is reduced by PP1 and Fyn... [/fig_ref]. Additionally, IFNcstimulated p120-catenin phosphorylation was also reduced in T84 cells treated with Fyn siRNA [fig_ref] Figure 3: IFNc-stimulated phosphorylation of E-cadherin and p120-catenin is reduced by PP1 and Fyn... [/fig_ref]. Consistent with the results presented in and C, control (non-immune) mouse IgG antisera did not immunoprecipitate E-cadherin, validating immunoblot experiments (data not shown). ## E-cadherin ubiquitinylation is stimulated by ifnc and is dependent upon fyn and hakai Next we wanted to identify factors that may promote the internalization of tyrosine phosphorylated E-cadherin. Research examining the mechanisms of epithelial cell infection by the enteric pathogen Listeria monocytogenes has indicated that the internalization of E-cadherin may occur in part via ubiquitindependent mechanisms. Here, we found increased ubiquitinylation in whole cell lysates at 6-24 h post-IFNc treatment . Immunoprecipitated E-cadherin from T84 cells treated 24 h previously with IFNc was ubiquitinylated (Ub), but this was only apparent in samples from epithelia in which proteasome activity was inhibited by MG132 . Conversely, treatment of T84 cells with chloroquine, an inhibitor of lysosomal activity, did not prevent the degradation of Ub-Ecadherin induced by IFNc. As an indicator of chloroquine activity, we observed a decreased amount of lysosomal-associated Ecadherin fragmentation. Cleavage of lysosomal-targeted E-cadherin is evidenced by a marked E-cadherin C-terminal 30 kDa peptide [bib_ref] Raf plus TGFb-dependent EMT is initiated by endocytosis and lysosomal degradation of..., Janda [/bib_ref]. The lower panel of shows the soluble cytoplasmic fragments of E-cadherin, including an approximately 85 kDa peptide fragment released by cleavage of the extracellular domain and a 30 kDa fragment consistent with the size of the lysosomal-generated E-cadherin peptide. IFNc-stimulated T84 cell lysates demonstrated greater levels of 30 kDa E-cadherin, but less of the protein fragment was present in cell lysates from IFNc+chloroquine treated cultures . Additionally, only Ub-E-cadherin, not 'unmodified' E-cadherin levels, were markedly reduced by MG132. We then focused on candidate ubiquitinylation mechanisms that could direct E-cadherin to the proteasome for degradation. Hakai is a phosphorylation-dependent RING-type E3 ligase with a reported specificity for Ecadherin [bib_ref] Hakai, a ccbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex, Fujita [/bib_ref]. Hakai-mediated ubiquitinylation of E-cadherin is Src-dependent and promotes the proteasomal degradation of Ecadherin [bib_ref] Hakai, a ccbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex, Fujita [/bib_ref] [bib_ref] Successive posttranslational modifications of E-cadherin are required for InlA-mediated internalization of Listeria..., Bonazzi [/bib_ref]. Thus, we sought to determine whether IFNcstimulated E-cadherin phosphorylation by Fyn was required for Hakai mediated ubiquitinylation of E-cadherin. The upper panels of presented ubiquitinylation immunoblotting from RIPA-isolated T84 whole cell lysates. Irrespective of treatment, whole cell ubiquitin levels were only slightly modulated. However, from E-cadherin immunoprecipitates (shown in the lower panels) occludin, in low-salt buffer compared to high salt-1% SDS lysis buffer. B) E-cadherin is associated with p120-catenin and beta-catenin and complex solubility is increased by IFNc (10 ng/ml, 6 h) treatment as shown by SDS-PAGE of detergent-free (low-salt) lysates immunoprecipitated with anti-E-cadherin antibody. Inclusion of PP1 (125 nM) inhibits increased solubilization of E-cadherin/p120-catenin/beta-catenin caused by IFNc. Data shown are representative of three independent experiments. C) Immunoprecipitation of E-cadherin and p120-catenin following lysis in high-salt-containing buffer shows only marginal increase in expression in the IFNc treated epithelia. IgH and actin are included as loading controls. doi:10.1371/journal.pone.0038441.g002 we observed that levels of ubiquitinylated E-cadherin following 24 h IFNc exposure were significantly increased. In contrast, IFNc-stimulated T84 cells treated with Fyn siRNA demonstrated a significant reduction of ubiquitinylation, consistent with a requirement for tyrosine phosphorylation of E-cadherin prior to ubiquitinylation . Hakai co-immunoprecipitated with E-cadherin following IFNc treatment, and this association was reduced in Fyn siRNA-treated epithelia. We then assessed the effects of Hakai siRNA upon ubiquitinylation of E-cadherin . Similarly to Fyn siRNA treatment, whole cell lysate immunoblots (upper panels) showed marginal changes to overall ubiquitinylation levels, but immunoprecipitation of E-cadherin (lower panels) indicated an IFNc-stimulated increase in Ub-Ecadherin. Hakai siRNA-treated T84 epithelia displayed significantly reduced levels of Ub-E-cadherin following IFNc treatment. ## Fyn-and hakai-specific sirna reduce ifnc-stimulated ecadherin internalization Initial studies indicated that IFNc treatment of T84 epithelia resulted in a relatively rapid internalization of E-cadherin , but what of the effects, if any, in a longer timeframe? To address this, we used the proteasome inhibitor, MG132, to prevent the degradation of E-cadherin and so enhance its detection. When using low-salt lysis buffer, we found that the levels of soluble cytosolic E-cadherin were still elevated 48 h after IFNc-treatment and that this was substantially reduced by Fyn but not control siRNA treatment of the epithelia . This contrasted with the detection of E-cadherin in T84 cell extracts produced by lysis with a high-salt, Triton X-100-based buffer, where only slight increases in E-cadherin protein were observed following IFNc treatment relative to either controls or IFNc+Fyn siRNA-treated epithelia , lower panel). Consistent with the effects of Fyn, Hakai siRNA reduced the IFNc-stimulated solubilisation of E-cadherin . Again, there was only a modest increase in E-cadherin detected in Triton X-100, high salt lysis buffer-extracted lysates of IFNc-treated T84 cells. presents representative confocal immunofluorescence images showing membrane distribution of E-cadherin and occludin (images were collected at a consistent z-plane depth for E-cadherin and occludin, based on the first observable occludin immunoreactivity). Treatment with Fyn or Hakai siRNA reduced the loss of adherens junction-localized E-cadherin following 48 h of exposure to IFNc. Conversely, IFNc-stimulated disruption of tight junctions, indicated by dissociation of occludin, was neither affected by Fyn nor Hakai siRNA. ## T84 cell dissociation stimulated agitation of ifnc-treated monolayer is inhibited by fyn and hakai sirna A key functional measure of E-cadherin stability is the integrity of cell-cell contacts. The drop in TER that is consistently observed 48 h after IFNc-treatment (measured before the gentle flushing) was unaffected by PP1 co-treatment or knock-down of Fyn kinase or Hakai with siRNA [fig_ref] Figure 6: Increased cell detachment evoked by fluid shear in IFNc-treated T84 epithelial cell... [/fig_ref] , which is consistent with the inability of either treatment to prevent the disruption in the pattern of the tight-junction protein occludin by IFNc and the inability of PP1 to prevent the subtle changes in ZO-1 distribution . However, while the monolayer remained intact it was more fragile as demonstrated by the substantial increase in cell detachment evoked by a gentle, consistent flushing with warm PBS which could mimic some aspects of shear stress or fluid transit along the intestine. As shown both by enumeration of detached cells [fig_ref] Figure 6: Increased cell detachment evoked by fluid shear in IFNc-treated T84 epithelial cell... [/fig_ref] and quantification of total protein in cells collected from the culture medium [fig_ref] Figure 6: Increased cell detachment evoked by fluid shear in IFNc-treated T84 epithelial cell... [/fig_ref] , PPI inhibition of Src kinases significantly inhibited IFNc-stimulated cell detachment, as did siRNA knock-down of Fyn kinase or Hakai in T84 epithelia. # Discussion The enteric epithelial layer is an important active participant in mucosal immunity through the secretion of anti-microbial peptides, promotion of oral tolerance and formation of a barrier to the entry of lumen-derived material [bib_ref] Autophagy, microbial sensing, endoplasmic reticulum stress, and epithelial function in inflammatory bowel..., Kaser [/bib_ref] [bib_ref] Intestinal mucosal barrier function in health and disease, Turner [/bib_ref]. Epithelial injury can contribute to the exacerbation of inflammatory responses and affect the rate of restoration of homeostasis following infection, potentially leading to prolonged inflammation [bib_ref] Autophagy, microbial sensing, endoplasmic reticulum stress, and epithelial function in inflammatory bowel..., Kaser [/bib_ref]. Indeed, disruption of the integrity of the gut epithelium (or its function) is associated with chronic inflammatory diseases, such as Crohn's disease and ulcerative colitis [bib_ref] Epithelial barriers in homeostasis and disease, Marchiando [/bib_ref]. In addition to the direct effects of long-term injury to the epithelium, chronic inflammation can contribute to the development of cancer, a relationship that is increasingly recognized in the context of inflammatory bowel diseases and colorectal cancer [bib_ref] Inflammatory bowel disease as a risk factor for colorectal cancer, Lukas [/bib_ref]. Thus, there is considerable value in understanding the full impact of inflammatory signals such as IFNc on the control of enteric epithelial cell-cell contacts. The molecular and cellular mechanisms that program the gut to remain in a chronic diseased state are poorly defined. IFNc is an established immune effector molecule associated with intestinal inflammation in humans and animal models. Aside from its key function as an immune-stimulatory cytokine, in vitro study (and a lesser number of in vivo observations [bib_ref] Interferon-c is causatively involved in experimental inflammatory bowel disease in mice, Ito [/bib_ref] [bib_ref] Pro-inflammatory cytokines in the pathogenesis of inflammatory bowel diseases, Strober [/bib_ref] indicate that IFNc can significantly disrupt epithelial barrier function [bib_ref] Interferon-c regulation of intestinal epithelial permeability, Beaurepaire [/bib_ref] , thereby potentially exacerbating inflammation by facilitating a breach of the epithelial layer and entry of antigen and microbes into the mucosa. Therefore, IFNc is of particular interest since elucidation of the signal transduction pathways that promote immune function as opposed to those which elicit alterations in cell-cell interactions and decrease epithelial barrier function may uncover unique targets for therapeutic intervention. Extensive research efforts are revealing the molecular assembly of the epithelial tight junction, the structure primarily responsible for restricting the movement of material between adjacent cells, and how pro-inflammatory cytokines, including IFNc, affect the tight junction [bib_ref] Intestinal mucosal barrier function in health and disease, Turner [/bib_ref] [bib_ref] Interferon-c regulation of intestinal epithelial permeability, Beaurepaire [/bib_ref] [bib_ref] Signalling to and from tight junctions, Matter [/bib_ref]. Comparatively little attention has been directed towards assessing . Ubiquitinylation of E-cadherin stimulated by IFNc is inhibited by siRNA targeting Fyn kinase and Hakai. A) Representative immunoblot showing a time-dependent increase in total ubiquitinylation induced in T84 epithelial cells treated with IFNc (10 ng/ml). B) Upper panels demonstrate that treatment with the proteosome inhibitor, MG132 (500 nM), but not chloroquine (CQ, 5 mM; reduces lysosomal acidification) prevents the IFNc-evoked degradation of E-cadherin as shown by the presence of ubiquitinylated E-cadherin. Lower panels show cytoplasmic cleavage products of E-cadherin. Levels of the 30 kDa lysosomal fragment (arrowhead) were increased following IFNc treatment but were reduced in IFNc+chloroquine-treated samples. C) Immunoprecipitated E-cadherin demonstrated ubiquitinylation following IFNc stimulation which was reduced following Fyn siRNA. E3 ubiquitin ligase Hakai is co-immunoprecipitated with E-cadherin following IFNc stimulation but is reduced in Fyn siRNA treated epithelia. Densitometric analysis (performed in triplicate) is shown below a representative immunoblot. Whole lysates (input) demonstrate knockdown of Fyn by siRNA. D) Hakai siRNA inhibits E-cadherin ubiquitinylation; whole cell lysates indicate Hakai knockdown achieved by siRNA. Densitometry is shown below a representative immunoblot (mean6SEM; *, p,0.05 compared to controls (Unstim) and IFNc+Fyn (or Hakai) siRNA treated epithelia; actin and IgH are included as loading controls). doi:10.1371/journal.pone.0038441.g004 cytokine regulation of the adherens junction. Positioned directly beneath the tight junction, the formation of the adherens junction is considered as a critical forerunner to development of the tight junction and hence epithelial monolayer formation. Here, using a series of molecular analyses and the human T84 epithelial cell line (a model often used to define principles of the control of epithelial permeability [bib_ref] Mechanism of interferon-c-induced increase in T84 intestinal epithelial tight junction, Boivin [/bib_ref] [bib_ref] Enteroaggregative Escherichia coli disrupt epithelial cell tight junctions, Strauman [/bib_ref] [bib_ref] Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic..., Lewis [/bib_ref] , we have confirmed that E-cadherin expression is affected by IFNc [bib_ref] Proinflammatory cytokines disrupt epithelial barrier function by apoptosisindependent mechanisms, Bruewer [/bib_ref] and provide evidence in support of a requirement for Fyn kinase in IFNcevoked loss of E-cadherin from the adherens junction. These data add to growing awareness that Src-kinase, including Fyn, activity affects the stability of E-cadherin at the cell surface: Src kinases promote the phosphorylation of E-cadherin at C-terminal residues associated with removal of E-cadherin from the cell membrane . Hakai siRNA inhibits IFNc-stimulated internalization of E-cadherin. Low salt, detergent-free lysis of MG132 (500 nM)-pretreated T84 cells reveal that IFNc-stimulated (10 ng/ml, 48 h) internalization of E-cadherin is reduced in presence of A) Fyn and B) Hakai siRNA. High-salt lysates do not demonstrate as dramatic an increase in solubility relative to low-salt lysis (lower panels). Data are representative of two experiments for each lysis preparation. C) Confocal photomicrographs of immunofluorescent detection of E-cadherin and occludin localization. IFNc treatment disrupts the pattern of E-cadherin (adherens junction) and occludin (tight junction) distribution, and treatment with Fyn kinase and Hakai siRNA partially abrogates the displacement of E-cadherin but not occludin from the peri-junctional region. Data shown are representative of three experiments for Fyn, two experiments for Hakai. Scale bar = 10 mm. doi:10.1371/journal.pone.0038441.g005 [bib_ref] Tyrosine phosphorylation and cadherin/catenin function, Daniel [/bib_ref] [bib_ref] Dynamic and static interactions between p120 catenin and E-cadherin regulate the stability..., Ishiyama [/bib_ref] ; Src has been shown to regulate p120-catenin binding to the juxtamembrane domain of E-cadherin, a critical cytoplasmic region that mediates E-cadherin membrane localization [bib_ref] Dynamic and static interactions between p120 catenin and E-cadherin regulate the stability..., Ishiyama [/bib_ref] ; the E-cadherin binding partners, p120-catenin and beta-catenin, are phosphorylated by Src kinases [bib_ref] Identification of src phosphorylation sites in the catenin p120ctn, Mariner [/bib_ref] ; and p120-catenin, a factor required for stable integration of E-cadherin into the cell membrane, is phosphorylated by Fyn [bib_ref] Specific phosphorylation of p120-catenin regulatory domain differently modulates its binding to RhoA, Castano [/bib_ref]. In addition, phosphorylation of p120-catenin facilitates the association of E3-like ligase Hakai. Consistent with these findings, our data support a model whereby IFNc causes removal of E-cadherin from the epithelial surface via the mobilization of Fyn kinase and subsequent targeting for degradation via the E3-like ligase, Hakai. Thus, these novel data highlight an additional mechanism through which epithelial cell-cell adhesion may be disrupted during ongoing inflammatory reactions involving IFNc. The impact of destabilization of the adherens junction on an epitheliums barrier function once tight junctions have formed is unclear [bib_ref] Ecadherin is essential for in vivo epidermal barrier function by regulating tight..., Tunggal [/bib_ref]. Certainly TER is a direct reflection of the tight junction as perseverance of E-cadherin did not ablate the ability of IFNc to reduce TER; however, E-cadherin does provide a level of cell-cell stability since its loss leads to a more friable monolayer. Structural components of the tight junction, such as claudins and occludin, cycle rapidly into and out-of the epithelial cell membrane and are anchored to the cytoskeleton via adaptor proteins, principally isoforms of zonula occludens (ZO) [bib_ref] MLCKdependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction..., Yu [/bib_ref]. ZO-1 and the adherens junction proteins can interact and both play important roles in establishing cell polarity [bib_ref] Tumor necrosis factor-a and inflammation disrupt the polarity complex in intestinal epithelial..., Mashukova [/bib_ref] , which is essential for proper function of the enteric epithelium. For example, Ecadherin can physically interact with polarity-promoting and regulatory factors, such as the tumour suppressing phosphatase PTEN [bib_ref] Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression..., Kotelevets [/bib_ref] [bib_ref] Par-3-mediated junctional localization of the lipid phosphatase PTEN is required for cell..., Feng [/bib_ref] and Par3/Bazooka [bib_ref] PAR-3 mediates the initial clustering and apical localization of junction and polarity..., Achilleos [/bib_ref] , respectively. Consequently reduced surface expression of E-cadherin may affect the maintenance of the epithelial tight junction, the ability of enterocytes to spread and heal a wound, and to restore a polarized monolayer with the ability to vectorially transport electrolytes. An intriguing alternative possibility is that IFNc-stimulation of E-cadherin internalization might have a protective function. For instance, the bacterial pathogen Listeria monocytogenes can use Ecadherin as a receptor for entry into the enterocyte and hence removal of E-cadherin from the adherens junction could limit L. monocytogenes invasion [bib_ref] Subversion of cellular functions by Listeria monocytogenes, Pizarro-Cerda [/bib_ref]. Src-family kinases possess oncogenic properties: Src-mediated destabilization of E-cadherin has been presented as a molecular mechanism for malignancy in tissues including the colonic epithelium [bib_ref] The SRC-induced mesenchymal state in late-stage colon cancer cells, Avizienyte [/bib_ref] [bib_ref] Prognostic significance of the wnt signalling pathway molecules APC, beta-catenin and Ecadherin..., Lugli [/bib_ref] [bib_ref] Src family kinases as potential therapeutic targets for malignancies and immunological disorders, Benati [/bib_ref] and emerging data suggest that Fyn kinase participates in the malignant transformation of prostate epithelium [bib_ref] Fyn: a novel molecular target in cancer, Saito [/bib_ref] [bib_ref] A multivariate analysis of adhesion molecules expression in assessment of colorectal cancer, Ngan [/bib_ref]. Further, though unrelated to its ubiquitin ligase activity, Hakai may be considered an oncogenic factor via its ability to modify RNA splicing [bib_ref] Hacking RNA: Hakai promotes tumorigenesis by enhancing the RNA-binding function of PSF, Figueroa [/bib_ref]. Also, Hakai, C-terminal fragments of E-cadherin, p120-catenin and beta-catenin have been localized to the nucleus in in vitro cell systems [bib_ref] Hacking RNA: Hakai promotes tumorigenesis by enhancing the RNA-binding function of PSF, Figueroa [/bib_ref] [bib_ref] A role for the cleaved cytoplasmic domain of E-cadherin in the nucleus, Ferber [/bib_ref]. So while focusing on IFNc regulation of structural elements of the epithelial barrier we should not overlook IFNc-Fyn-Hakai activity in the contexts of cell signalling, gene regulation and cancer. As the field of inflammation-driven cancer (e.g. colorectal cancer) gains momentum we speculate that IFNc regulation of E-cadherin and associated signalling molecules, such as beta-catenin, is worthy of substantive investigation and has the potential to yield key insights into the regulation of the malignancy. In conclusion, our data suggest the Src kinase Fyn acts as a pivotal signal in specific aspects of IFNc control of epithelial function. Not only is it central to the regulation of macromolecular permeability [bib_ref] Interferon-c-induced increases in intestinal epithelial macromolecular permeability requires the src kinase fyn, Smyth [/bib_ref] , but via its affect on E-cadherin it can modify cellcell interactions, intracellular signalling pathways and possibly also oncogenic processes as well. These effects contrast with the accepted role of IFNc as an anti-cancer factor due to its activation of anti-tumour cell types, namely cytotoxic T cells, macrophages and natural killer cells [bib_ref] Interferon-c and interleukin-12 pathway defects and human disease, Dorman [/bib_ref]. Thus, we propose that Fyn kinase could be exploited to inhibit many of the pathological effects of IFNc on intestinal epithelial cells, and possibly epithelia in general. # Author contributions [fig] Figure 3: IFNc-stimulated phosphorylation of E-cadherin and p120-catenin is reduced by PP1 and Fyn kinase siRNA. A) Time course analysis reveals a generalized increase in tyrosine phosphorylation in T84 whole cell lysates treated with IFNc (10 ng/ml). B) Tyrosine phosphorylation of E-cadherin following IFNc stimulation (6 h) was reduced by PP1 (125 nM) as demonstrated in a representative immunoblot of E-cadherinimmunoprecipitated T84 cell lysates and quantified by densitometric assessment conducted on the result of three representative experiments. C) Increased E-cadherin and p120 catenin tyrosine phosphorylation evoked by IFNc was reduced in cells in which Fyn expression (lowest panels) had been knocked-down by siRNA. Actin and IgH are included as loading controls. Graph depicts densitometry analysis of phospho-E-cadherin immunoblots from three experiments (mean6SEM; *, p,0.05 compared to control (Unstim) and IFNc+Fyn siRNA treated epithelia). doi:10.1371/journal.pone.0038441.g003 [/fig] [fig] Figure 6: Increased cell detachment evoked by fluid shear in IFNc-treated T84 epithelial cell monolayers is reduced by siRNA knock-down of Fyn kinase and Hakai. A) Electrically confluent T84 cell monolayers treated with IFNc (10 ng/ml, 48 h, control siRNA) displayed the expected drop in transepithelial resistance (TER) that was unaffected by PP1 (125 nM) co-treatment or by siRNA knockdown of Fyn kinase or the E3-like ligase, Hakai. In contrast, cell detachment from the monolayer caused by gentle fluid shear stress evoked by IFNc, and assessed by (B) cell counts and (C) total protein from suspended cells was statistically significantly reduced by PP1, and to a lesser extent by siRNA targeting Fyn kinase or Hakai (measurement of cell detachment as detected by amount of cellular protein from washed cells (mean6SEM; n = 426 monolayer preparations from 2 experiments; * and #, p,0.05 compared to control (Unstim) mono- [/fig]

