key: cord-347816-e2xkzps9 authors: wesemann, u.; hadjamu, n.; willmund, g.; dolff, s.; vonderlin, n.; wakili, r.; vogel, j.; rassaf, t.; siebermair, j. title: influence of covid-19 on general stress and posttraumatic stress symptoms among hospitalized high-risk patients date: 2020-08-14 journal: psychological medicine doi: 10.1017/s0033291720003165 sha: doc_id: 347816 cord_uid: e2xkzps9 nan to the editor: a recently published study found a 96.2% prevalence rate for significant posttraumatic stress symptoms in quarantined patients diagnosed with covid-19 (bo et al., 2020) . in addition, various psychiatric disorders have been found in patients in the early stages of the covid-19 outbreak (talevi et al., 2020) . thereby, the rates for posttraumatic stress disorder (ptsd) in covid-19 affected patients appear higher when compared to ptsd rates in the general population or in emergency service staff after terrorist attacks (11% to 25%; berninger et al. 2010; brackbill, hadler, digrande, ekenga, & farfel, 2009; schlenger et al. 2002; wesemann et al., 2018b; wesemann, mahnke, polk, & willmund, 2020) . in addition, the rates established by bo et al. were significantly higher than those in previous studies by fang, zhe, and shuran (2004) or mak, chu, pan, yiu, and chan (2009) on patients with the severe acute respiratory syndrome (sars). the aim of this study was to determine the prevalence of 'general stress' and severe posttraumatic stress in patients who were hospitalized with the initial suspect of a covid-19 infection. the suspicion of covid-19 was considered a critical event for mental health, since the vast majority of the patients also had comorbidities that were known to translate in a worse outcome together with a covid-19 infection (clark et al., 2020) . the second goal was to compare patients with and without definitive confirmation of a covid-19 diagnosis for general stress and posttraumatic stress. our hypothesis was that there was no difference in patients with and without covid-19, since we assumed that risk perception was the same in both groups. all patients with suspect of a covid-19 infection who were admitted to the university hospital of essen between 8 march and 26 may were encouraged to participate in the study. after hospital admission, they were provided with the ptsd checklist for dsm-5 (pcl-5; weathers et al., 2013) and the patient health questionnaire (phq) stress module (löwe, spitzer, zipfel, & herzog, 2002) . at this stage, patients and treating medical staff were not aware of their covid-19 infection status which resulted in a double-blinded design of the study. written informed consent was given by all participants and the study was approved by the local ethics committee of the university of essen medical school (irb number 20-9263-bo). a total of n = 60 patients with an average age of 59 ± 17.8 years were included (n = 33, men). the diagnosis of covid-19 was later confirmed in n = 19 (31.7%). about half of the patients included presented with dyspnea, especially dry coughing, one of the leading symptoms of the sars-cov-2 infection (jiang et al., 2020; tabata et al., 2020) . chi-squared testing showed no sample bias between the groups with and without covid-19. a detailed list of comorbidities is provided in table 1 . the prevalence of significant posttraumatic stress symptoms was 37.9% [95% confidence interval (ci) 35.5-40.3 in all patients and 42.1% (95% ci 37.2-47.0) in the subgroup of patients with later confirmation of a covid-19. the 2 × 2 chi-squared tests were performed to test the second goal: regarding patients with v. without covid-19 there was no significant difference in the dichotomized severity of posttraumatic stress symptoms: χ 2 (1, n = 58) = 0.21, p = 0.647. accordingly, there were no significant correlations between the groups with or without covid-19 and the pcl-5 scales. however, there was a significant correlation (r = 0.35; p = 0.019) between the diagnosis of covid-19 and stress of the phq stress module. in a subsequent chi-squared test with the dichotomized values, there was a significant difference between the groups: χ 2 (1, n = 50) = 5.56; p = 0.018 with more patients with covid-19 in the high general stress group. we could not replicate the findings of bo et al. (2020) with prevalence rates of 'significant posttraumatic stress symptoms' far above 90%. nevertheless, our established prevalence rates are still high when compared to other studies (berninger et al., 2010; brackbill et al., 2009; schlenger et al., 2002) . this can be partly explained by the highrisk population examined in our study. chronic lung diseases, severe cardiovascular diseases or oncological diseases in combination with suspected covid-19 may have had a negative impact on the severity of ptsd symptoms in our patients. these results clearly support the hypothesis that patients with covid-19 develop a higher level of general stress compared to patients without covid-19. in this case, stress would not only be a psychological factor, but could also have an organic correlate. it is also conceivable that the high level of stress in patients with covid-19 could be a predictor of the later onset of posttraumatic stress symptoms after the diagnosis has been received. routine screening for mental fitness in this high-risk population is recommended, as is the case in the military (peterson, park, & castro, 2011; wesemann et al., 2018a) . the influence of covid-19 on general and posttraumatic stress should receive a special focus and might be adapted to diseases with a similar pandemic character. longitudinal study of probable post-traumatic stress disorder in firefighters exposed to the world trade center disaster posttraumatic stress symptoms and attitude toward crisis mental health services among clinically stable patients with covid-19 in china asthma and posttraumatic stress symptoms 5 to 6 years following exposure to the world trade center terrorist attack … centre for the mathematical modelling of infectious diseases covid-19 working group (2020). global, regional, and national estimates of the population at increased risk of severe covid-19 due to underlying health conditions in 2020: a modelling study survey on mental status