The Relationship between Different Large-Sided Games and Official Matches on Professional Football Players’ Locomotor Intensity # Introduction One of the major challenges in football is building training exercises that allow players to be confronted with real match scenarios while continuously enhancing their performance (e.g., tactical, technical, physical, physiological, psychological). In this sense, small-and large-sided games (LSG), also known as small-and/or large-sided conditioned games, are very popular training resources commonly used by coaches to replicate those scenarios [bib_ref] Physiology of small-sided games training in football: A systematic review, Hill-Haas [/bib_ref] [bib_ref] Effects of pitch area-restrictions on tactical behavior, physical, and physiological performances in..., Gonçalves [/bib_ref] [bib_ref] Physical and technical comparisons between various-sided games within professional soccer, Owen [/bib_ref]. The design of sided games, considering the formal game format, allows the chaotic effect of football to be simplified without compromising the fundamental characteristics of the match or its dynamic and complex quality. Besides promoting the strategical-tactical side, they also simulate the physiological/physical part of the game [bib_ref] Small-sided games in team sports training: A brief review, Halouani [/bib_ref]. The configuration of these training exercises is strictly related to manipulating task constraints. For a typical task, constraints could include changes in format (e.g., number of players involved in the game and numerical relationships), scoring method (e.g., having or not having goalkeepers, using or not using goals or targets), strategical-tactical missions (e.g., coach-specific instructions), training regimen (e.g., work-to-rest ratio, sets, repetitions) and/or pitch configuration (e.g., dimensions of the playing area, area per player, the shape of the pitch, width-to-length ratio) [bib_ref] Physiology of small-sided games training in football: A systematic review, Hill-Haas [/bib_ref] [bib_ref] Small sided games in soccer-A systematic review, Sarmento [/bib_ref]. The management of these variables is well documented in the literature. In fact, in recent years, the interaction between these task constraints has been widely investigated to modify players' acute and chronic load responses to levels that elicit physiological match-specific adaptations [bib_ref] A systematic review on small-sided games in football players: Acute and chronic..., Bujalance-Moreno [/bib_ref] [bib_ref] Small-sided games: An umbrella review of systematic reviews and meta-analyses, Clemente [/bib_ref]. Nowadays, the association between training and match monitoring has been one of football's most common research topics [bib_ref] Training load and player monitoring in high-level football: Current practice and perceptions, Akenhead [/bib_ref]. The technological and analytical method evaluations have provided to coaches, members of technical staff and sports scientists several load measures obtained throughout the use of global positioning systems (GPS), among other microtechnologies (e.g., accelerometers, heart rate monitors, etc.) [bib_ref] Load measures in training/match monitoring in soccer: A systematic review, Miguel [/bib_ref]. Within the load measures categorization, the locomotor intensity by means of GPS has been widely used for training load assessment and monitorization. The evidence around these locomotor activities is clear, since the internal load is strongly associated with the amount of running completed rather than the several other locomotor intensity measures typically monitored in team-sport players [bib_ref] The relationships between internal and external measures of training load and intensity..., Mclaren [/bib_ref]. The most common metrics given by GPS include distances, accelerations, decelerations, impacts and total load. Within distances, one of the most used locomotor intensity measures in the assessment of the amount of work developed by the players in training and games is total distance covered (TD), which is measured in absolute (m), and relative values (m/min, m/15 min, m/h) as a percentage of the highest data reached in the match [bib_ref] Load measures in training/match monitoring in soccer: A systematic review, Miguel [/bib_ref]. Within the accelerations, mechanical work (MW), is one of the most used variables to describe locomotor intensity. This metric was created to quantify the total load that players are exposed to and is based on the acceleration data recorded by triaxial accelerometers [bib_ref] Accelerometry-based external load indicators in sport: Too many options, same practical outcome?, Gómez-Carmona [/bib_ref] with high reliability and validity [bib_ref] Mechanical Player Load™ using trunk-mounted accelerometry in football: Is it a reliable,..., Barreira [/bib_ref] [bib_ref] PlayerLoad™: Reliability, convergent validity, and influence of unit position during treadmill running, Barrett [/bib_ref]. It can be measured in absolute (arbitrary units (AU) and g) and relative (AU/ min, g/min and AU/m) values [bib_ref] Load measures in training/match monitoring in soccer: A systematic review, Miguel [/bib_ref] and as a percentage of the highest data reached in the match. Several research works have focused on these two metrics for analyzing the physiological/physical demands placed on professional football players [bib_ref] Wellbeing perception and the impact on external training output among elite soccer..., Malone [/bib_ref] [bib_ref] Examining the external training load of an English premier league football team..., Akenhead [/bib_ref] [bib_ref] Physical performance differences between starter and non-starter players during professional soccer friendly..., Giménez [/bib_ref] [bib_ref] Characterization of the weekly external load profile of professional soccer teams from..., Clemente [/bib_ref] [bib_ref] Training/match external load ratios in professional soccer players: A full-season study, Clemente [/bib_ref] [bib_ref] Quantifying the physical loading of five weeks of pre-season training in professional..., Clemente [/bib_ref]. Although LSG appears to be a beneficial means to comprehend the players' performance in official matches, it remains a relatively unpopular topic in the literature [bib_ref] Effects of pitch area-restrictions on tactical behavior, physical, and physiological performances in..., Gonçalves [/bib_ref]. Namely, there is a shortage of consistency in the design of LSGs, the age and level of ability of the players and the pitch size variation used by researchers. Moreover, the generality of research in this area is conducted with young players [bib_ref] Acute effects of the number of players and scoring method on physiological,..., Clemente [/bib_ref] [bib_ref] Physiological responses according to rules changes during 3 vs. 3 small-sided games..., Halouani [/bib_ref] , while studies conducted with senior players [bib_ref] Validity and reliability of 6-a-side small-sided game locomotor performance in assessing physical..., Stevens [/bib_ref] [bib_ref] Timescales for exploratory tactical behaviour in football small-sided games, Ric [/bib_ref] , and particularly with professional players, are rare. Further, when it comes to applying the optimal constraints to the training task and the appropriate management of players' training load, match activity plays a key role [bib_ref] A contemporary multi-modal mechanical approach to training monitoring in elite professional soccer, Owen [/bib_ref] [bib_ref] Quantification of in-season training load relative to match load in professional Dutch..., Stevens [/bib_ref]. To the best of our knowledge, only very few studies have explored the relationships between GPS locomotor intensity indicators and official matches, especially with large formats (e.g., GK + 10 vs. 10 + GK) and with the highest data reached in the game regarding key locomotor intensity indicators, such as TD and MW. This novel information will be crucial to identify which LSG conditions, in terms of pitch sizes, provide professional football players with a better level of readiness for competition. Therefore, this study aimed to compare the locomotor demands imposed by LSG and the official match, and to compare the effect of different pitch sizes in the locomotor demands. # Materials and methods ## Experimental approach The study followed an observational study design. Microelectromechanical systems monitored locomotor demands in three consecutive matches and LSG applied in different training sessions. The observational period occurred over four weeks. The data collection started six weeks after the season began (corresponding to the first half of the season). The official matches occurred at the weekends. The LSG were monitored over 3 days after the match. The data collection in training sessions occurred between 04:00 and 05:30 p.m. on natural grass field with sunny weather conditions and similar temperatures. In the case of the official matches, the data collection occurred between 06:00 and 10:30 p.m. on natural grass field with sunny weather conditions and similar temperatures. ## Participants A priori sample calculation was determined based on T-Test Family-Wilcoxon signedrank test (i.e., matched pairs). This indicated that to detect a large effect size of r = 0.90 with an alpha probability of 0.05, a power of 0.95, the sample size would need to comprise 16 participants. GPower, (Heinrich Heine University, Düsseldorf, Germany; 3.1.9.7 software) was used in the calculations [bib_ref] Power 3: A flexible statistical power analysis program for the social, behavioral,..., Faul [/bib_ref]. Sixteen male professional outfield football players from the same team (i.e., age: 26.3 ± 3.0 years old; height: 181.6 ± 5.2 cm; body mass index: 23.8 ± 1.5 kg/m 2 ; percentage fat mass: 10.4 ± 2.9) participating in the Portuguese premier league were included in this analysis. Of the players, 7 were defenders (DF), 6 were midfielders (MF), and 3 were forwards (FW). The data were collected from daily player locomotor demands in which player activities were routinely measured during training sessions and matches. The following inclusion criteria were used: (1) players were included in the analysis if they participated in all training sessions of the week where the LSG and official match occurred; and (2) players also had to have participated for at least 45 min in one of the official matches, as suggested by previous research [bib_ref] Training/match external load ratios in professional soccer players: A full-season study, Clemente [/bib_ref] [bib_ref] Small-sided games in elite soccer: Does one size fit all?, Lacome [/bib_ref] [fig_ref] Table 1: Players' individual descriptive of the official matches [/fig_ref]. The players were informed of the study design and the benefits and consequences of their participation and freely signed an informed consent form. All procedures were approved by the Ethical Committee of Faculty of Human Kinetics, University of Lisbon (CEIFMH n.º: 35/2021) and followed the ethical standards of the Declaration of Helsinki for a study in humans. ## Large-sided games The most used LSG played during the period of the study (i.e., GK + 10 vs. 10 + GK) were compared with the 10 most demanding minutes reached in the official matches. Keeping the same format, the LSG ranged in the pitch's size as follows: (1) ## Task objectives The main objective for the three LSG conditions was to score as many as goals as possible and not to to give any possible chance for the opponent to score. ## Task rules All the official game rules were maintained for the three LSG, except for the offside rule and the start and restart of play rule. Every time one of the teams won a free kick (direct and indirect), a penalty, a throw-in or a corner kick, the restart of the game was performed by the GK of the team to whom the goal kick belonged. All LSG were also played with free touch rule per player. ## Sets 1 Minutes per set 10 All LSG lasted 10 min and integrated a standard training session (e.g., tactical, technical and physical factors were amalgamated) within a typical training week compound of 5 field sessions. The training week consisted of a typical microcycle structure using the following schedule: previous match-day (MD); MD + 1 and MD + 2: recovery period; MD-4 and MD-3: acquisition period; MD-2 and MD-1: tapering period; and next MD. All the official game rules were maintained for the three LGS, except for the offside rule and the start and restart of play rule. Every time one of the teams won a free kick (direct and indirect), a penalty, a throw-in or a corner kick, the restart of the game was performed by the GK of the team to whom a goal kick belonged. There were four balls always placed next to the goal of the goalkeeper to whom the ball belonged to make this replacement process quick. The verbal encouragement of the coach remained the same throughout the three LGS. ## Official matches Official match activities were assessed from data collected over three official 11-aside games. Three different official matches were selected according to opponents' level. Considering the classification in the championship, three different levels were selected: lower, middle and upper position on the table. Each microcycle throughout the study finished with an official match. The team's management remained the same throughout the study. The team systematically played in a 1-4-3-3 formation, with 4 DF, 3 MF and 3 FW. The same warm-up protocol was conducted before each official match, including moderate running, dynamic stretching, mobility and balance exercises, accelerations and decelerations, and ball possession drills in a 5 vs. 5 configuration. The average playing area per player across the three official matches was approximately 325 m 2 . ## Locomotor demands Each player's movements were recorded by 10-Hz GPS Unit (EVO, Catapult, Melbourne, Australia) during each training session and official match. The GPS unit also included an accelerometer, a gyroscope and a magnetometer (100 Hz, 3 axes ± 16 g). The GPS device was put in a skin-tight bag in the thoracic region between the scapulae. Data were collected during what was good weather and satellite conditions for GPS training sessions and matches [bib_ref] Training/match external load ratios in professional soccer players: A full-season study, Clemente [/bib_ref]. The following measures were collected during each training session and official match: (1) total distance (TD: consisting of the total distance covered by each player); and (2) mechanical work (MW: total load players are exposed to and is obtained from the acceleration in the three axes recorded by the GPS accelerometers, measured in arbitrary units . Catapult open field cloud was then used to compute a moving average over each criterion variable (TD and MW) for the official matches, using a 10 min duration, and the maximum value per player was recorded. These data were then averaged for each one of the three competitive matches. The LSG conditions lasted for 10 min and were also analyzed for each player and averaged as three different training drills (corresponding to each pitch dimension used). Descriptive statistics and analysis were then calculated based on this design. ## Statistics First, descriptive statistics were calculated (means, standard deviation and confidence intervals) for age, body composition and physical fitness variables. Second, the Friedman test was used to identify TD and MW changes across the three LSG formats and the three competition moments. Third, a Wilcoxon signed-rank test was conducted to analyze individual differences in the scores of the locomotor intensity between each LSG condition and the official full game. Statistical analysis was performed using IBM SPSS Statistics v.26.0 (IBM, Armank, NY, USA). The significance level was set to p < 0.05. # Results First, descriptive statistics of age, body composition and physical fitness variables are presented in [fig_ref] Table 3: Descriptive statistics of age, body composition and physical fitness variables [/fig_ref]. Second, the results of the Friedman test indicated that there was a significant difference in the total distance (TD) scores [χ2 (2, n = 16) = 15.50, p < 0.001, [fig_ref] Condition 1: CD1 [/fig_ref] ] and mechanical (MW) scores [χ2 (2, n = 16) = 14.00, p = 0.001, [fig_ref] Figure 2: Changes in mechanical work across the three LSG formats and the three... [/fig_ref] ] between the three different pitch size conditions in GK + 10 vs. 10 + GK. Inspection of the median values showed that the GK + 10 × 10 + GK (291 m 2 ) format presented the highest score in TD and MW. Third, a Wilcoxon signed-rank test was conducted to analyze a change in TD and MW from training conditions and to official matches. ## Total distance The Wilcoxon signed-rank test revealed a significant difference in TD between GK + 10 vs. 10 + GK (i.e., smallest pitch size) and the Official Match 1 (i.e., against a team with a low position on the table) z = −2.40, p = 0.017 (r = 0.60) and Official Match 2 (i.e., against a team with a middle position on the table) z = −2.51, p = 0.012 (r = 0.63). The median score on the TD revealed lower scores in the training condition when compared with the official match [fig_ref] Table 4: Individual differences in the scores of the locomotor intensity between each LSG... [/fig_ref]. There was a significant difference in TD between GK + 10 vs. 10 + GK (i.e., medium pitch size) and the Official Match 1 (i.e., against a team with a low position on the table) z = −3.52, p < 0.001 (r = 0.88), Official Match 2 (i.e., against a team with a middle position on the table) z = −3.36, p < 0.001 (r = 0.84) and Official Match 3 (i.e., against a team with a upper position on the table) z = −2.64, p = 0.008 (r = 0.66). The median score on the TD revealed lower scores in the training condition when compared with all official matches [fig_ref] Table 4: Individual differences in the scores of the locomotor intensity between each LSG... [/fig_ref]. Finally, there was a statistically significant difference in TD between GK + 10 vs. 10 + GK (i.e., large pitch size) and the Official Match 1 (i.e., against a team with a low position on the table) z = −2.22, p = 0.026 (r = 0.56) and Official Match 3 (i.e., against a team with an upper position on the table) z = −2.54, p = 0.011 (r = 0.63). The median score on the TD revealed higher scores in the training condition when compared with the official match [fig_ref] Table 4: Individual differences in the scores of the locomotor intensity between each LSG... [/fig_ref]. [fig_ref] Figure 3: Players' individual variation of locomotor demands [/fig_ref] depicts the players' individual variation of locomotor demands (i.e., total distance and mechanical work) across the three official matches and GK + 10 vs. 10 + GK LSG conditions. ## Mechanical work The Wilcoxon signed-rank test revealed a significant difference in MW between GK + 10 vs. 10 + GK (i.e., smallest pitch size) and the Official Match 1 (i.e., against a team with a low position on the table) z = −3.06, p = 0.002 (r = 0.77), Official Match 2 (i.e., against a team with a middle position on the table) z = −2.95, p = 0.003 (r = 0.74) and Official Match 3 (i.e., against a team with a upper position on the table) z = −2.69, p = 0.007 (r = 0.67). The median score on the MW revealed lower scores in the training condition when compared with all official matches [fig_ref] Table 4: Individual differences in the scores of the locomotor intensity between each LSG... [/fig_ref]. There was a significant difference in MW between GK + 10 vs. 10 + GK (i.e., medium pitch size) and the Official Match 1 (i.e., against a team with a low position on the table) z = −3.06, p <.001 (r = 0.88), Official Match 2 (i.e., against a team with a middle position on the table) z = −2.95, p = 0.001 (r = 0.83) and Official Match 3 (i.e., against a team with a upper position on the table) z = −2.69, p = 0.001 (r = 0.80). The median score on the MW revealed lower scores in the training condition when compared with all official matches [fig_ref] Table 4: Individual differences in the scores of the locomotor intensity between each LSG... [/fig_ref]. Finally, no significant differences in MW were seen between GK + 10 vs. 10 + GK (i.e., large pitch size) and any official match. # Discussion The novel findings of the present study showed a difference in locomotor intensity metrics when pitch sizes change in LSG. Bigger pitch sizes caused an increase in TD and MW. There was a significant difference in locomotor intensity metrics between opponents from different positions on the table (i.e., lower, medium or upper). Competition against teams from the upper position caused a lower TD. The biggest LSG (291 m 2 per player) was the only one that required similar levels of locomotor intensity as required in the official match. This study gives important information for coaches to plan LSG formats with similar locomotor intensity to those they will find in the official match. First, keeping a similar LSG format, we analyzed the pitch size variation in total distance covered and the MW. A recent umbrella review of systematic reviews and metaanalyses on SSG in team ball sports revealed that pitch configuration (i.e., mainly the pitch size and the influence of different relative areas of play) was currently one of the main game constraints discussed in the literature [bib_ref] Small-sided games: An umbrella review of systematic reviews and meta-analyses, Clemente [/bib_ref]. However, it is claimed more research should investigate the effect of manipulation of playing field dimensions on the emerged outcomes of the games [bib_ref] How manipulating task constraints in small-sided and conditioned games shapes emergence of..., Ometto [/bib_ref] , since some studies differed significantly in their results and interpretations of the findings. In addition, the information about LSG (e.g., GK + 9 vs. 9 + GK; GK + 10 vs. 10 + GK) in professional football is scarce. Our results reinforce the idea that larger pitches cause increases in the TD and the MW. It means that the larger the playing field dimensions during practice, the greater the distances between players in the same team and between those players and the opposing team. Since LSG are widely used by coaches, the present study results have important practical implications and give helpful information to guide methodologies used in professional football training. Secondly, in this study, we investigated the variation of TD and MW considering different levels of opponents (i.e., low, middle and upper position on the table). Clemente et al. [bib_ref] Small-sided games: An umbrella review of systematic reviews and meta-analyses, Clemente [/bib_ref] concluded that greater competitive levels increase locomotor intensity during games. The present study contradicts these findings, since it was found that the competition moment against the team from the upper position caused a lower total distance run. Despite the tactical-technical approach of the players that may influence the team's behavior, it is comprehensive that playing against an opponent from the upper position on the table generates a bigger concern about the cohesion of the defensive process that may cause a huge contention of the team and a lower distance covered. Third, we compared the different LSG (i.e., in terms of pitch sizes) and the real moment of competition on TD and the MW, considering the levels of the opponents. This information allows us to understand better which LSG more accurately simulate the official full match. Previous studies have compared the relative physical demands of SSG (medium and large) in official matches and in non-professional football players [bib_ref] Physical and technical comparisons between various-sided games within professional soccer, Owen [/bib_ref] [bib_ref] A Comparison of GPS workload demands in match play and small-sided games..., Beenham [/bib_ref] [bib_ref] Comparing the physical demands of friendly matches and small-sided games in semiprofessional..., Casamichana [/bib_ref] [bib_ref] External load variations between mediumand large-sided soccer games: Ball possession games vs..., Clemente [/bib_ref]. However, to our knowledge, this is the first study that considers the levels of opponents when comparing the LGS. This opens new and important perspectives about the teams' preparation for specific matches. In the present study, independently of the opponents' position on the table, the largest LGS (i.e., 291 m 2 ) simulates the official match more accurately than other sided games in terms of TD and MW metrics. Similar results were seen by authors of Ref [bib_ref] External load variations between mediumand large-sided soccer games: Ball possession games vs..., Clemente [/bib_ref] , who concluded that GK + 9 vs. 9 + GK simulates the official match more accurately than other large-sided and/or conditioned games in sprinting and loading demands. These differences in TD between official matches and large-sided and conditioned games also agree with previous reports [bib_ref] Physical and technical comparisons between various-sided games within professional soccer, Owen [/bib_ref] [bib_ref] Comparing the physical demands of friendly matches and small-sided games in semiprofessional..., Casamichana [/bib_ref]. It is also important to underline that the largest LSG format required a bigger mean TD covered (i.e., 1273.4 ± 101.9 m) when compared to the official match against a team with a lower position on the table (i.e., 1203.6 ± 74.7 m) and the official match against a team with an upper position on the table (i.e., 1156.5 ± 87.1 m). It means that the largest GK + 10 vs. 10 + GK (i.e., 291 m 2 ) was the format studied that better prepared the team for the official match. Based on these results, coaches should consider this format to better simulate the TD and the MW required by the official matches. We acknowledge that the differences in constraints between large-sided and conditioned games in previous studies make the results difficult. However, it is believed that LSG better simulates the official match in terms of TD and MW, which is an important message for coaches. This is in line with the ecological dynamics approach that supports LSG as a practice methodology that ensures task constraints during training are similarly demanding to a competitive context [bib_ref] Nonlinear pedagogy: A constraints-led framework for understanding emergence of game play and..., Chow [/bib_ref] [bib_ref] The role of nonlinear pedagogy in supporting the design of modified games..., Chow [/bib_ref]. It means that, probably, LSG forces players in a better way to adapt their overall actions to a changing performance environment, similarly to competitive performance conditions. It is also important to acknowledge that once we are analyzing the players' performance, the temperatures and relative humidity for each training session and official match should be registered in the future, since hydration status and ambient conditions are important variables that may affect the players' performance. # Conclusions The present study demonstrates that pitch size variation (i.e., 195 m 2 ; 241 m 2 ; 291 m 2 ) in LSG requires different locomotor intensity in professional football players. A bigger pitch size causes an increase in TD and MW. In addition, the level of opponents, considering their position on the table, induces a differently covered TD. The competition moment against teams from the upper position causes a lower TD run. Finally, the largest LSG format more accurately simulates the official full match. These novel results have important implications and give precious detailed information for coaches to better plan LSG formats with similar locomotor intensity to those they will find in the competition moment. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from all players to publish this paper. # Data availability statement: The data presented in this study are available on request from the corresponding author. [fig] Condition 1: CD1): 67 × 64 m, average area per player = 195 m 2 ; (2) CD2: 78 × 68 m, average area per player = 241 m 2 ; and (3) CD3: 100 × 64 m, average area per player = 291 m 2 (Table 2) [/fig] [fig] Figure 1: Changes in total distance across the three LSG formats and the three official matches. [/fig] [fig] Figure 2: Changes in mechanical work across the three LSG formats and the three official matches.For the three official matches, there was a significant difference in the TD scores [χ2 (2, n = 16) = 13.88, p = 0.001]. The match against the best-classified team presented the lowest score in TD. No significant difference was seen for MW [ [/fig] [fig] Figure 3: Players' individual variation of locomotor demands (i.e., total distance and mechanical work) across the three official matches and GK + 10 vs. 10 + GK LSG conditions. The columns represent the average score for the group. [/fig] [fig] Author: Contributions: Conceptualization, R.C. and É.R.G.; Methodology, R.C., É.R.G. and R.H.; Validation, H.L., F.M.C. and H.S.; Formal analysis, É.R.G.; Investigation, R.C. and É.R.G.; Resources, R.C., É.R.G. and R.H.; Writing-original draft preparation, R.C. and É.R.G.; Writing-review and editing, A.I., A.M., F.M.C., R.H. and H.S.; Visualization, A.I., A.M., F.M.C., R.H. and H.S.; Project administration, É.R.G. and H.L.; Funding acquisition, É.R.G., H.L., A.I. and H.S. All authors have read and agreed to the published version of the manuscript. [/fig] [fig] Funding: This work was supported by the Swiss National Centre of Competence in Research LIVES, "Overcoming Vulnerability: Life Course Perspectives," granted by the Swiss National Science Foundation (Grant Number: 51NF40-185901). A.I. acknowledges support from the Swiss National Science Foundation (Grant Number: 10001C_189407). R.C. and E.R.G. acknowledge support from LARSyS-Portuguese national funding agency for science, research, and technology (FCT) pluriannual funding 2020-2023 (Reference: UIDB/50009/2020). This study is framed in Marítimo Training Lab Project. The project received funding under application no. M1420-01-0247-FEDER-000033 in the System of Incentives for the Production of Scientific and Technological Knowledge in the Autonomous Region of Madeira-PROCiência 2020. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Faculty of Human Kinetics, (CEIFMH N. º 34/2021) and followed the ethical standards of the Declaration of Helsinki for Medical Research in Humans (2013) and Oviedo Convention (1997). [/fig] [table] Table 1: Players' individual descriptive of the official matches. [/table] [table] Table 2: Large-sided games characteristics. [/table] [table] Table 3: Descriptive statistics of age, body composition and physical fitness variables. [/table] [table] Table 4: Individual differences in the scores of the locomotor intensity between each LSG condition and the official match. [/table]

Fortunella margarita Transcriptional Reprogramming Triggered by Xanthomonas citri subsp. citri Background: Citrus canker disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc) has become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative Fortunella margarita Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in Fortunella and citrus at the molecular level. Results: cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus. Conclusion: Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases. # Background Citrus trees are susceptible to a number of diseases with different degrees of economic impact. One of the most severe in terms of economic losses is citrus canker disease (sometimes referred to as Asiatic citrus canker) caused by Xanthomonas citri subsp. citri, (synonym, Xanthomonas axonopodis pv. citri strain A; Xac-A). Xcc is a biotrophic bacterial phytopathogen that belongs to the genus Xanthomonas of the α-subdivision v within Proteobacteria. Susceptibility to citrus canker disease varies among citrus types and relatives, but most of the commercially grown citrus types are susceptible hosts to Xcc. Disease symptoms include canker lesions on the green aerial parts of the plant as well as fruit; infections can result in both foliar and fruit abscission, thereby decreasing the productivity of affected trees. In addition there can be reduced profitability as a result of blemished fruit that can be harvested but not sold in the fresh market. Plants have evolved multiple defence mechanisms to survive pathogen attacks [bib_ref] R proteins as fundamentals of plant innate immunity. Cellular &amp, Głowacki [/bib_ref]. The first branch of the indispensable plant innate immunity system is triggered by pathogen-associated molecular patterns (PAMPs) such as the lipopolysaccharides (LPSs), peptidoglycan and bacterial flagellin, as well as the chitin and glucan from fungi. The second branch utilizes the nucleotidebinding site-leucine-rich repeat (NBS-LRR) encoded by R (resistance) genes named the effector-triggered immunity (ETI) [bib_ref] The plant immune system, Jones [/bib_ref]. The Xanthomonas spp. phytobacterial pathogens have evolved unique pathogenesis mechanisms to avoid host recognition and suppress host defences [bib_ref] Avoidance of host recognition by alterations in the repetitive and C-terminal regions..., Yang [/bib_ref]. Bacterial effector proteins are delivered via the bacterial type III secretion system (TTSS) into the plant cell to evade recognition by the different plant surveillance systems [bib_ref] The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins,..., Alfano [/bib_ref]. These effectors in general contribute to host resistance or susceptibility as well as to modifying host responses. A fundamental element of the ETI in resistant plants is a localized cell collapse or a hypersensitive response (HR) at infection sites in an attempt to restrict the growth of the pathogen [bib_ref] Self/nonself perception and recognition mechanisms in plants: a comparison of self-incompatibility and..., Sanabria [/bib_ref] [bib_ref] Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify..., Gottig [/bib_ref]. This is a common feature of disease resistant responses in incompatible plant-pathogen, and occasionally some non-host, interactions [bib_ref] Localization of hydrogen peroxide accumulation during the hypersensitive reaction of Khalaf et..., Bestwick [/bib_ref] [bib_ref] Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE..., Ali [/bib_ref]. Some of the Xanthomonas spp. effector proteins, for instance PthA/AvrBs3, are essential to elicit citrus canker symptoms and if expressed by itself inside host cells, pthA is sufficient to cause symptoms of citrus canker disease [bib_ref] A pthA homolog from Xanthomonas axonopodis pv. citri responsible for host-specific suppression..., Shiotani [/bib_ref] [bib_ref] Xanthomonas citri: breaking the surface, Brunings [/bib_ref] [bib_ref] Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance..., Romer [/bib_ref] [bib_ref] Diverse members of the AvrBs3/PthA family of type III effectors are major..., Yang [/bib_ref] [bib_ref] Expression of a single, host-specific, bacterial pathogenicity gene in plant cells elicits..., Duan [/bib_ref]. In the meantime however, other recent studies show that other types of proteins are injected through the Xcc TTSS and do not necessarily alter the physiological and transcriptional responses to the pathogen in citrus [bib_ref] Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify..., Gottig [/bib_ref] [bib_ref] Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE..., Ali [/bib_ref] [bib_ref] Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis..., Tasic [/bib_ref] [bib_ref] The type III effectors of Xanthomonas, White [/bib_ref]. While certain genes involved in systemic acquired resistance (SAR) have been characterized and used as markers for studying plant defence mechanisms [bib_ref] Systemic gene expression in Arabidopsis during an incompatible interaction with Alternaria brassicicola, Schenk [/bib_ref] , crosstalk between signals and hormone pathways has also been proposed [bib_ref] Making sense of hormone crosstalk during plant immune responses, Spoel [/bib_ref] [bib_ref] ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant..., Lorenzo [/bib_ref] [bib_ref] Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses, Nemhauser [/bib_ref]. Consequently, plant resistance is correlated with the activation of a complex network of defence pathways and the response of the host plant to a microbial assault is therefore expected to result in drastic changes in the patterns of gene expression throughout the plant [bib_ref] Use of microarray analysis to dissect the plant defense response, Glazebrook [/bib_ref] [bib_ref] Ethylene Modulates the Role of NPR1 in Cross-Talk Between Salicylate and Jasmonate..., Spoel [/bib_ref]. Kumquats (Fortunella spp.), close relatives to citrus species, are reported to have high levels of field resistance to citrus canker. Previously, we have shown a sharply contrasting phenotype in grapefruit and kumquat when both plants were challenged with a high concentration of Xccr (OD600 nm = 0.3). Grapefruit (Citrus paradisi Macf. cv. Duncan), considered to be highly susceptible to the bacterium, showed the characteristic sequence of canker lesion development. Initially lesions appeared as water soaking, followed by the development of a raised corky form; each such lesion is a reservoir of new bacterial inoculum. Bacterial exudates were visible between 10 and 21 days post-inoculation. In contrast, PCD was observed in kumquat leaves in the form of a HR 3-5 days after inoculation with the canker-causing bacterium. Only necrotic lesions were observed and the bacterial population over time was shown to have an 'avirulent' incompatible growth pattern where bacterial multiplication ceased upon the development of necrosis [bib_ref] Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify..., Gottig [/bib_ref] [bib_ref] Bacterial pathogens in plants: Life up against the wall, Alfano [/bib_ref]. New tools have been developed in recent years through advances in genomics, proteomics, and bioinformatics that have particular utility for examining pathogen: host interaction complexities [bib_ref] Use of microarray analysis to dissect the plant defense response, Glazebrook [/bib_ref] [bib_ref] ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology..., Alba [/bib_ref] [bib_ref] High-throughput functional annotation and data mining with the Blast2GO suite, Gotz [/bib_ref] [bib_ref] Citrus genomics, Talon [/bib_ref]. The purpose of this study was to examine simultaneous changes in expression profiles for genes differentially expressed in the early stages (6-72 hpi) of citrus canker infection in kumquat, particularly those previously implicated in PCD-related responses such as HR. # Results and discussion In this study, identification of differentially expressed kumquat genes during its interaction with Xcc was pursued in an attempt to unravel the nature of the resistance mechanism(s) employed by the plant. Previously, kumquat suppression subtractive hybridization (SSH) cDNA libraries were constructed from Xcc-inoculated vs. mock inoculated leaves. Since SSH allows differential amplification of rare target sequences due to the elimination of more abundant house-keeping cDNA transcripts found in common from both samples, the technique has the potential of uncovering pertinent cDNA sequences. Subtraction was done in both directions, forward (inoculated-mock) and reverse (mockinoculated) and the resulting cDNAs were subsequently sequenced. Preliminary screening macroarrays were used to confirm enrichment of the subtracted libraries with differentially expressed genes (data not shown). ## Microarray experimental design Kumquat microarray chip hybridization data were assessed for overall signal intensity and consistency of the expression ratio over all time points, which resulted in the exclusion of chips with inconsistent results. [fig_ref] Figure 1: Scatter plot analysis of the M-values from two microarray hybridizations using RNA... [/fig_ref] is a scatter plot showing M-values from two different biological replicate-hybridizations with Xcc-inducible targets (Cy5-labeled) and mock inoculated non-infected targets (Cy3-labeled) confirming high data consistency levels (R 2 = 0.921). ## Functional annotation and an overview of global gene expression The B2GO program [bib_ref] Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics..., Conesa [/bib_ref] was used to assign GO (Gene Ontology) terms for hits obtained through eBLAST homology searches in NCBI. A general view of the similarity of the query set with the NCBI database, the distribution of the cut off for the e-value as well as the distribution of species with similar sequences are shown in Additional files 1, 2, and 3. The GO annotation score is considered to be more intuitive than regular blast evalues since GO annotation is carried out by applying an annotation rule (AR) on the ontology terms. Additionally, query sequence descriptions are obtained by applying a language processing algorithm that extracts informative names and avoids low-content terms such as "hypothetical protein" or "expressed protein". Using Blast2Go suite default parameters, 1042 probes were provided with GO annotations (Additional file 4). Approximately 25% of the transcripts on the array do not show similarity to proteins present in public databases. Some of these could represent exclusive genes of the citrus or kumquat lineages, but a fraction of these uncharacterized sequences may possibly represent low quality or 3'UTR sequences. Similar percentages of unknown sequences have been reported in other small-scale EST projects [bib_ref] Pseudomonas syringae pv. tomato type III effectors AvrPto and AvrPtoB promote ethylene-dependent..., Cohn [/bib_ref] [bib_ref] Regulation of the Arabidopsis transcriptome by oxidative stress, Desikan [/bib_ref] [bib_ref] An investigation of the storage and biosynthesis of phenylpropenes in sweet basil, Gang [/bib_ref] and therefore this pattern can be considered characteristic of this approach. Since a citrus genome sequence is now available, future studies will have a wealth of citrus genomic sequence information that can be utilized to identify kumquatspecific as well as novel citrus genes involved in diverse defence mechanisms [bib_ref] Citrus genomics, Talon [/bib_ref]. Gene ontology analysis provided an extremely informative snapshot of the Xcc/kumquat interaction. The hierarchical structure for the gene ontology of a group of sequences can be visualized as a tree by means of directed acyclic graphs (DAG) [bib_ref] Blast2GO: A comprehensive suite for functional analysis in plant genomics, Conesa [/bib_ref]. For instance, the molecular functions of the network implicated in the kumquat response to Xcc infection is illustrated in the DAG presented in [fig_ref] Figure 2 A: Directed Acyclic Graph [/fig_ref]. The graph demonstrates a tree controlled by the Seq filter that organizes the number of nodes to be displayed. Seq is the number of different sequences annotated at the child GO term. On the whole, the biological meaning for different sequences in the data set was best illustrated in terms of three GO gene categories; the biological processes [fig_ref] Figure 3: A multilevel pie chart showing the distribution of probes on the chip [/fig_ref] underlying molecular functions (Additional file 5), and the cellular compartments where proteins were localized (Additional file 6). ## Kumquat transcriptional changes in response to xcc infection An important aspect of the data was that, for many genes, transcript abundance varied over time points, and a number of genes were only up-or down-regulated at one or two time points [fig_ref] Figure 4: Venn diagram demonstrating the number of upregulated genes [/fig_ref]. Two approaches were used to identify patterns of gene expression. First; the ASCA-gene analysis methodology revealed that most of the total variability in the data was related to time-associated changes [bib_ref] Discovering gene expression patterns in time course microarray experiments by ANOVA-SCA, Nueda [/bib_ref]. According to ASCA, 289 probes were selected as differentially expressed, 172 of which were at statistically significant levels (Additional file 7). Moreover, the time-associated variation could be divided into two main variability patterns. One pattern (accounting for 20% of the variation) represented genes whose expression levels changed significantly at 24 hpi from their levels at 6 hpi and then recovered to values similar to the starting values (or to even greater values in the opposite direction) at 72 hpi. However, the major pattern (80% of the time-associated variation) indicated a strong gene expression change between 6 and 24 hpi followed by preservation of expression levels at 72 hpi. This indicates that the strongest response to infection occurred at 6 to 24 hpi, and the majority of genes maintained their change for up to 3 days with a smaller percentage reverting to initial values. The second approach, maSigPro analysis, indicated that 317 probes were differentially expressed throughout time, (adjusted p value < 0.05 and R 2 of the model fit >= 0.6; Additional file 8). The results of both approaches were combined into 433 probes that were then filtered using more stringent conditions to provide a unique Children that represent a more specific instance of a parent term have 'is a' relationship to the parent. The darker the color of the node the more number of Blast hits and the higher annotation score it has. All nodes contain the hit annotation scores in numbers. result. The union rather than the intersection of the two approaches was taken because the two methods reveal different aspects of the data and are thus complementary. The ASCA-gene methodology focuses on shared gene expression changes to find important genes, while maSigPro treats genes independently and evaluates significant time dependent-changes. Although ASCA-gene methodology may miss some genes whose expression pattern is rare but significant, these will be captured by the gene-wise maSigPro approach. Alternatively, maSig-Pro can miss genes with less pronounced changes, which can be recovered by ASCA-gene if their profile is abundant within the dataset. The use of both approaches together resulted in the identification of 437 differentially expressed genes 312 of which with acceptable p-values that could be divided into 4 clusters according to their expression patterns [fig_ref] Figure 5: Cluster patterns [/fig_ref] , Additional file 9). The criterion for this division is as follows. From the ASCA analysis we obtained the main patterns of variation: Cluster pattern A indicates a strong change in expression between 6 hpi and 24 hpi, which is then maintained at time 72 hpi. Cluster B pattern is comprised of genes differentially expressed at 6 hpi as compared to either 24 hpi or 72 hpi. For each pattern, the correlation of the mean value of each gene at each time point with the profiles indicated by ASCA-gene was calculated; subsequently genes were divided into 4 clusters depending on whether expression levels changed in positive or negative directions. In this analysis, genes cannot be classified simply as induced or repressed, because this depends on the time points considered; for example, genes in cluster pattern C are repressed at 24 hpi and then induced at 72 hpi. ## Functional categorization of transcripts underlines key elements in kumquat response to xcc infection Based on the assumption that altered gene profiles during plant-microbe interactions can be correlated with symptoms, gene ontology and annotation, we believe that Xcc represents a typical example of how the bacterial pathogen can manipulate the host systems in its favour as elucidated previously in different studies [bib_ref] The plant immune system, Jones [/bib_ref] [bib_ref] A pthA homolog from Xanthomonas axonopodis pv. citri responsible for host-specific suppression..., Shiotani [/bib_ref] [bib_ref] A Bacterial Effector Acts as a Plant Transcription Factor and Induces a..., Kay [/bib_ref] [bib_ref] Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens..., Cernadas [/bib_ref]. Information on all of the specific transcripts discussed in the subsequent paragraphs is given in [fig_ref] Table 1: Functional categorization of cDNAs identified from microarray analysis [/fig_ref]. Cernadas et al. inoculated 'Pera' sweet orange with either Xcc, which causes typical canker symptoms on this citrus type, or Xanthomonas axonopodis pv. aurantifolii pathotype C (Xaa), which only produces symptoms on Mexican lime, followed by a detailed transcriptional analysis for the sweet orange plants [bib_ref] Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens..., Cernadas [/bib_ref]. Although the analyses done in that study cannot be directly compared with our study because of differences in methodology, some generalizations are noted below. The distribution of functions within the significantly expressed genes in Xcc infected kumquats indicates that the highest number of transcripts (~30%) was associated with response to stress, electron transport, and/or oxidative stress (as shown in [fig_ref] Figure 3: A multilevel pie chart showing the distribution of probes on the chip [/fig_ref] , an indication of an early regulatory changes in the plant immune system by Xcc. Earlier studies, such as that of Cernadas et al., have come to the same perception [bib_ref] Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens..., Cernadas [/bib_ref]. Each identified cluster was subjected to functional analysis by either studying the distribution of GO terms or performing enrichment analysis to see if there were functional categories that were significantly represented. A total of 137 genes, which makes up more than 30% of the genes that were significantly expressed, were down-regulated in the interval between 6 hpi and 24 hpi,. Most of them were grouped in Clusters A and C [fig_ref] Figure 6: Blast2GO directed acyclic graph showing "molecular function" after Xcc inoculation among transcripts... [/fig_ref]. The expression levels of the genes in both of these clusters reached a minimal expression level at 24 hpi followed by either a minor (Cluster A) or major (Cluster C) recovery by 72 hpi [fig_ref] Table 1: Functional categorization of cDNAs identified from microarray analysis [/fig_ref]. For instance, the expression of the thioredoxin f gene homologue (KLLFI3-F09) that belongs to cluster A reached its maximum level of expression (+1.8 fold) by 72 hpi after slight decrease at 24 hpi.The lipoxygenase gene homologue (KSLFII1-F07) that belongs to cluster C was 1.5 fold down-regulated at 6 hpi followed by a 3 fold increase in expression when compared to the its expression level at 6 hpi sample. Genes in Cluster A and C were frequently related to oxidative stress response. Most of the activity for genes in these clusters is located in the mitochondria, the cell membrane and the chloroplast [fig_ref] Figure 6: Blast2GO directed acyclic graph showing "molecular function" after Xcc inoculation among transcripts... [/fig_ref]. Cluster B was the largest cluster and included 235 genes with up-regulated expression levels between 6 hpi to 24 hpi followed by sustained expression until 72 hpi [fig_ref] Figure 5: Cluster patterns [/fig_ref] , [fig_ref] Figure 7: Blast2GO directed acyclic graph showing "molecular function" [/fig_ref]. Cluster D contained 61 members that had a low steady expression up to 24 hpi, and were subsequently upregulated [fig_ref] Figure 5: Cluster patterns [/fig_ref] , [fig_ref] Figure 7: Blast2GO directed acyclic graph showing "molecular function" [/fig_ref]. This cluster includes genes, such as the glycosyltransferase-like gene (KSLFI7-F12), that mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), a key mechanism in determining the diversity, activity and chemical complexity of plant natural products. In plants, UGTs (uridine diphosphate sugar glycosyl transferases) generally use UDP-glucose and occasionally UDP-xylose for glucosylation of phenylpropanoid aglycones. Albrecht and Bowman [bib_ref] Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter..., Albrecht [/bib_ref] proposed using UGTs and other glycosyltransferases as prospective genetic engineering candidates due to their important role in resistance and tolerance to citrus tristeza virus (CTV) as well as citrus huanglongbing (HLB) in trifoliate oranges (Poncirus trifoliata L. Raf.). Phenolics are mainly synthesized in plants via the phenylpropanoid pathway and are incorporated into many important compounds including plant hormones, secondary metabolites involved in stress, defence responses, and xenobiotics such as herbicides [bib_ref] Higher plant glycosyltransferases, Ross [/bib_ref]. In addition, phenylpropanoid pathway intermediates, for example pcoumaric acid, caffeic acid, ferulic acid and sinapic acid, and pathway derivatives, including flavonoid aglycones and glycosides, exhibit antimicrobial activity [bib_ref] Natural products and plant disease resistance, Dixon [/bib_ref] [bib_ref] A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and..., Mugford [/bib_ref]. ## Kumquat transcriptional changes in response to xcc infection ros vs ros scavenging In order to maintain homeostasis and overcome the damaging effects of ROS (reactive oxygen species), a balance between SODs (superoxide dismutase) and the different H 2 O 2 -scavenging enzymes is considered to be critical in determining the levels of O 2 and H 2 O 2 in plant cells [bib_ref] Reactive Oxygen Species during plant-microorganism early interactions, Nanda [/bib_ref]. Accordingly, there is a constant interplay between the antioxidant state and processes generating ROS. ROS are produced in chloroplasts, peroxisomes, and mitochondria in response to biotic as well as abiotic stresses [bib_ref] Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between..., De Pinto [/bib_ref] [bib_ref] Signal transduction during oxidative stress, Vranova [/bib_ref]. Accordingly, the expression of different enzymes that produce ROS were evidently stringently controlled and coordinated during the kumquat/Xcc interaction. For instance, while formate dehydrogenase (FDH; KLLRI2-G05), a mitochondrial NAD dependent enzyme, was 1.5 fold upregulated by 6 hpi, amine oxidase (KSLFI3-G05) that contributes to the synthesis of H 2 O 2 and secondary metabolites was downregulated by 1.5 and 1.6 fold at 6 and 24 hpi respectively in response to Xcc challenge [fig_ref] Table 1: Functional categorization of cDNAs identified from microarray analysis [/fig_ref]. Concurrently, Xcc-inoculated kumquat plants overexpressed genes related to ROS scavenging to restrict damage to the inoculated parts of the plant, in this case the leaves. For instance, CuZnSOD (KLLF13-A03) expression in kumquats was increased~1.5 fold at 6 hpi and was stabilized at 24 hpi and 72 dpi [fig_ref] Table 1: Functional categorization of cDNAs identified from microarray analysis [/fig_ref]. The same phenomenon was observed previously in tomato infected with Botrytis cinerea, a sign of increased ROS production by the host as part of the defence response to infection [bib_ref] The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria..., Kuzniak [/bib_ref]. Furthermore, while the expression of some of the genes linked to protease inhibitors and endopeptidase activities such as protease inhibitor homologue (KLLFI2-D02) was suppressed by the bacteria, other serine-type endopeptidase inhibitors such as an ATP-dependent ion protease (KSLFIV1-H05) was >2 fold up-regulated as early as 6 hpi subsequent to Xcc inoculation. In the same context, the redox coupling ascorbate-glutathione cycle, known to be responsible for peroxide detoxification [bib_ref] Ascorbic acid, metal ions and the superoxide radical, Halliwell [/bib_ref] , was repressed by 6 hpi in the kumquat dataset; examples include dehydroascorbate reductase (KSLRI1-F02) and glutathione peroxidase (KLLFII3-G07). Ascorbate and glutathione are non-enzymatic antioxidant molecules that have a role in other cycles, including those that synthesize and in some cases modulate flavonoids, alkaloids, phenolic compounds, α-tocopherol and carotenoids, all of which contribute in scavenging ROS [bib_ref] Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and..., Foyer [/bib_ref]. Dehydroascorbate reductase activity is indispensible when the ascorbate peroxidase (APX) levels are higher than normal under certain conditions to ensure preservation of the reduced form of ascorbate. Both proteins in addition to certain types of trypsin inhibitors might also catalyze a plant response [bib_ref] A novel-dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor, Trumper [/bib_ref]. A similar study to investigate the Xanthomonas-grapefruit compatible interaction might present a platform to compare gene expression profiles of some genes of interest in both plants. Accumulating evidence indicates that protein ubiquitination and degradation, last steps in protein turnover, are involved in plant defence responses. A number of recent studies have investigated a possible role of Ubox E3 ubiquitin ligases in PTI (PAMPS-triggered immunity), ETI (effector-triggered immunity), as well as plant cell death and defence [bib_ref] The E3 ubiquitin ligase activity of Arabidopsis PLANT U-BOX17 and Its functional..., Yang [/bib_ref] [bib_ref] Negative regulation of PAMPtriggered immunity by an E3 ubiquitin ligase triplet in..., Trujillo [/bib_ref]. In the present study, 6 ubiquitination pathway-related genes, for example ubiquitin-conjugating enzyme ucb7 (CSL1-A02), were isolated in the kumquat forward subtracted libraries; more investigation of their expression levels after infection will follow. Other induced genes that are involved in the proteolysis process are present in clusters A, B and C. ## Genes involved in photosynthesis A distinct down-regulation in the expression of ribu-lose1,5-bisphosphate carboxylase/oxygenase at 6 hpi, followed by an increase in expression that reaches maximum expression at 24 hpi, was observed in the microarray dataset (Rubisco small and large subunits; for example KLLFIII3-G09 and KSLRII2-F01) [fig_ref] Table 1: Functional categorization of cDNAs identified from microarray analysis [/fig_ref]. Rubisco, the most abundant protein in leaves, is the main source of energy production in plant cells. A decrease in photosynthesis was previously shown in Arabidopsis leaves as early as 3 h after challenge with the P. syringae avirulent strain, while after 48 h the rate of photosynthesis was lower with the virulent strain [bib_ref] Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and..., Bonfig [/bib_ref]. Most of the photosynthetic machinery in challenged kumquat leaves was repressed at 6 hpi, including chlorophyll A/B binding protein (KLLFIII3-A06 and KLLFIII3-E08). Three photosynthesis-related genes were differentially down-regulated during the first 24 hours. In Pto-mediated resistance, 30 photosynthesis-related genes and 12 genes encoding chloroplast-associated proteins were suppressed [bib_ref] Overexpression of the disease resistance gene Pto in tomato induces gene expression..., Mysore [/bib_ref]. These results show that plants reduce photosynthetic potential to induce HR following pathogen attack. Further, Quirino et al. [bib_ref] Molecular aspects of leaf senescence, Quirino [/bib_ref] suggested that HR and senescence are two programs that involve biochemical similarities as well as an overlap. The research reinforced the idea of a connection between defence response and senescence. Evidently, the down-regulation of genes involved in photosynthesis during the Xcc/kumquat interaction represents a cost for the plant fitness where energy resources were redirected to defence response. M-value is the base two logarithm of the ratio between the background-subtracted foreground intensity measured in the red and the green channels. These ESTs were identified as having a cy5 cy3 ratio > ± 1.5 for four out of six spots on the microarrays. -Putative function determined with the Gene ontology sequence description -Cluster: The cluster to which the putative gene belongs according to Blast2 GO functional analysis. -P-value associated to the statistical analysis for differential expression adjusted for multiple comparisons. ## Cell wall remodelling Xcc inoculation of kumquat was followed by the downregulation of various genes related to cell wall remodeling and rapid expansion such as endoglucanases. The expression level of a kumquat homologue of this wall loosening protein (KLLFI2-C10) was insignificant. On the other hand, genes related to cell wall reorganization, for example xyloglucan endotransglycosylase/hydrolase (XET,-an enzyme involved in cell wall elongation and restructuring), were significantly up-regulated by 24 hpi (KSLFIII1-H08). In 'Pera' sweet orange, a major difference in the response to inoculation of the two bacterial strains was that Xcc strongly upregulated several cell wall remodelling enzymes, while Xaa upregulated genes related to endoglucanase inhitors and lignin biosynthesis. A phenomenon that we observed in kumquat plants is the development of a few minute necrotic flecks on the leaves when inoculated with low concentrations of the bacterium (Xcc). Neither leaf abscission nor water soaked lesions were observed on the leaves later under our conditions. It is also worth mentioning that although Cernadas et al. used a relatively high concentration of Xaa (OD600 nm = 0.6,~double what we used for Xcc with kumquat) only pustles were recorded in sweet orange inoculated with Xaa that were not followed by necrosis. Using light microscopy, we have previously shown mesophyl collapse in kumquat leaves which was followed by leaf abscission 72 hrs post inoculation with Xcc. Alternatively, grapefruit mesophyl cells from inoculated leaves showed enlargement (hypertrophy) and division (hyperplasia) followed by raised circular lesions that became raised and developed into white or yellow spongy pustules. These pustules then darkened and thickened into brown corky canker lesions. Pustule formation and hypertrophy were linked previously to the PthA effector in Nicotiana benthamiana [bib_ref] A Bacterial Effector Acts as a Plant Transcription Factor and Induces a..., Kay [/bib_ref]. Alternatively, accumulation of the tomato XTH (xyloglucan endotransglucosylase/hydrolase LeXTH1) protein 6 hours after attachment of the parasite has provided evidence for a role of XTH in defence reactions associated with the incompatible tomato-Cuscuta interaction as was presented in Albert et al. [bib_ref] The cell wallmodifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction..., Albert [/bib_ref]. ## Resistance genes and related proteins Most of the differentially expressed genes in the kumquat/Xcc interaction have also been identified in plantinsect interactions [bib_ref] Transcriptional regulation of sorghum defense determinants against a phloem-feeding aphid, Zhu-Salzman [/bib_ref] , non-host resistance [bib_ref] EST and microarray analyses of pathogen-responsive genes in hot pepper (Capsicum annuum..., Lee [/bib_ref] and Rgene mediated resistance [bib_ref] Overexpression of the disease resistance gene Pto in tomato induces gene expression..., Mysore [/bib_ref] , suggesting a high level of convergence between different types of resistance mechanisms. The expression levels of some homologues to the PR3 (endochitinase) (KFII1-B11) and ß-galactosidase (BG1) (KRI2-D05) genes were found to be modestly activated; 2 and 1 fold up-regulated respectively 6 hpi, although both genes were down-regulated by 24 hpi as shown by the microarray and the qRT-PCR (quantitative real-time PCR) data , Additional File 10). Chitinase expression is a plant defence strategy typically used against wall components of fungi and insects [bib_ref] Substrate specificities of tobacco chitinases, Brunner [/bib_ref]. According to the qRT-PCR, the kumquat chitinase gene was >2-fold up-regulated by 6 hpi, after which it was suppressed at 24 hpi, and then its expression increased. Other studies have also reported the induction of chitinases in response to bacterial pathogens but their function is not well known [bib_ref] Plant chitinases-regulation and function, Kasprzewska [/bib_ref]. Interestingly, the expression levels of the kumquat PR1 (Pathogenesis -related gene1) gene homologue, normally a marker of salicylic acid-induced systemic acquired resistance (SAR) that is usually up-regulated after pathogen infections, was lower in the infected samples as early as 6 hpi compared to control mock inoculated samples (KLLFII2-A04) (according to the microarray results and non-published data). The region upstream of the PR1 promoter, W-box sequences, was shown previously to act as a negative cis-acting element in the expression of defence related genes [bib_ref] Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis, Lebel [/bib_ref]. This implies a different basal defence response and SAR regulation mechanism from that of Arabidopsis and other dicotyledonous plants in kumquat after Xcc infection. PR genes that were identified in citrus vary in their responses to different pathogens as shown in this study and others where variation in expression levels of different members of the PR gene family was dependent on the nature of different elicitors [bib_ref] PR gene families of citrus: their organ specific-biotic and abiotic inducible expression..., Campos [/bib_ref] [bib_ref] Tomato RAV Transcription Factor Is a Pivotal Modulator Involved in the AP2/..., Li [/bib_ref]. The biological activity of a large majority of PR genes in plants during biotic stress is yet to be revealed. More interestingly, according to the microarray results, expression levels of a number of homologues to other defence related genes such as the NDR1 gene (KLLFII2-E03) were also repressed 24 hpi after Xcc infection. LRR proteins are known to be a part of the early signal transduction cascade involved in the recognition of pathogen Avr products [bib_ref] Six amino acid changes confined to the leucine-rich repeat beta-strand/beta-turn motif determine..., Dodds [/bib_ref]. Sequences for a number of homologues known to be part of different hormonal defence pathways (for instance transcription factors, receptor like and receptor-like kinases) were found to be differentially expressed in kumquat after Xcc inoculation. ## Key molecular features of kumquat pcd A number of genes homologous to known resistant response-specific genes were expressed in the kumquat transcriptome concurrently following Xcc inoculation, listed and discussed below: (i) KSLFI3-C10 is homologous to hsr203J , a carboxylesterase (CXE) gene implicated previously in the incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. Its promoter is highly, rapidly, and specifically activated in response to HR inducing bacterial inoculation, does not respond to various stress conditions, and is strongly dependent on hrp (hypersensitive response and pathogenicity) genes of the pathogenic bacterium [bib_ref] hsr203J, a tobacco gene whose activation is rapid, highly localized and specific..., Pontier [/bib_ref]. It has been proposed that its expression should be a useful marker for programmed cell death occurring in response to diverse pathogens. (ii) KLLRI2-B05 shares homology with DND1 (DEFENCE NO DEATH 1), which encodes a cyclic nucleotide-gated ion channel that allows passage of Ca2 +, K+ and other cations. The Arabidopsis thaliana dnd1 mutant failed to produce HR cell death in response to an avirulent pathogen infection [bib_ref] The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotide-gated..., Clough [/bib_ref]. (iii) During programmed cell death or apoptosis cytochrome c is released to the cytoplasm from the intermembrane space of the mitochondrion [bib_ref] Mitochondria and apoptosis, Green [/bib_ref] [bib_ref] The dynamic plant chondriome, Logan [/bib_ref]. Once in qRT-PCR analysis of genes expressed in response to Xcc inoculation a (5 × 10 8 cfu/ml) concentration of the Miami strain X04-59. the cytoplasm, it activates caspases (cysteine aspartatespecific proteases), killer proteins that dismantle the cell [bib_ref] Cytochrome c release from mitochondria: all or nothing, Martinou [/bib_ref]. Two key proteins known to be core components of the apoptic machinery in animals, caspase and Bax-Inhi-bitor1 gene homologues, were identified in our dataset. According to our data, a homologue that has caspase activity KLLRI2_A12 was slightly up-regulated by 6 hpi in kumquat challenged leaves. Bax is a member of the Bcl2 family that plays a regulatory role preventing apoptosis by inhibiting adapters needed for the activation of caspases [bib_ref] The cell wallmodifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction..., Albert [/bib_ref]. A kumquat homologue of the Bax-Inhibi-tor1 gene (KLLFIII2-E06) was shown to be slightly up regulated 6 hpi in response to Xcc challenge as was previously shown with Arabidopsis thaliana Bax Inhibitor-1 (AtBI-1), isolated during a differential screen of plants challenged with the phytopathogen Pseudomonas syringae [bib_ref] AtBI-1, a plant homologue of Bax inhibitor-1, suppresses Bax-induced cell death in..., Sanchez [/bib_ref]. In the same context, Bax inhibitor has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo in animals. (iv) Endopeptidase inhibitors are often part of an inducible, jasmonic acid associated defence pathway that accumulates upon wounding, pathogen, or herbivore damage in leaves [bib_ref] ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology..., Alba [/bib_ref]. The antagonistic interaction between proteases and endopeptidase inhibitors is considered to be a cell death control mechanism [bib_ref] Multiple mediators of plant programmed cell death: Interplay of conserved cell death..., Hoeberichts [/bib_ref]. [bib_ref] Kunitz trypsin inhibitor: an antagonist of cell death triggered by phytopathogens and..., Li [/bib_ref] demonstrated that a serine protease (Kunitz trypsin) inhibitor (KTI1) of Arabidopsis is involved in modulating PCD in plant-pathogen interactions [bib_ref] Kunitz trypsin inhibitor: an antagonist of cell death triggered by phytopathogens and..., Li [/bib_ref]. RNAi silencing of the AtKTI1 gene resulted in enhanced lesion development after infiltration of leaf tissue with the PCD-eliciting fungal toxin fumonisin B1 (FB1) or the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 carrying avrB (Pst avrB). Trypsin inhibitor (KSLFIII1-H12 and KLLFIII3-F03) and a miraculin serine type endopeptidase inhibitor (FI2-A05) sequences were found in original early subtraction libraries representing transcripts expressed during early infection (30 min.pi-24 hpi). While KLLFIII3-F03 (trypsin homologue) gene expression was significantly suppressed 6 hpi, the expression of KSLIII1-H12 was slightly suppressed and then 2 fold upregulated 24 hpi. Further analysis should be done to study the difference between the mechanism(s) of action of these two genes. ## Suppression of defence responses A very evident down-regulation of a considerable number of genes was recorded by 6 hpi which may be caused by defence suppression imposed by Xcc effectors (Clusters A and C; [fig_ref] Figure 6: Blast2GO directed acyclic graph showing "molecular function" after Xcc inoculation among transcripts... [/fig_ref] and 6B). It has been shown previously that Xcc exploits the Type III secretion system (T3SS) to inject different effector proteins into citrus plants in order to avoid host recognition and subsequently MAMPS/PAMP-triggered immunity. The bacterial effector proteins suppress plant defences including basal defence, gene-for-gene resistance, and nonhost resistance. There was no accumulation of any SAR gene transcripts including PR1, a marker for enhanced defence; in addition some other key elements in the SA defence pathway were suppressed. On the other hand, the S-adenosyl-l-methionine:benzoic acid salicylic acid carboxyl methyltransferase gene (KLLRI2-C03) was at least 1-fold upregulated in kumquat leaves by 6 hpi in response to Xcc inoculation; the gene is known to play a role in plant defence responses [bib_ref] OsBISAMT1, a gene encoding S-adenosyl-Lmethionine: salicylic acid carboxyl methyltransferase, is differentially expressed..., Xu [/bib_ref]. In addition, the SA-binding protein 2 (SABP2 KLLRI2-G01), a lipase protein that belongs to the hydrolase super family, was found to be up-regulated at 6 hpi by at least 2 fold; the gene was previously found to be required for the plant immune response in tobacco [bib_ref] High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and..., Kumar [/bib_ref]. ## Realtime quantitative polymerase chain reaction validation Validation of the presented microarray dataset was carried out using TaqMan gene expression assay for a number of homologues on the array. Genes that were implicated in plant defence including basic leucine zipper transcription factor (KLLFII2-C05), a putative chitinase protein (CHI1)(KLLRI2-D05), a putative disease resistance leucine rich protein (CSL1-D05), a receptor like protein kinase (KLLRI2-H10), a beta galactosidase like protein (KLLFII1-B11) and a putative mitogen-activated protein (CSL2-A02) were selected for validation using q RT-PCR. As summarized in and Additional file 10, the qRT-PCR data correlated with the microarray results confirming the up-or down-regulation of all analyzed genes although as expected the qRT-PCR was more sensitive. # Conclusions In this study, a F. margarita custom microarray representing 1024 unigenes was used to study the response to inoculation with X. axonopodis pv. citri. A very distinct though delayed HR was observed in Xcc-inoculated kumquat plants where initially the bacterium grew exponentially, followed by a sudden leaf tissue collapse (necrosis with no canker lesions) 2-5 days after inoculation. A comparable delayed HR was observed in tomato resistance response to race T3 mediated by AvrXv3 effector and RxvT3 R protein [bib_ref] Identification and expression profiling of tomato genes differentially regulated during a resistance..., Gibly [/bib_ref]. The current kumquat analysis allowed simultaneous investigation of the expression of more than one group of genes known to be linked to more than one biological process and cellular compartment in relation to the HR caused by Xcc infection. A large number of genes were found to be differentially expressed after infection. Most of the genes involved in defence mechanisms in kumquat appear to be associated with the phenomena that precede the HR including oxidative burst, protein degradation, and regulation of photosynthesis as well as the production of ROS that is associated with the oxidative burst. One very distinct observation was that some of the defence genes such as PR1 and NDR1 were downregulated in kumquats in response to Xcc inoculation as early as 6 hpi, a phenomenon currently under further examination. What clearly appears to be a resistant response and a drastic decrease in the bacterial population, in addition to the activation of genes involved in ROS production as well as and programmed cell death, seems to be a common mechanism that is pursued by more than one citrus bacterial pathogen with no associated-resistance genes yet identified [bib_ref] Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens..., Cernadas [/bib_ref]. Future work will compare differences in gene response in both resistant and susceptible citrus types. [bib_ref] Ethylene regulates the susceptible response to pathogen infection in tomato, Lund [/bib_ref]. The bacterial strain used was Xanthomonas citri subsp. citri A; Miami X04-59 (Xcc). The inoculum was adjusted to 5 × 10 8 cfu/ml. A similar set of plants was mock-inoculated using sterile tap water as controls. Leaves from the two sets of plants were used in subsequent experiments. # Methods ## Plant material and inoculation with bacteria ## Microarray platform The kumquat microarray chip was developed and printed at the University of Florida (Gainesville, FL, USA). The array included ESTs chosen from 4 previously constructed Nagami kumquat forward and reverse leaf subtraction cDNA libraries. The cDNA libraries were constructed using RNA extracted from leaf tissue collected at different intervals post inoculation with Xcc (see below) and pooled into early and late library sets to capture a wide spectrum of differentially expressed transcripts. Random DNA sequencing was performed from 5'and 3' ends of randomly selected clones using universal primers, generating sequence information from 2788 and 1655 clones from the early and late leaf subtraction libraries respectively. The initial dataset was reduced to a total of 2304 transcripts that were selected according to sequence alignment similarities with proteins in the Genbank database. Sequences were selected based on quality and length. The dataset included 2254 kumquat ESTs comprising 738 contigs and 1516 singletons, in addition to 50 cDNA control elements. Each probe was printed in 3 locations on the array using the Omnigrid Microarrayer (Gene Machines, San Carlos, CA, USA) so that all clones had 3 technical replicates on each slide, generating a total of 6912 spots. Post-printing slide processing was performed as described in [bib_ref] Discovery and analysis of inflammatory disease-related genes using cDNA microarrays, Heller [/bib_ref] [bib_ref] Discovery and analysis of inflammatory disease-related genes using cDNA microarrays, Heller [/bib_ref] with some modifications. In brief, a combination of sequential baking and UV crosslinking was implemented where slides were baked for 80 min at 80°C in a drying oven without vacuum. The slides were then washed twice in 0.1% SDS for 5 minutes each to remove any unbound DNA. ## Experimental plan To identify genes that are considered to be differentially expressed in kumquat, a time-course experiment was designed utilizing the kumquat/Xcc pathosystem. Six independent Xcc or mock-inoculated kumquat plants were used; each plant was considered an independent biological replicate. Since citrus canker is a non-systemic disease, 6-10 leaves per treated kumquat plant were independently infiltrated using 5 × 10 8 cfu/ml Xcc. All RNA samples isolated from healthy mock-or Xcc inoculated (infected) leaves were processed independently. It is unlikely that differential gene expression observed was caused by the pressure infiltration inoculation method used, since this factor was normalized by treating the mock inoculated plants in the exact same way as infected plants. Individual leaves were harvested from the inoculated and mock-inoculated plants at specific time-points postinoculation (pi) according to designated conditions for each experiment; there were 3 time points for the microarray experiment and 5 for subsequent real-time PCR assays. For the microarray experiment, the three time points (6 hpi, 24 hpi, and 72 hpi) were chosen based upon the internal bacterial populations previously detected at these times following inoculation and the knowledge that kumquat leaves abscised 3-5 days after inoculation. In addition, previous experiments revealed that there were some transcripts differentially expressed as early as 30 min post-inoculation with Xcc. Finally, the RNA yield and the abundance of cellular transcripts decreased as the leaves approached total PCD, as has been shown previously by others [bib_ref] Molecular aspects of leaf senescence, Quirino [/bib_ref]. Time points of 0 and 120 hpi were added for the real time PCR assays. The healthy mock-inoculated and Xcc-inoculated leaf samples were immediately frozen in liquid nitrogen. For each respective time point, total RNA was extracted using RNeasy columns (QIAGEN, Valencia, CA, U.S.A.) according to the manufacturer's protocol. RNA purity, concentration and quality were assessed using a spectrophotometer and a BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA). ## Fluorescent probe, hybridization, and scanning Prior to slide hybridization with probe, slides were prehybridized in a solution containing 5× SSC, 0.1% SDS and 1% bovine serum albumin at 42°C for 45 min to eliminate nonspecific binding of the probe to the slide. Slides were washed using MilliQ RNase free water, then isopropanol, once each, and air-dried. Slides were maintained at the hybridization temperature until loaded with probe. cDNA labeling was performed using the Genisphere (Hatfield, PA, USA) 3DNA Array50 ® Expression Array Detection Kit according to the manufacturer's protocol for total RNA. For each time-point 125 μg of DNA-free total RNA isolated from an independent plant (biological replicate) per slide was reverse transcribed for each of the mock inoculated and the infected leaf samples separately using Ambion reverse transcriptase (Ambion; LaJolla, CA) in the presence of Genisphere dT primers. Two-step hybridization was performed as follows. The first hybridization, carried out at 48°C overnight, contained 10 μl of the concentrated cDNA (heat denatured probe) made using either the Cy5-RT primer capture sequence or the Cy3-RT primer capture sequence, in Genisphere 2× formamide-based hybridization buffer. Three successive post hybridization washes were performed, first in 2X SSC, 0.2% SDS at 55°C for 10 min, then 2× SSC for 10 min and finally in 0.2× SSC for 10 min at room temperature. The second hybridization (for addition of the dye) was performed using 2.5 μl of either Cy3 or Cy5 dendrimer, 2 μl of high-end differential buffer and 58 μl of hybridization buffer. For each time point, three mock-inoculated samples from three individual plants were labeled with Cy-3 and the Xcc infected samples were labeled with Cy-5 and both were hybridized to the same array. Post-hybridization washes were conducted as performed earlier following the primary hybridization, with the addition of 0.1 ml dithiothreitol (DTT) into the first and second wash solutions to reduce oxidation of fluorescent dyes. ## Cdna microarray setup and quality control Control measures such as the detection sensitivity level were determined using internal control probes and nonspecific control elements. Human genomic DNA, the green fluorescent protein gene, and the lambda control template DNA fragment were included as negative controls. Additionally, cDNAs previously implicated in pathogen defence such as PR1 and NPR1 from Arabidopsis, NDR1 from citrus, were printed 3 times on the array to test the ability of the microarray method to detect changes in gene expression. These were considered to be specific positive controls. In addition, the microarray ratio for each gene analyzed was normalized against the microarray ratio obtained for 18 S. # Transcriptome data analysis Agilent's Feature Extraction Software (Agilent Corp., Palo Alto, CA) was used to analyze the microarray data. Data were uploaded into the statistical platform Rfor statistical analysis and the Limma package was used for pre-processing. Data were Lowes transformed followed by scaling between arrays [bib_ref] Normalization for cDNA microarray data: a robust composite method addressing single and..., Yang [/bib_ref]. Two array slides were chosen for each time-point experiment based on the consistency of the signal across the replicates. The fold difference in expression was computed as: 2٨average ratio (2 to the power of the average ratio). cDNAs with an average ratio of 1.0 or higher were considered differentially expressed, which represents a 1.5 fold or higher difference in expression. Statistical analysis was performed using two different approaches. Time-dependent gene expression changes were analyzed by the maSigPro methodology [bib_ref] maSigPro: a method to identify significantly differential expression profiles in time-course microarray..., Conesa [/bib_ref]. Data were subsequently subjected to ASCA-gene analysis that combines ANOVA and multivariate methods to identify main and secondary patterns of gene expression associated with different experimental factors [bib_ref] Discovering gene expression patterns in time course microarray experiments by ANOVA-SCA, Nueda [/bib_ref]. Statistical analysis identified a number of selected genes that were further grouped into clusters. Functional information about the ESTs represented in the array was obtained by Blast2GO analysis using default parameters [bib_ref] Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics..., Conesa [/bib_ref]. Blast2GO uses Blast and an elaborated annotation algorithm to assign Gene Ontology (GO), Enzyme Code and InterPro functional labels to a set of uncharacterized sequences [bib_ref] High-throughput functional annotation and data mining with the Blast2GO suite, Gotz [/bib_ref]. The functional characterization of these clusters was done by applying the Functional Enrichment (FE) method included in Blast2GO which implements the Gossip algorithm. FE methods assess which functional categories are over-represented within a group of genes in relation to a broader list, in this case the whole kumquat array. Finally, the major induced transcriptional changes considered functional classes as a whole were studied with the PCA-maSigFun method [bib_ref] Functional assessment of time course microarray data, Nueda [/bib_ref]. This method combines Principal Component Analysis and maSigPro to characterize the "expression profiles" associated with cellular functions. Sequence data from this work have been deposited in the NCBI Genbank database libraries (http://www.ncbi.nlm.nih.gov/ GenBank/index.html), using the BankIt dbEST database, and accession numbers were obtained. [Genbank: GW687757to GW690680]. (See Additional File 11). ## Quantitative real-time quantitative pcr Kumquat leaves were infiltrated with Xcc (5 × 10 8 cfu per milliliter), then total RNA was isolated from inoculated leaves 0,6,24,72, 120 hpi for both the microarray and the quantitative real-time PCR experiment as previously stated in the 'experimental plan'. Total RNA was isolated separately for each respective time point using the TRIzol reagent (Invitrogen, Carlsbad,CA, U.S.A.) according to manufacturer's instructions. RNA samples were further purified using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) including DNaseI TURBO DNase (Ambion, Austin, TX, U.S.A.) treatment according to the manufacturer's instructions. RNA quality and quantity was then assessed using microspectrophotometry (Nanodrop Technologies, Inc., Wilmington, DE). cDNA was synthesized from 1 μg of total RNA using Applied Biosystems (High Capacity cDNA Reverse Transcription Kit PN 4368813, 4374966). A TaqMan gene expression assay was then used to validate the transcript accumulation levels of a specific subset of genes from the kumquat microarrays. Reactions were performed in the ABI Prism7900 HT sequence detector (Applied Biosystems, Foster City, CA, U.S.A.). Primers for qRT-PCR were designed using the Primer Express 3.0 software (Applied Biosystems), and data were normalized using a Taqman ribosomal RNA control, in addition to the kumquat 18S ribosomal gene that served as an internal control where each real-time PCR reaction was done in parallel with the 18S primers. For internal controls, a number of genes, for example actin showed inconsistencies. The 18s surprisingly showed coherency throughout the interaction, and this was noticed during the microarrays and was subsequently confirmed with the Realtime PCR (RT-qPCR) 18S expression curve. qRT-PCR was carried out at 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. [fig] Figure 1: Scatter plot analysis of the M-values from two microarray hybridizations using RNA samples from two independent kumquat plants inoculated with 5 × 10 8 cfu/ml Xcc. Each spot represents the normalized hybridization signal intensity for each transcript on the microarray. RNA samples from non-inoculated and inoculated leaf tissue were labeled with Cy3 and Cy5, respectively. (h6_1): 6 hours post inoculation hybridization results from slide # 1 hybridized to plant A-RNA samples vs. (h6_2): 6 hours post inoculation hybridization results from slide # 2 hybridized to plant B-RNA samples. [/fig] [fig] Figure 3: A multilevel pie chart showing the distribution of probes on the chip. Biological processes within all of the lowest nodes with the given number of sequences or score value plot jointly with an e-value cutoff (e-06). [/fig] [fig] Figure 2 A: Directed Acyclic Graph (DAG) visualizing the hierarchical structure of the Gene Ontology (GO) in inoculated kumquat leaves. [/fig] [fig] Figure 4: Venn diagram demonstrating the number of upregulated genes (numbers in red) vs the down-regulated (numbers in green) subsequent to Xcc inoculation. Results were based on the mean inductions of six experimental replicates. Genes with M-values>0.5 (1.5 fold) were considered up-regulated while M-values<0 were considered down-regulated. [/fig] [fig] Figure 5: Cluster patterns. The overall average gene expression profiles for genes from different functional clusters at each time-point. [/fig] [fig] Figure 6: Blast2GO directed acyclic graph showing "molecular function" after Xcc inoculation among transcripts representing the enriched functional categories (P < 0.25). (A) Cluster A. (B) Cluster C. [/fig] [fig] Figure 7: Blast2GO directed acyclic graph showing "molecular function" (P < 0.25) among transcripts induced at 24hpi. (A) Cluster B. [/fig] [fig] Fortunella: margarita (Lour.) Swingle (Nagami kumquat) plants were used in all of the experiments described in this study. Plants were approximately 2 years old at the time of the experiment and were maintained in the quarantine greenhouse facility at the Division of Plant Industry, Florida Department of Agriculture (Gainesville, FL, USA) under controlled conditions. Leaves from a set of six kumquat plants were infiltrated with bacterial cultures according to Lund et al. [/fig] [table] Table 1: Functional categorization of cDNAs identified from microarray analysis. [/table]

A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies Several lines of evidence indicate that antibody-dependent cellular cytotoxicity(Wren et al., 2013)is important in the pathogenesis of HIV-1 infection. Namely, ADCC is induced during natural HIV-1 infection or in HIV-1 vaccine studies, the latter demonstrated by the RV144 vaccine trial. To expedite the assessment of ADCC in studies of HIV, we have developed a high throughput assay. We have optimized the rapid fluorometric antibody-mediated cytotoxicity assay (RFADCC) by transfecting the EGFP-CEM-NKr cell line to constitutively express SNAP-tagged CCR5. This cell line can then serve as a source of HIV-specific targets when coated with monomeric gp120, spinoculated with inactivated intact virions, infected by cell-free viral diffusion or infected by cell-to-cell transmission of virus. The optimized strategy has two significant advantages over the original RFADCC method: First, the preparation of detectable target cells is less labor intensive and faster as it does not rely on multiple staining and washing steps for target cells. Second, because the target cell markers GFP and SNAP are constitutively expressed, the assay provides highly reproducible data. These strengths make the optimized RFADCC assay suitable not only for studies of HIV-1 specific cytotoxicity but also for studies of cell-cell transmission of virus. In conclusion, this assay provides a new generation T cell line that can expedite large clinical studies as well as research studies in humans or non-human primates. # Introduction ADCC in HIV-1 has been studied for over 20 years [bib_ref] Specific antibodydependent cellular cytotoxicity responses associated with slow progression of HIV infection, Wren [/bib_ref] , but interest in the HIV-specific response was prompted by findings from the recent RV144 clinical vaccine trial showing ADCC, together with low IgA, as a correlate of protection [bib_ref] Antibody-dependent cellular cytotoxicitymediating antibodies from an HIV-1 vaccine efficacy trial target multiple..., Bonsignori [/bib_ref]. Also, observations obtained in several natural HIV infection systems [bib_ref] Immune escape from HIV-specific antibody-dependent cellular cytotoxicity (ADCC) pressure, Chung [/bib_ref] [bib_ref] An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational..., Ferrari [/bib_ref] have highlighted a key role of ADCC activity in the immune response against the virus. A number of experimental assays have been standardized and utilized to characterize human or non-human primate antibodies for HIV-specific cytotoxicity. Many of the ADCC assays measure the potency of antibodies to mediate killing of virus-infected target T cells, mainly CEM NKr CCR5, by healthy, uninfected donor PBMC effector cells. These assays rely on the quantification of target cells that are prelabeled with traceable compounds, the loss of which indicates a decrease in membrane integrity or decrease in target cell viability. As ADCC readouts, these assays exploit critical steps of cytotoxicity, such as release of 51 chromium due to apoptotic killing of specific targets [bib_ref] Evidence for a correlation between antibody-dependent cellular cytotoxicitymediating anti-HIV-1 antibodies and prognostic..., Ahmad [/bib_ref] , release of granzyme B by activated effectors , loss of intracellular carboxyfluorescein diacetate succinimidyl ester (CSFE) due to disruption of target cell membrane integrity [bib_ref] A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity, Gomez-Roman [/bib_ref] or decrease in luciferase signal due to direct killing of virus-bearing luciferase targets [bib_ref] HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities, Pollara [/bib_ref]. In addition, a novel ADCC assay that incorporates a CD16 + NK effector cell line and a CD4 + T-cell line expressing HIV Tat-inducible luciferase has been utilized to identify the inverse correlation between ADCC titers and risk of infection in the RV144 trial [bib_ref] Antibody-dependent cellular cytotoxicitymediating antibodies from an HIV-1 vaccine efficacy trial target multiple..., Bonsignori [/bib_ref]. More recently, another ADCC assay based on the quantification of killed targets using the cell marker eFluor670 and a live/dead dye was reported [bib_ref] Flow cytometry-based assay to study HIV-1 gp120 specific antibody-dependent cellular cytotoxicity responses, Richard [/bib_ref]. Although these assays have provided important information about HIV pathogenesis or design and delivery of HIV vaccines, most are typically labor intensive and time consuming. Likewise, the RFADCC assay employed by our group to characterize mAbs specific for highly conserved regions of HIV-1 envelope exposed during viral entry [bib_ref] A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity, Gomez-Roman [/bib_ref] [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref] [bib_ref] Structural definition of an antibody-dependent cellular cytotoxicity response implicated in reduced risk..., Acharya [/bib_ref] is equally demanding. However, because flow cytometry analyses have allowed a detailed understanding of the phenotype of the cells involved, we modified the original RFADCC assay to streamline the manipulations and improve the inter-experimental reproducibility. To this end, we optimized our RFADCC assay to avoid the need for the cumbersome and multiple staining steps and washings, including eliminating the harsh target cell membrane staining with PKH26. The modified assay now involves only one rapid staining step and is highly useful for the systematic analysis of ADCC using target cells either sensitized with gp120, spinoculated with intact HIV virions, infected by cell-free virus or by cell-to-cell transmission of virus. # Material and methods ## Cell lines, viruses and monoclonal antibodies (mabs) The human T lymphoblastoid cell lines CEM NKr CCR5 [bib_ref] Natural killing target antigens as inducers of interferon: studies with an immunoselected,..., Howell [/bib_ref] and EGFP-CEM-NKr [bib_ref] A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity..., Kantakamalakul [/bib_ref] were obtained from the National Institute of Allergy and Infectious Diseases (NIAID) Reagent Repository. CEM NKr CCR5 cells were maintained in RPMI 1640 medium with glutamine and supplemented with 10% fetal bovine serum (FBS), HEPES buffer (Sigma) 10 mM, sodium pyruvate (Sigma) 500 μM, β-Mercaptoethanol (GIBCO) 50 μM and gentimicin (GIBCO) 50 μg/ml (termed R10 medium). The EGFP-CEM-NKr and EGFP-CEM-NKr-CCR5-SNAP cells were maintained in R10 medium supplemented with neomycin G418 (Gemini, Bio-Products) at 800 μg/ml and 1.5 mg/ml, respectively. For cell-bound virion studies, we employed AT-2 inactivated HIV-1 BalSuPT1-CCR5 CL.30 at a multiplicity of infection (MOI) of 5. This MOI was calculated using analytical information provided by Dr. Jeffery Lifson (National Cancer Institute at Frederick, Frederick, MD), who generously supplied this preparation. For infection by cell-free virus or cell-cell virus spread, we used HIV-1 Bal infectious molecular clone (NIH AIDS Reagent & Reference Reagent Program Lot. 9 021056) [bib_ref] The role of mononuclear phagocytes in HTLV-III/LAV infection, Gartner [/bib_ref] and HIV-1 Bal infectious molecular clone produced in SupT1-R5 cells [bib_ref] Antigenic properties of the HIV envelope on virions in solution, Ray [/bib_ref] respectively. The human mAbs included in the study were isolated from our Natural Virus Suppressors (NVS) cohort [bib_ref] HIV-1 natural viral suppressors: control of viral replication in the absence of..., Sajadi [/bib_ref] [bib_ref] Epidemiologic characteristics and natural history of HIV-1 natural viral suppressors, Sajadi [/bib_ref] and characterized to be specific for highly conserved regions of CD4-triggered gp120 [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref] : N5-I5 targets the mAb A32-like epitope surface around β2, β111-strands and α0and α1-helixes of layers 1 and 2 of the inner domain of gp120 [bib_ref] Immunological evidence for interactions between the first, second, and fifth conserved domains..., Moore [/bib_ref] [bib_ref] Topological layers in the HIV-1 gp120 inner domain regulate gp41 interaction and..., Finzi [/bib_ref] [bib_ref] Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and..., Pancera [/bib_ref] [bib_ref] Structural definition of an antibody-dependent cellular cytotoxicity response implicated in reduced risk..., Acharya [/bib_ref] [bib_ref] Cocrystal structures of antibody N60-i3 and antibody JR4 in complex with gp120..., Gohain [/bib_ref] ; C11 targets a distinct and discontinuous epitope on the seven-stranded βplatform and a residue in the extended C terminus of the gp120 molecule [bib_ref] Immunological evidence for interactions between the first, second, and fifth conserved domains..., Moore [/bib_ref] [bib_ref] Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and..., Pancera [/bib_ref] [bib_ref] Epitope specificity of human immunodeficiency virus-1 antibody dependent cellular cytotoxicity [ADCC] responses, Pollara [/bib_ref] [bib_ref] Cocrystal structures of antibody N60-i3 and antibody JR4 in complex with gp120..., Gohain [/bib_ref] ; N12-i12 targets the gp120 co-receptor binding site (CoRbs) [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref] ; N10-U1 targets the immunodominant domain of gp41 (unpublished result). The anti-respiratory syncytial virus mAb Synagis (MedImmune) was used as a negative control in most experiments. ## Production of egfp-cem-nkr cells stably expressing ccr5-snap EGFP-CEM-NKr cells (2 × 10 6 ) were transfected with 2 μg of Tag-lite pSNAP-CCR5 vector (pSNAPCCR5, htfr) with the Amaxa nucleofector and Amaxa Cell line Nucleofector Kit C (Cat. VCA-1004) according to the manufacturer's instructions. EGFP-CEM-NKr cells were maintained with R10 medium supplemented neomycin at 800 μg/ml during and after transfection due to the EGFP vector resistance. On day 2 posttransfection, the expression of CCR5-SNAP molecule on the cell surface was assessed by flow cytometry of cells stained with PE anti-CCR5 mAb 2D7 (BD Pharmingen Cat. 555993). The CCR5-positive EGFP-CEM-NKr cells were subjected to three rounds of selection by labeling with the 2D7 mAb and sorting with a FACSARIA II (Becton Dickinson, BD). Finally, when it was determined that CCR5 expression peaked on EGFP-CEM-NKr CCR5-SNAP cells, clones that showed distinct high levels of CCR5 expression were selected by limiting dilution. The expression of the SNAP tag was also assessed by indirect staining with rabbit anti-SNAP-Tag pAb (GenScript Corporation Cat. A00684) and PE F(ab′)2 donkey antirabbit IgG (eBioscience Cat. 12-4739) (data not shown). EGFP-CEM-NKr-CCR5-SNAP cells were passaged twice per week, with a higher neomycin dose (1.5 mg/ml) in order to better induce the CCR5-SNAP molecule expression on the cell surface and monitored weekly by flow cytometry. Quantibrite PE beads (Phycoerythrin Fluorescence Quantification Kit, BD Bioscience) were used to estimate the number of CCR5 and CD4 molecules present per EGFP-CEM-NKr-CCR5-SNAP cell, with CEM NKr CCR5 cells used as a reference. ## Optimized rfadcc assay and cell surface staining To optimize the RFADCC assay [bib_ref] A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity, Gomez-Roman [/bib_ref] for high throughput efficiency, the regular double staining with the membrane PKH-26 dye and the intracellular carboxyfluorescein diacetate, succinimidyl ester (CSFE) dye were replaced with the membrane staining of the CCR5-SNAP-tag and the constitutive intracellular expression of GFP. For the ADCC protocol [fig_ref] Figure 2: Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , EGFP-CEM-NKr-CCR5-SNAP target cells were stained with the fluorescent substrate SNAP-Surface Alexa Fluor 647 (New England BioLabs Cat. S9136S) for 20 min at 37°C with or without coating of monomeric HIV-1 Bal gp120 (50 μg/ml). For the studies with spinoculated virus, the cells were first stained with the SNAP-Surface dye and then spinoculated with the AT-2 inactivated Bal HIV-1 virus at 2000 RPM for 2 h at 12°C. Gp120-sensitized, virus-spinoculated or infected EGFP-CEM-NKr-CCR5-SNAP target cells were then washed twice with cold R10 medium and added to a 96-well V-bottom plate (5000 cells/well). A final volume of 100 μl/well of antibody dilution was added and incubated with sensitized targets for 15 min at room temperature. A total of 250,000 purified human effector PBMC isolated by Ficoll-Paque from the whole blood of healthy human donors cells were added to each well at an effector/target (E/T) ratio of 50:1. After incubation at 37°C in 5% CO 2 for 2 h (gp120-coated) or 3 h (virusspinoculated or infected cells), cells were washed with phosphatebuffered saline (PBS) containing 1% FBS (wash buffer), and fixed in 1% paraformaldehyde. Samples were analyzed at approximately 35,000 events per sample on an LSRII Fortessa flow cytometer (BD Biosciences). Doublets were excluded by forward-scatter area (FSC-A) versus forward-scatter height (FSC-H). Data were analyzed by FlowJo software (Tree Star, Ashland, OR). ADCC activity (=% cytotoxicity) is defined as the percentage of EGFP-CEM-NKr-CCR5-SNAP target cells that lose GFP staining but retain the CCR5-SNAP tag dye. For cell surface staining, HIV-1 gp120-sensitized and infected EGFP-CEM-NKr-CCR5-SNAP target cells were incubated for 30 min at RT with 1 and 5 μg/ml, respectively, of Alexa-Fluor 647-conjugated mAbs C11, N5-i5 or N12-i2 in PBS. Target cells spinoculated with Bal AT-2 inactivated virus were incubated for 30 min at RT, respectively with 2 μg/ml of Alexa-Fluor 594-conjugated mAbs C11, N5-i5, N12-i2 or N10U1 in PBS. Cells were then washed once with wash buffer and fixed in a 1% paraformaldehyde. ## Hiv-1 infection of egfp-cem-nkr-ccr5-snap cells To prepare the EGFP-CEM-NKr-CCR5-SNAP cells for virus infection assays, the cells were split 1:2 the previous day. Spinoculation was performed in a 96-well U-bottom plate by resuspending 5 × 10 5 target cells in a medium containing 240 ng of infectious virus (control cells were incubated without virus), as measured by HIV-1 p24 antigen capture ELISA. Mixtures of target cells and virus were centrifuged for 2 h at 2000 RPM at 12°C. Afterward, the viral inoculum was diluted 1:2 in R10 medium containing G418 1.5 mg/ml, and the cells and virus were placed into one well of a 12-well flat-bottom plate. The cells were then cultured adding fresh medium every 2 days. At 5 days postinfection, EGFP-CEM-NKr-CCR5-SNAP cells were harvested, washed twice with R10 medium, counted and divided into two tubes: one was stained for RFADCC as previously described; the second one was used to quantify the efficiency of infection and mAb binding. For intracellular p24 staining, the cells were washed once with PBS and stained with Live/Dead Fixable Near-IR Dead Cell Stain (Molecular Probes) and with 5 μl of (APC)-conjugated mouse anti-CD4 OKT4 mAb (eBioscience) for 30 min at RT. After a wash in wash buffer, the cells were then fixed and permeabilized using the Cytofix/Cytoperm Kit (BD-PharMingen, San Diego, Calif.) for 20 min at 4°C. Subsequently, the permeabilized cells were washed once with the buffer provided by the manufacturer, resuspended and stained for 30 min at RT with 5 μl (PE)-conjugated mouse anti-p24 mAb (KC57-RD1; Beckman Coulter, Inc.). After two additional washes, HIV-1-or mock-infected PBMC was analyzed with an LSRII Fortessa flow cytometer (BD Biosciences) and data analysis was performed with FlowJo software (Tree Star, Inc., San Carlos, Calif.). Live and GFP positive cells were analyzed for intracellular p24-Ag and surface CD4. ## Hiv-1 cell-cell transmission in egfp-cem-nkr-ccr5-snap cells Cell-cell virus transmission experiments were performed using CEM NKr CCR5 cells as virus-infected donor cells and EGFP-CEM-NKr-CCR5-SNAP cells as virus-acceptor targets. Accordingly, CEM NKr CCR5 cells were infected with HIV-1 Bal Molecular Clone and frozen at 3 days post-infection at the peak of infectivity (23% p24 + cells, [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] Panel C 0 h). The day before the co-culture experiment, the donor cells were thawed, rested overnight in R10 medium at 37°, 5% CO 2 and then washed twice with PBS, keeping the supernatant as a control [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] Panel A-Free virus Transwell-FVT). The infected CEM NKr CCR5 donors were plated with the acceptor EGFP-CEM-NKr-CCR5-SNAP targets at a ratio 1:1 (1 × 10 6 donor: 1 × 10 6 acceptor) for 7 days [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] Panels A-C). For reference, the donor and acceptor cells were cultured at the same ratio in a separated trans-well chamber (12 mm Transwell® with 3.0 μm pore polycarbonate membrane insert (Corning #3402) to evaluate the ability of the donor-produced virus to infect the acceptor cells in a cell-free manner [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] Panel A-Co-Culture Transwell-CCT). The readout of the newly infected targets was the count of GFP + p24 + CD4 + cells over various time points. Every 24 h, an aliquot of the donor/acceptor cell combination was harvested and stained for the expression of surface CD4 and intracellular p24, as previously described in the previous section. # Results ## Generation and characterization of the levels of ccr5 molecules on the egfp-cem-nkr-ccr5-snap cell line Keeping the original RFADCC assay format, we developed a more reliable and rapid approach to label the CEM target cells. To eliminate CSFE staining, we employed EGFP-CEM-NKr cells [bib_ref] A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity..., Kantakamalakul [/bib_ref] that constitutively express cytosolic GFP but are negative for CCR5 expression [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. Then, we engineered the EGFP-CEM-NKr cells to stably express the fusion protein N-terminal SNAP-tagged CCR5 on the cell surface. After three rounds of sorting and limiting dilution culture, we successfully isolated a stable GFP + clone that expressed CCR5-SNAP tag on the surface, as shown in [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel C. Of note, the transfection of CCR5-SNAP did not affect the level of GFP expression [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , compare panels B vs. C). More importantly, the CCR5-SNAP tag construct provided the advantage of using an anti-SNAP-tag (linked to CCR5) dye, which is relatively bright, non-toxic and specific for cell-surface [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , eliminating the background from non-CCR5-SNAP tag expressing cells [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel D and E). The SNAP tag provided the opportunity to use a second fluorochrome (SNAP-Surface Alexa Fluor 647) needed to identify the killed target cells in the RFADCC assay [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. We then determined the levels of CCR5 and CD4 molecules expressed on the cell surface with calibration beads and found that the new cell line expressed a physiologic level of CCR5 [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. Additionally, we selected for cells with relatively high CD4 levels, which is typically useful for studying HIV-1 infection [fig_ref] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. ## Optimized rfadcc assay with gp120-sensitized egfp-cem-nkr-ccr5-snap cells To compare the original RFADCC with the modified RFADCC, CEM NKr CCR5 cells were stained with PKH26 and CSFE, while EGFP-CEM- NKr-CCR5-SNAP cells were stained with Alexa-Fluor 647 surface SNAP dye as summarized in [fig_ref] Figure 2: Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. As shown in [fig_ref] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1... [/fig_ref] Panel A, the two cell lines behaved similarly, having a comparable percentage of target cells migrating from the double stained population into the region containing the killed target cell population, positive only for PKH-26 or SNAP-Alexa Fluor 647 respectively. Subsequently, the new target cells were employed to measure the ADCC activity of two additional human anti-gp120 mAbs. As a negative control, we used an unrelated mAb, Synagis, specific for respiratory syncytial virus (RSV). The assay was performed by adding the double-labeled target cells to mAbs dilutions, allowing them to bind, and then adding PBMC as effectors. As shown in [fig_ref] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1... [/fig_ref] Panel B, the new cellular system was able to detect the specific anti-gp120 ADCC activity of C11, N5-i5 and N12-i2 mAbs, with values comparable to the original RFADCC [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref]. As expected, the RSV mAb was negative in the induction of cytotoxicity. In addition, we evaluated the ability of the gp120-coated EGFP-CEM-NKr-CCR5-SNAP cell line as a tool to study the binding of the same mAbs. As shown in [fig_ref] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1... [/fig_ref] , we detected a very strong signal for all the Alexa Fluor 647-labeled antibodies (C11, N5-i5 and N12-i2), specific for the binding of gp120 molecules on EGFP-CEM-NKr-CCR5-SNAP cells (Panels C--E), compared to the negative control of uncoated cells (dashed greygray curve). Interestingly, the intensity of the binding detected in the new cell line was comparable to the standard gp120-coated CEM NKr CCR5 cells [fig_ref] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1... [/fig_ref] Panels F-H). ## Optimized rfadcc assay with virion bound-egfp-cem-nkr-ccr5-snap cells In order to evaluate the possibility of using the new cell line to identify mAbs that mediate ADCC against cell-bound intact HIV, we spinoculated the EGFP-CEM NKr CCR5-snap cells with AT-2 inactivated HIV-1 Bal, as described in the Materials and Methods section [bib_ref] Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational..., Rossio [/bib_ref] [bib_ref] Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding, O&apos;doherty [/bib_ref]. This approach allows the evaluation of immunogenicity of envelope epitopes exposed upon binding of cell surface CD4. As shown in [fig_ref] Figure 4: ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions [/fig_ref] (left panel) in the modified system, the reference antibodies were able to induce strong cytotoxicity against cell-bound virus. The ADCC values were comparable to those obtained in the original RFADCC with CEM NKr CCR5 cells bound with the same virus [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref]. Moreover, we quantified the efficiency of the spinoculation-mediated virus binding by testing binding with the reference mAbs labeled with Alexa Fluor-594. We were able to detect a strong signal for all the antibodies tested [fig_ref] Figure 4: ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions [/fig_ref] right Panels A-C). Of note for the virus-bound cells, the values for ADCC activity and binding ability were lower than with the gp120-coated cells because of the A. For gp120-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP cells were stained with SNAP-Surface Alexa Fluor 647 for 20 min at 37°C with or without monomeric HIV-1 gp120. B. For AT-2 inactivated virus-based ADCC assay EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye first and then spinoculated with the inactivated virus. C. For IMCinfected targets-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP were spinoculated with IMC, cultured for 5 days, washed twice and then stained with Alexa Fluor 647-SNAP tag dye. Sensitized cells were incubated with dilutions of antibodies for 15 min at room temperature (RT) and subsequently with PBMC as effector cells for 2 or 3 h at 37°C. Cells were then washed and fixed in 1% paraformaldehyde. The readout is the loss of GFP, as a direct measure of the percentage of target cells cytotoxicity mediated by the mAbs. After coating with monomeric HIV-1 Bal gp120 and adding PBMC as effector cells, we measured the ADCC activity of a reference HIV-1 mAb, C11. The cytotoxicity readout in the original RFADCC was measured as loss of CSFE, while in the modified RFADCC it was the loss of GFP. relatively low number of envelope spikes per virus and the low numbers of cells that can be bound synchronously by the virus in vitro (compare [fig_ref] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1... [/fig_ref] [gp120] vs [fig_ref] Figure 4: ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions [/fig_ref]. ## Infection and optimized rfadcc assay with egfp-cem-nkr-ccr5-snap cells In order to determine whether the modified cell line was suitable for virus infection and thus use in an infected-cell RFADCC assay, we spinoculated the EGFP-CEM-NKr-CCR5-SNAP cells with HIV-1 subtype B Bal infectious molecular clone (IMC), as previously described . At 5 days post-infection Bal-spinoculated EGFP-CEM-NKr-CCR5-SNAP cells presented a high cellular viability (approximately, 90%), comparable to control uninfected cells (data not shown). The rate of infection was about 20% (as shown by the presence of p24 in [fig_ref] Figure 5: ADCC assay using cells infected with HIV-1 BaL IMC [/fig_ref]. As expected, a high percentage of p24 + cells down-regulated the surface expression of the CD4 molecule (Veillette [bib_ref] Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on..., Zhong [/bib_ref]. CEM NKr CCR5 cells, infected with HIV-1 Bal molecular clone, were used at the peak of infection as virus donors. EGFP-CEM-NKr-CCR5-SNAP cells were used as virus acceptor targets of the infection transmitted cell to cell. Donors and targets were plated together to allow the spread of the virus, and the readout of the newly infected targets was the count of GFP + /P24 + cells. Background of cell-free virus spread was determined with donors and targets separated in Transwell chambers and with targets subjected to supernatants harvested from infected donor cells. B. and C. The count of GFP + /p24 + /CD4 + or − cells was monitored for 7 days from the beginning of the co-culture by flow cytometry. . We employed the infected cells as targets for our high throughput RFADCC. Interestingly, we were able to detect binding and cytotoxicity of the CD4i reference mAbs C11 and N5-i5, the epitopes of which are normally masked in an unbound, native Env trimer [bib_ref] Structural definition of an antibody-dependent cellular cytotoxicity response implicated in reduced risk..., Acharya [/bib_ref] [bib_ref] Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated..., Veillette [/bib_ref] [fig_ref] Figure 5: ADCC assay using cells infected with HIV-1 BaL IMC [/fig_ref] left panel and right panels A-B); this is most likely due to the residual surface membrane CD4 expression in a fraction of p24 + infected cells, rendering them sensitive to ADCC with these CD4i mAbs . In addition, we revealed low level binding and ADCC activity for N10-U1, which is specific for the immunodominant domain of gp41 [fig_ref] Figure 5: ADCC assay using cells infected with HIV-1 BaL IMC [/fig_ref] left panel and right Panel C). As expected, Synagis was negative for the ADCC activity and for binding to Bal infected EGFP-CEM-NKr-CCR5-SNAP cells (data not shown). This result constitutes additional evidence that the chimeric CCR5-SNAP tag is able to act as a functional co-receptor for HIV-1 during virus entry and fusion with the cellular membrane. ## A new tool to determine cell-cell virus spread HIV-1 has been shown to be capable of two modes of propagation: direct infection by cell-free virions and cell-to-cell transmission [bib_ref] Avoiding the void: cell-to-cell spread of human viruses, Sattentau [/bib_ref]. Notably, cell-to-cell spread from infected to noninfected cells occurs through the formation of virological synapses [bib_ref] Recruitment of HIV and its receptors to dendritic cell-T cell junctions, Mcdonald [/bib_ref] [bib_ref] Avoiding the void: cell-to-cell spread of human viruses, Sattentau [/bib_ref] [bib_ref] Quantitative 3D video microscopy of HIV transfer across T cell virological synapses, Hubner [/bib_ref] and has been shown to be a more rapid and efficient mechanism compared to free virus-cell diffusion. This feature supports the hypothesis that cellcell transmission may be a relevant mode of virus dissemination in infected individuals [bib_ref] Quantitation of human immunodeficiency virus type 1 infection kinetics, Dimitrov [/bib_ref] [bib_ref] Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived..., Carr [/bib_ref] [bib_ref] Predominant mode of human immunodeficiency virus transfer between T cells is mediated..., Chen [/bib_ref] [bib_ref] Inefficient human immunodeficiency virus replication in mobile lymphocytes, Sourisseau [/bib_ref]. Thus, it is important to have a tool that allows the methodical evaluation of mAbs or other molecules compatible with this type of infection. Accordingly, we designed an experimental method based on [bib_ref] Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on..., Zhong [/bib_ref] system in which the authors compared the efficiency of HIV-1 transmission by cell-free virus vs. cell-cell transmission in co-cultures. To evaluate it with our EGFP-CEM-NKr-CCR5-SNAP cell line, we persistently infected CEM NKr CCR5 cells with HIV-1 Bal molecular clone, using these cells as donors of replication-competent virus at the peak of the infection, as found by the levels of p24 expression [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel C, Donor-0 h). With the EGFP-CEM-NKr-CCR5-SNAP cells as the virus infectable targets, these cells were directly co-cultured with the virus infected donors or separated from the donors by a trans-well membrane for 168 h. An additional control was the supernatant harvested from the infected CEM NKr CCR5 cells just before the experiment (summarized in scheme [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel A). The readout of the assay was the count of GFP + /p24 + cells, representing newly infected target cells. As shown in [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel B, the co-culture of infected donors with uninfected targets allowed the transmission of the virus through cell-cell contacts, reaching a peak of GFP + /p24 + cell count at 48 h. Additionally, flow-cytometry analysis allowed the characterization of the levels of expression of CD4 on the surface of the newly infected GFP + /p24 + cells. In [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , Panel C, we show that by 48 h of co-culture, levels of CD4 on the cell surface had decreased from 100% to 5% and by 72 h, CD4 levels were further decreased to 1%. Thus, using this culture system, the physical contact of cells was much more efficient in spreading the infection [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref] , than culturing uninfected cells with cells actively producing virus or with cell-free virus-containing supernatant, as shown in [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. # Discussion The RFADCC assay has provided an important contribution in the characterization of antibodies that mediate strong cytotoxicity against HIV-1 sensitized cells. Here we describe an optimization of the original RFADCC that has been modified in the following ways: to replace the time consuming staining of target cells with two dyes and subsequent multiple washings, we took advantage of the cell line EGFP-CEM-NKr [bib_ref] A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity..., Kantakamalakul [/bib_ref] that constitutively expresses one of the two fluorochromes needed to identify target cells. Next, to include a second fluorochrome and replace the PKH26 dye, which is relatively toxic, we transfected cells with a SNAP tag-linked CCR5, and then stained the targets with an Alexa Fluor dye directed to the SNAP tag. The first part of the ADCC procedure now is dramatically faster and more reproducible (summarized in [fig_ref] Figure 2: Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. Moreover, as we show in Figs. 3-4, the results obtained both in gp120-coated or AT-2 inactivated virus bound-cells are very comparable to each other and consistent with what we had previously reported [bib_ref] Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein..., Guan [/bib_ref]. We also demonstrated that the new cell line can serve as a susceptible target for HIV-1 infection and subsequently be employed as targets for ADCC activity mediated by anti-HIV-1 antibodies [fig_ref] Figure 5: ADCC assay using cells infected with HIV-1 BaL IMC [/fig_ref]. These results are significant for highthroughput evaluation of antibody responses to epitopes exposed earlier during viral entry and later during viral budding post-infection. Finally, we assessed the possibility of utilizing EGFP-CEM-NKr-CCR5-SNAP cells to evaluate cell-cell spreading of HIV-1, which has been demonstrated to be the most effective for virus transmission. In this model, we exploited the expression of the GFP to identify the modified target cells that have been newly infected during the experiment [fig_ref] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells [/fig_ref]. Taken together, the use of the EGFP-CEM-NKr-CCR5-SNAP cell line makes the RFADCC a faster and more reliable tool for the evaluation of the cytotoxicity against cells infected with many human and nonhuman pathogens, such as HIV-1 and SIV viruses. [fig] Figure 1: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were generated by stable transfection of EGFP-CEM-NKr cells with Tag-lite pSNAP-CCR5 vector (pSNAP-CCR5, htfr). A-C. GFP and CCR5 expression in parental CEM-NKr-CCR5, EGFP-CEM-NKr and transfected EGFP-CEM-NKr-CCR5-SNAP cells. D-F. Labeling with surface SNAP tag-Alexa Fluor-647 dye vs. GFP expression in the three cell lines. Right panel. The levels of CCR5 and CD4 molecules expressed on EGFP-CEM-NKr-CCR5-SNAP cells in comparison to CEM NKr CCR5 were assessed by flow cytometry with Quantibrite PE calibration beads. [/fig] [fig] Figure 2: Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells. The optimized assay was modified from Gomez-Roman et al. (2006). [/fig] [fig] Figure 3: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1 Bal gp120. A. Comparison of RFADCC assay layout with CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells. CEM NKr CCR5 were stained with PKH-26 and CSFE (Gomez-Roman et al., 2006), while EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye. Killing by C11 mAb (1 μg/ml) is determined as loss of CSFE in PKH26-positive CEM NKr CCR5 cells or loss of GFP in SNAP-positive EGFP-CEM-NKr-CCR5-SNAP cells. B. Cytotoxicity curves for gp120coated EGFP-CEM-NKr-CCR5-SNAP. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. The binding of 1 μg/ml Alexa-Fluor 647-conjugated mAbs C11 (C and F), N5-i5 (D and G) or N12-i2 (E and H) was compared in gp120-coated-CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells. [/fig] [fig] Figure 4: ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were labeled with Alexa Fluor 647-SNAP tag and then spinoculated with HIV-1 Bal AT-2 virus at 2000 rpm for 2 h at 12°C. After two washes, cells were incubated with dilutions of mAbs (C11, N5-i5, N12-i2 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37°C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. Right panel. The efficiency of the spinoculation was evaluated by cell surface staining with 2 μg/ml Alexa Fluor-594-conjugated mAbs C11 (Panel A), N5-i5 (Panel B) or N12-i2 (panel C). [/fig] [fig] Figure 5: ADCC assay using cells infected with HIV-1 BaL IMC. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were spinoculated with HIV-1 Bal molecular clone at 2000 rpm for 2 h at 12°C. After 5 days of co-culture with the virus, cells were washed twice, labeled with Alexa Fluor 647-SNAP tag dye and incubated with dilutions of mAbs (C11, N5-i5, N10-U1 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37°C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC data represent the typical results obtained in three independent experiments. Upper right panel. The efficiency of the infection was evaluated by staining of the cells with live/dead (not shown), CD4-APC and p24-PE. Lower right panel. Binding of infected EGFP-CEM-NKr-CCR5-SNAP cells with 5 μg/ml Alexa Fluor-647-conjugated mAbs C11 (panel A), N5-i5 (panel B) or N10U1 (panel C). [/fig] [fig] Figure 6: EGFP-CEM-NKr-CCR5-SNAP cells: New tool to study the cell-cell virus spread. A. Schematic representation of the experimental design (modified from [/fig]

Therapeutic Interventional Endoscopic Ultrasound Based on Rare Cases in Indonesia: A Single-Center Experience in Unselected Patients Background: Endoscopic ultrasound (EUS) is still not widely available and has a barrier in most Southeast Asian countries due to lack of training program, high cost, and hospital investment. In this study, we would like to show the impact of therapeutic interventional EUS procedures in gastroenterology practice in Indonesia, which represents the biggest Southeast Asian country. Methods: Patients who underwent interventional EUS procedure in Medistra Hospital were prospectively recruited within 1 year. Results: Of 147 patients who underwent EUS procedures, 39 patients underwent fine needle aspiration. Most of the cases suffered from pancreatic cancer (47.5%) followed by ampullary cancer (20%), gastric subepithelial mass (10%), and other conditions. There were 4 rare cases that underwent therapeutic interventional EUS procedures. Patients with large mesenteric cyst attached to the gastric wall and large left liver lobe cyst with gastric compression who were previously suspected with gastrointestinal stromal tumor were successfully managed by cyst aspiration. One patient with a large pseudocyst due to chronic pancreatitis was successfully managed by plastic Case stent placement. Another patient with duodenal duplication cyst causing duodenal obstruction was managed by inserting a plastic stent through the cyst. No complications were observed during and after the therapeutic EUS procedures. Conclusions: Innovation in interventional EUS has a high impact in gastroenterology practice as well as in a developing country like Indonesia, which represents the biggest Southeast Asian country. Further developments are needed regarding the cost, investment, and especially the necessary training curriculum to make this technology available in tertiary referral centers. # Background Endoscopic ultrasound (EUS) has been shown to be a highly favorable diagnostic and therapeutic development in Western countries as well as in some developed Asian countries. In most Southeast Asian countries including Indonesia, EUS development is still in its infancy and encounters many barriers related to the lack of proper training facilities, expensive hospital investment, insurance coverage, and high procedural cost [bib_ref] Impact of endoscopic ultrasound procedures in various pancreatobiliary disorders in Indonesia based..., Lesmana [/bib_ref]. The development of therapeutic interventional EUS has replaced some surgical procedures such as EUS-guided biliary drainage, EUS-guided stenting of pancreatic pseudocysts, and EUS radiofrequency ablation for preventing recurrent liver cyst or pancreatic cancer [bib_ref] Therapeutic endoscopic ultrasound, Cheriyan [/bib_ref] [bib_ref] Interventional endoscopic ultrasound: therapeutic capability and potential, Tarantino [/bib_ref] [bib_ref] Endoscopic ultrasound-guided biliary drainage: a review, Iwashita [/bib_ref]. In the field of hepatology, EUS has also given a new paradigm in managing patients with gastric varices in liver cirrhosis through so-called EUS-guided vascular intervention [bib_ref] Endoscopic ultrasound guided vascular access and therapy, Sexena [/bib_ref]. However, in view of high costs and lack of expertise, most gastroenterologists would still prefer to refer such patients to surgeons. However, some surgical procedures such as pancreaticoduodenectomy require a high-experience center, which might not be available in most Asian developing countries. Therefore, to illustrate the availability of suitable patients for EUS procedures and to report the impact of therapeutic interventional EUS procedures in unselected patients, we like to report a rare case series in a private referral hospital to document the need for therapeutic EUS facilities also in a developing country. # Materials and methods Patients who underwent therapeutic interventional EUS procedures were recruited from the EUS hospital database. The therapeutic interventional EUS procedures were performed by a consultant gastroenterologist experienced in advanced therapeutic endoscopic procedures including endoscopic retrograde cholangiopancreatography (ERCP), interventional transabdominal ultrasound procedures, and EUS-fine needle aspiration (FNA) procedures. The procedures were supervised by a senior consultant experienced in interventional gastroenterology procedures. The EUS equipment was an Olympus JF UCT 180 EUS scope which was connected to an Aloka IPF-1701C ultrasound machine (Tokyo, Japan). The EUS needles used were Olympus 19-G aspiration needle NA-200H-8022 and the EUS Boston 19-G aspiration needle 17462614. The guide wire was a 0.35-inch Tracer Metro Direct Wire Guide (METII-35-480/Wilson Cook). Double pigtail stents were used (7-Fr Zimmon Biliary Stent). # Results Of 147 patients who underwent EUS procedures from the hospital database, 39 patients (20 males and 19 females) underwent EUS-FNA procedures and 4 of them underwent therapeutic interventional EUS procedures. Based on the EUS-FNA database, pancreatic malignancy (47.5%) was the most common etiology of the diseases followed by ampullary cancer (20%), gastric subepithelial mass (10%), intraductal papillary mucinous neoplasm (5%), benign pancreatic cyst (5%), large intra-abdominal cyst attached to the gastric wall, large left liver lobe cyst compressing the gastric wall, duodenal duplication cyst (DDC) with chronic recurrent pancreatitis, and distal common bile duct mass. Therapeutic interventional EUS procedures were successfully performed in 4 rare cases without complications. The first case was a 26-year-old female referred from another hospital with incidental finding of a large intra-abdominal cyst suspected of a giant pancreatic pseudocyst, which was located between the gastric wall and the corpus to tail area of the pancreas based on the abdominal CT scan. The patient previously underwent evaluation for history of chronic hepatitis B infection. Transabdominal ultrasound found an incidental cyst and therefore, the patient was suggested to undergo abdominal CT scan examination. However, the patient did not have any history of pancreatitis or a history of alcohol consumption. Cyst aspiration was done [fig_ref] Figure 1: A large intra-abdominal benign mesenteric cyst successfully managed by aspiration needle through... [/fig_ref] and the result of the fluid analysis corresponded to a benign cyst. Then the patient underwent surgery because of recurrent cyst and was shown to have a benign solitary mesenteric cyst. After operation, there was no further recurrence. The second case was a 63-year-old female who was referred from another hospital with abdominal fullness because of compression of the gastric wall due to a large submucosal mass suggestive of a gastrointestinal stromal tumor based on upper gastrointestinal endoscopy examination. EUS examination revealed a large liver cyst in the left liver lobe just beside the heart, which compressed the gastric wall. Cyst aspiration was successfully done without complication. The third case was a 37-year-old male referred from another hospital with a history of heavy alcohol consumption, abdominal pain and distention, and recurrent pancreatitis during the last months. The amylase and lipase blood levels before procedure were 823 and 1,892 U/L. EUS examination revealed pancreatic alterations compatible with chronic pancreatitis with a large pancreatic pseudocyst compressing the gastric wall. A 7-Fr double pigtail stent was placed into the pseudocyst under EUS guidance [fig_ref] Figure 2: A large infected pancreatic pseudocyst successfully managed by 7-Fr double pigtail stent [/fig_ref]. After 1 month, the cyst had resolved and after 3 months, the stent was removed. His last amylase and lipase blood levels were 22 and 40 U/L. His clinical condition came back to normal and the patient remained free of pancreatitis attacks. The last case was a 20-year-old male referred from another hospital with recurrent abdominal pain due to recurrent pancreatitis. Amylase and lipase were 145 and 1,008 U/L, respectively, and the CA 19-9 was 3.41 U/mL. Based on the abdominal CT scan, there was a large hypodense mass with a diameter of 55 × 44 × 72 mm at the head of the pancreas area suggestive of a cystadenoma. EUS examination revealed a bulging cyst at the postpyloric area compressing and obstructing the inside of the duodenal cavity suggestive of a picture of DDC. This finding was compatible with the abdominal CT scan examination. The cyst was suspected to be due to a lot of the preserve inside the cyst cavity [fig_ref] Figure 3: Duodenal duplication cyst with duodenal obstruction successfully managed by 7-Fr double pigtail... [/fig_ref]. The patient and the family were advised to undergo surgery, but the family refused. Then, a 7-Fr double pigtail stent was placed into the cyst cavity. The amylase and lipase from the cyst fluid analysis were 27,016 and 115,450 U/L, respectively. The culture from the cyst fluid revealed Klebsiella pneumoniae, which was treated by meropenem. One month after the procedure, the cyst had resolved and there were no further recurrent attacks of pancreatitis. His last amylase and lipase values were 51 and 86 U/L. # Discussion To our knowledge, this is the first study in Indonesia, which represents the biggest Southeast Asian country, to document the usefulness of diagnostic EUS and therapeutic interventional EUS in gastroenterology practice. The study indicates that the indications for the procedure mimic those in Western countries and draw attention to rare findings. The development of EUS procedures in most Southeast Asian country is still questioned and is hampered because of lack of training facilities, necessity of hospital investment, and high cost. The purpose of this study was to show the major impact of therapeutic interventional EUS procedures in difficult and rare cases in gastroenterology practice because EUS development is still lacking in most Asian developing countries. This study confirms that the role of EUS procedure in pancreatic cysts or pseudocysts is superior to other imaging studies (transabdominal ultrasound, abdominal CT scan, and abdominal MRI) [bib_ref] Endoscopic ultrasound in the evaluation of pancreatobiliary disorders, Larghia [/bib_ref]. The advantage of EUS procedure is the possibility for cyst aspiration for fluid analysis; this can be combined with therapeutic interventions such as stenting for drainage [bib_ref] Pancreatic pseudocyst -when and how to treat?, Aghdassi [/bib_ref]. Another common therapeutic EUS procedure is EUS-guided biliary drainage when ERCP has failed in common bile duct cannulation and stenting [bib_ref] Multicenter study on endoscopic ultrasound-guided expandable biliary metal stent placement: choice of..., Dhir [/bib_ref]. Surgical intervention remains possible for internal and external drainage, especially in complicated cases with infected pseudocyst, pancreatic duct stricture, bile duct stenosis, and perforation. Even though a randomized controlled trial has shown that there were no differences in the success rate, complications, and re-intervention between surgery and endoscopic drainage, however, the right time for surgical action, the need of resection (partial leftsided pancreatectomy or Whipple's procedure), and the surgeon's experience remain major issues not only among the physicians and surgeons, but also for the patient and his family [bib_ref] Equal efficacy of endoscopic and surgical cystogastrostomy for pancreatic pseudocyst drainage in..., Varadarajulu [/bib_ref] [bib_ref] Stent versus surgery, Gouma [/bib_ref]. The first two cases described above show that simple cyst aspiration could solve the problem without the need for immediate surgery even though long-term follow-up is needed in case of possible recurrent cyst. Increasingly, surgical avoidance has become the preferred therapy for whenever there is no suspicion or evidence of malignancy. The other two rare cases (large pancreatic pseudocyst and DDC) were also successfully managed by therapeutic interventional EUS. Follow-up again is necessary to find out whether therapeutic EUS was the final solution or whether additional surgery by an experienced team will be advisable [bib_ref] A rare case of duodenal duplication treated surgically, Uzun [/bib_ref] [bib_ref] Endoscopic treatment of a duodenal duplication cyst, Kurien [/bib_ref]. This study has given important messages for the gastroenterological practice in Indonesia by showing that EUS procedures have an important role in managing some difficult cases in daily practice. However, these procedures require high skill and experience and the presence of a high-quality multidisciplinary team capable of handling potential complications inherent to the endoscopic procedure itself. [fig] Figure 1: A large intra-abdominal benign mesenteric cyst successfully managed by aspiration needle through EUS procedure. [/fig] [fig] Figure 2: A large infected pancreatic pseudocyst successfully managed by 7-Fr double pigtail stent. [/fig] [fig] Figure 3: Duodenal duplication cyst with duodenal obstruction successfully managed by 7-Fr double pigtail stent. [/fig]

The Development and Validation of Hundred Paisa Pain Scale for Measuring Musculoskeletal Pain The reduction in the pain intensity is one of the most important outcome measures in musculoskeletal disorders. The assessment of pain required reliable and valid scale. The aims of this prospective observational study were to develop and evaluate concurrent validity and test-retest reliability of hundred paisa pain scale (HPPS) for measuring musculoskeletal pain.A consecutive 74 patients with musculoskeletal pain with a wide variety of diagnoses were enrolled. Patients reported their intensity of pain on the following scale: HPPS, ''visual analog scale (VAS),'' and ''numerical rating scale (NRS).'' Patients were asked to complete another HPPS, VAS, and NRS after 2 days to determine the reproducibility of the scales. Spearman rank correlation coefficients between the HPPS and the NRS, and VAS were used to determine the validity of the scales. The correlation between the change score of HPPS, VAS, and NRS was used to determine the responsiveness of HPPS.Results of test-retest indicate that the reproducibility of HPPS was good to excellent with the intraclass correlation coefficient (ICC) value of 0.85 (95% confidence interval [CI], 0.76-0.91). The standard error of measurement (SEM) was 5.24. The minimum detectable change based on the SEM for test-retest was 14.52. The reproducibility of VAS is moderate to good with the ICC value of 0.82 (95% CI, 0.72-0.88). The reproducibility of NRS is good to excellent with the ICC value of 0.88 (95% CI, 0.81-0.92). There was a strong correlation between the HPPS and the VAS, and NRS (P < 0.01), which confirm the validity. The HPPS was responsive as the correlation of the change score of HPPS with the change score of VAS, and NRS were good (0.80 and 0.86, respectively).The HPPS is a valid and reliable scale to assess musculoskeletal pain, with psychometric properties in agreement with other comparable scale. (Medicine 94(29):e1162) Abbreviations: HPPS = hundred paisa pain scale, NRS = numerical rating scale, VAS = visual analog scale, VRS = verbal rating scale. # Introduction T he evaluation of treatment benefits for individuals with musculoskeletal pain requires outcome measures that are reliable, valid, and responsive. [bib_ref] A comparison of the ability of two upper extremity assessments to measure..., Lehman [/bib_ref] The assessment of pain intensity is vital in clinical practice and treatment outcome research. [bib_ref] Is pain a vital sign, Salcido [/bib_ref] [bib_ref] American pain society recommendations for improving the quality of acute and cancer..., Gordon [/bib_ref] There are varieties of scale, including the ''visual analog scale (VAS),'' ''verbal rating scale (VRS),'' and ''numerical rating scale (NRS),'' are used to quantify intensity of pain. [bib_ref] Studies comparing numerical rating scales, verbal rating scales, and visual analogue scales..., Hjermstad [/bib_ref] [bib_ref] Self-report scales and procedures for assessing pain in adults, Jensen [/bib_ref] The psychometric properties of these scales have been investigated, but no single scale has consistently been shown better to the others. [bib_ref] Studies comparing numerical rating scales, verbal rating scales, and visual analogue scales..., Hjermstad [/bib_ref] [bib_ref] Self-report scales and procedures for assessing pain in adults, Jensen [/bib_ref] These 3 scales are different in a variety of aspects, for example, the number of response categories, patient preference, and administration requirements. [bib_ref] Core outcome measures for chronic pain clinical trials: IMMPACT recommendations, Dworkin [/bib_ref] Previous studies indicate that the application of the VAS in the elderly patients is difficult than the NRS, furthermore, that the elderly prefer NRS over the VAS. [bib_ref] Self-report scales and procedures for assessing pain in adults, Jensen [/bib_ref] [bib_ref] Assessment of pain in elderly people, Gagliese [/bib_ref] For example, the application of VAS requires a person to imagine his pain in terms of a mathematical dimension, a task that may be difficult, [bib_ref] Methodological problems in the measurement of pain: a comparison between the verbal..., Ohnhaus [/bib_ref] [bib_ref] Some relationships between subjective measures of pain, Woodforde [/bib_ref] especially for the elderly. In addition, previous studies found 7% to 16% higher failure rates for VAS than those for NRS or VRS. [bib_ref] The measurement of pain in intensive care unit: comparison of 5 self-report..., Chanques [/bib_ref] [bib_ref] Comparing quantification of pain severity by verbal rating and numeric rating scales, Dijkers [/bib_ref] [bib_ref] The numeric rating scale for clinical pain measurement: a ratio measure?, Hartrick [/bib_ref] The correlation between the NRS and VAS are good and both are equally sensitive for the assessment of pain, but clinical studies indicate that the NRS is superior to others, especially with elderly, less educated, and chronic pain patients. [bib_ref] The measurement of musculoskeletal pain intensity: a comparison of four methods, Gallasch [/bib_ref] [bib_ref] Psychometric evaluation of selected pain intensity scales for use with cognitively impaired..., Taylor [/bib_ref] [bib_ref] Clinical importance of changes in chronic pain intensity measured on an 11-point..., Farrar [/bib_ref] Indian population, the majority of uneducated patients describes their pain on the amount of 100 paisa. For example, 20 paisa pain out of 100 paisa. It is easy for a person to imagine his pain intensity in terms of a given paisa as they know the magnitude of the money, for example, how much pain they feel on 100 paisa. Chakraborty and Mathur have published an anecdotal report on ''Rupee scale: For measurement of pain in India.'' 17 They reported their personal experiences of assessment of postoperative pain using the ''Rupee scale.'' In addition, Kapadia-Kundu and Dyalchand has developed a numerical scale, the ''Pachod paisa scale'' to measure attitudes, intentions, emotions, patient's satisfaction, and pain.However, both these studies did not report the psychometric properties such as the validity and reliability of these scales. In addition, the psychometric analysis of these scales to assess musculoskeletal pain intensity is not reported anywhere else. Therefore the present study intended to develop and validate a hundred paisa pain scale (HPPS) for measuring intensity of musculoskeletal pain. # Methods ## Scale development The HPPS consists of 11 point horizontal scale on a sequence of paisa. The left and right of the scale is anchored by 2 end; 0 paisa means no pain at all, whereas 100 paisa describes worst pain ever. The intervening points are represented by proportionally the increasing amount of 10 paisa [fig_ref] FIGURE 1: Hundred paisa pain scale [/fig_ref]. The patients can indicate the amount of paisa that most close to the magnitude of their pain intensity. ## Participants The patients with musculoskeletal pain who were treated in the outpatient orthopedic department at the Pushpanjali Hi-tech Rehab Centre, Kolkata, India were selected in this prospective observational study by convenience sampling method. Both male and female subjects with symptomatic musculoskeletal pain, age older than 21 years were included. Subjects were excluded if they had known diabetes mellitus, neurological disease, inflammatory joint disease, and uncooperative. Initially a total of 90 subjects screened and 74 participants fulfill the inclusion criteria. The study had an approval of the Institutional Ethics Committee (IEC) of the Rehabilitation Research Chair of King Saud University and each participant had given a written informed consent. ## Procedures A brief instruction about the procedure before completing the scales was given to the participants. The demographic data, including age, gender, height, weight, and body mass index (BMI), was recorded. The baseline measurement of pain intensity of each patient was taken by the HPPS, VAS, and NRS. The correlation between the baseline score of the HPPS, VAS, and NRS were used to determine the concurrent validity of the scale. The order of administration of 3 scales for each patient was determined by computerized randomization performed before the commencement of the study. Patients were asked to complete another HPPS, VAS, and NRS after 2 days to determine the reproducibility of the scales. The correlation between the change score of HPPS, VAS, and NRS was used to determine the responsiveness of HPPS. Usually in the reliability study, the responsiveness was assessed using the prevalidated instruments as an external criterion of change. [bib_ref] The validation of visual analogue scales as ratio scale measures for chronic..., Price [/bib_ref] In the present study, the VAS and NRS measurements were used as an external validation tool. Two independent investigators were involved in both sessions. During the 2nd and 3rd assessment, the participants were unaware about their first scores. # Statistical analysis SPSS for Windows version 19 (Statistical package for the Social Sciences, IBM, Inc. Chicago, IL, USA) was used to analyze the data. The normality of the data was tested using the Shapiro-Wilk test. The data were not normally distributed (P < 0.05). The test-retest reliability of an HPPS, VAS, and NRS were assessed using intraclass correlation coefficients (ICC 2, 1). The Bland-Altman plot method was used to assess the agreement between 2 readings. The average of 2 readings on the x-axis was plotted with the difference of each pair of readings on the y-axis [fig_ref] FIGURE 2: Bland-Altman plot [/fig_ref]. Data were visually interpreted to determine the consistency of 2 scores. The concurrent validity was tested using the Spearman correlation coefficients between the baseline scores of HPPS and the VAS and NRS. The responsiveness was tested using the Spearman correlation coefficients between the change score (end of intervention À baseline) of HPPS, VAS, and NRS. The calculation of the standard error of measurement (SEM) and the minimum detectable change (MDC) was done based on the results of the reliability analyses. The SEM was calculated as SDH(1 À r), where r is the reliability coefficient and SD is the standard deviation of the scores. 20,21 MDC was calculated as 1.96H2 (SEM).The level of significance in all tests was P < 0.05 with 95% confidence interval (CI). # Results ## Test-retest reliability Test-retest results are given in [fig_ref] TABLE 3: Test-Retest Reliability of [/fig_ref] , showing that the reproducibility of HPPS is good to excellent with the ICC value of 0.85 (95% CI, 0.76-0.91). The reproducibility of VAS is moderate to good with the ICC value of 0.82 (95% CI, 0.72-0.88). The reproducibility of NRS is good to excellent with the ICC value of 0.88 (95% CI, 0.81-0.92). The Bland-Altman limit of agreement of HPPS is depicted in [fig_ref] FIGURE 2: Bland-Altman plot [/fig_ref] showing a reasonable agreement between the test-retest when differences between the 2 readings is plotted against the mean of 2 readings. ## Concurrent validity A good positive correlation between HPPS and the VAS (r ¼ 0.855), and excellent positive correlation between HPPS and the NRS (r ¼ 0.918) were noted. This is shown in [fig_ref] TABLE 4: Validity Analysis [/fig_ref]. ## Responsiveness The change scores (end of intervention À baseline) of HPPS, VAS, and NRS were used to determine the responsiveness of the scale. The correlation between change scores of HPPS and VAS were 0.80 (rho) (P < 0.01). The correlation between change scores of HPPS and NRS were 0.86 (rho) (P < 0.01). ## Measurement error and minimum detectable change The SEM was 5.24, based on repeated measurements for test-retest. The MDCs based on the SEM for test-retest was 14.52 [fig_ref] TABLE 5: SEM, MDC, and Bland and Altman Tests for HPPSSEM MDCBland and Altman... [/fig_ref]. # Discussion The aim of the present study was to examine the psychometric properties including reliability and validity of HPPS and compare them with 2 other commonly used scales, namely the VAS and NRS. The result of the present study indicates that the reproducibility of the HPPS, VAS, and NRS were good to excellent, moderate to good, and good to excellent, respectively. In the present study, the reliability of HPPS had an ICC value 0.85, which was lower than the NRS (ICC ¼ 0.88) and higher to that of the VAS (ICC ¼ 0.82). However, in the previous study of musculoskeletal pain the VAS and NRS had little higher ICC values (0.97 and 0.99, respectively). 14 In the latter, the sample consisted of group of orthopedics patients and limited to low education level. However, in the present study, there was no limitation of educational level. Ferraz et al 23 had reported a high ICC values for literate as compared to illiterate patients with rheumatoid arthritis. A correlation coefficient of more than 0.75 is required to consider a valid instrument. [bib_ref] The validation of visual analogue scales as ratio scale measures for chronic..., Price [/bib_ref] In the present study, the HPPS had high correlation coefficients (r ¼ 0.85 À 0.91) with the NRS and VAS [fig_ref] TABLE 4: Validity Analysis [/fig_ref] , indicates strong consistency in pain scores between these scales. The results of present findings indicate that the HPPS is a valid measure of the musculoskeletal pain level. The HPPS appears to measure pain intensity similar to the VAS and NRS and may provide additional advantages for the Indian population as this scale is easy to understand by this population. In previous studies, the correlation between the NRS and VAS was high in patients with rheumatic and other chronic pain conditions. [bib_ref] Reliability of pain scales in the assessment of illiterate patients with rheumatoid..., Ferraz [/bib_ref] [bib_ref] Studies with pain rating scales, Downie [/bib_ref] In the present study, we found a strong correlation between the NRS and VAS (r ¼ 0.91). In the present study, the correlation between the change score of HPPS, VAS, and NRS was used to determine the responsiveness of HPPS. The results of the present study indicate that the correlation of change score of HPPS with the change score of VAS and NRS was good (0.80 and 0.86, respectively). A correlation score of 0.70 or more is acceptable for instruments that measure the same construct. [bib_ref] Self-report outcome measures for low back pain-searching for international cross-cultural adaptations, Costa [/bib_ref] Therefore HPPS give similar responses as compared to VAS and NRS in assessing musculoskeletal pain. # Study limitations The present study involves some potential limitations. The generalizability of present results may be limited due to the heterogeneity in the samples (eg, participants had pain of different etiologies). In addition, the present study did not consider the chronicity of the pain (ie, acute vs. subacute vs. chronic). Therefore, we recommend that future studies be conducted to compare the chronicity of pain using HPPS and use a homogeneous patient population. .08 À19.28 to 24.14 95% CI for p ¼ 95% confidence interval for the mean difference, p ¼ mean difference, HPPS ¼ hundred paisa pain scale MDC ¼ minimal detectable change, SD diff ¼ standard deviation of the differences, SEM ¼ standard error of measurement. [fig] FIGURE 1: Hundred paisa pain scale (HPPS). [/fig] [fig] FIGURE 2: Bland-Altman plot: intraindividual differences (n ¼ 74) between the hundred paisa pain scale on test and retest, plotted against the average of the 2 scores. The central line represents the mean difference and the dashed lines display the 95% LOA. [/fig] [table] Table 1: details the participants' characteristics. The mean age and standard deviation of the sample was 46.08 and 16.15 years, respectively. The sample consisted of 32 female and 42 male participants.Table 2details the baseline score of HPPS, VAS, and NRS. [/table] [table] TABLE 2: Descriptive Statistics of Baseline Scores HPPS ¼ hundred paisa pain scale, NRS ¼ numerical rating scale, SD ¼ standard deviation, VAS ¼ visual analog scale. [/table] [table] TABLE 3: Test-Retest Reliability of (HPPS), VAS, and NRS ICC (95% CI) 95% CI ¼ 95% confidence interval, HPPS ¼ hundred paisa pain scale, ICC ¼ intraclass correlation coefficient, NRS ¼ numerical rating scale, VAS ¼ visual analog scale. [/table] [table] TABLE 4: Validity Analysis: Correlation Matrix of HPPS, VAS, and NRS Similarly, Bijur et al 25 have demonstrated strong correlation between the NRS and VAS (r ¼ 0.94). [/table] [table] TABLE 5: SEM, MDC, and Bland and Altman Tests for HPPSSEM MDCBland and Altman Test [/table]