of subjects recovered from sars (in chinese) review of the clinical characteristics of coronavirus disease 2019 (covid-19) phq-d gesundheitsfragebogen für patienten longterm psychiatric morbidities among sars survivors assessment for the u.s. army comprehensive soldier fitness program: the global assessment tool psychological reactions to terrorist attacks: findings from the national study of americans' reactions to clinical characteristics of covid-19 in 104 people with sars-cov-2 infection on the diamond princess cruise ship: a retrospective analysis. the lancet. infectious diseases mental health outcomes of the covid-19 pandemic the ptsd checklist for dsm-5 (pcl-5) long-term effects of the terror attack in berlin in 2016 on paranoid ideation in female emergency personnel assessing psychological fitness in the military -development of an effective and economic screening instrument burdens on emergency responders after a terrorist attack in berlin. occupational medicine acknowledgements. we would like to thank the hospital staff that was involved in data collection for the present study.financial support. this research received no specific grant from any funding agency, commercial or not-for-profit sectors.conflict of interest. none. key: cord-030805-3imi63xz authors: lodha, surabhi; gupta, rashmi title: book review: stress less, accomplish more: meditation for extraordinary performance date: 2020-08-07 journal: front psychol doi: 10.3389/fpsyg.2020.01830 sha: doc_id: 30805 cord_uid: 3imi63xz nan while browsing through the internet, the authors tried to find a brief meditation technique to practice during the covid-19 pandemic. they came across the videos of emily fletcher teaching ziva meditation online. it drove them to her book titled "stress less, accomplish more." emily fletcher is the founder of ziva meditation and the creator of zivaonline, the world's first online meditation training program. a powerful meditation practice transitioned her from being a young but anxious and insomniac broadway actress to a dedicated practitioner and a flourishing meditation professional. in this book, she introduces a new technique called "z-technique, " adapted from her live, in-person ziva meditation. this technique is specially designed for high-performing, outcome-driven individuals, teaching them to be less stressed while increasing productivity and achieving success. the first chapter points out that people often cannot meditate because of super-busy schedules, fear of failure (the meditation shame spiral), or vagueness about the practice. it educates on how and how not to approach meditation. while introducing the z-technique, the author argues that investing only 25 min twice a day to this technique could significantly improve the remaining hours qualitatively. chapters two and three explain that meditation gives us the control to regulate our response to perceived stress. the long-term effect of stress from work, family, relationships, etc., is toxic and debilitating, manifested in decreased work performance, reduced attention span, irritability, mood swings, etc. the concept of adaptation energy is reintroduced here (gorban et al., 2016) as a psychological measure of one's stress-coping capacity. this reservoir could be detoxified from accumulated stress and abundantly replenished with energy by de-exciting the nervous system through meditation. the fourth chapter further argues that this internet era, which equates rest or relaxation with laziness and stagnancy, has seen a spike in sleep-related problems like insomnia and restlessness. the author asserts that both sleep and meditation are equally crucial for a healthy life. while sleep cleans our brain by cleaning out toxic by-products (xie et al., 2013) , meditation rests our entire body, including the brain. adding to the further benefits of mediation, in the fifth and the sixth chapters, it is suggested that meditation revamps the body by eliminating the built-up stress in the form of disturbed sleep, organ inflammation, chronic acidity, dullness, and pain. moreover, regular meditation practice improves immune functions and treats disorders like burnout and chronic fatigue, depression, anxiety, infertility, irritable bowel syndrome, migraines, parkinson's disease, pain, etc. it improves longevity and quality of life combined with ayurveda-proper diet, exercise and yoga practices, and environmental harmony. the above claims are supported by providing neurological evidence like changes in the right and left-brain, amygdala, insula, corpus callosum, and telomere. the seventh chapter posits that, unfortunately, contemporary culture is built on the "i'll be happy when syndrome, " which is an abstract idea that one's happiness is based on future achievements. this far-fetched pursuit is exhausting and bewildering. but luckily, meditation helps us pull out of the future, settles into the present, and instills the fact that bliss and contentment are within us, independent of external situations. the explanation of the harmful effects of stress and how meditation eradicates them is followed by the description of ziva or the z-technique in chapter eight. ziva is born out of nishkam karma yoga of indian spiritual traditions, which requires no focused or effortful concentration or a forcible mind clearing (diwan and kamra, 2018) . the z-technique is a sequenced combination of three "ms": mindfulness, meditation, and manifesting. this simple 25-min technique begins with mindfulness of 2-3 min (aware and completely present), similar to the "open awareness meditation" style (lutz et al., 2008) . it is followed by 14-16 min of meditation (healing from the past), which includes the sub-vocalization of an impersonal word like "one." the author calls this "whisper of an echo, " which lets our body and mind drift into a deeply relaxed state spontaneously. the technique ends with 2-3 min of manifesting (consciously creating and planning our future). one offers gratitude to everything, accompanied by seeing dreams as unfolding in the present, and not merely magical thinking. the ninth and tenth chapters further explain that the ztechnique enables us to detect the subtleties and patterns in our daily lives as it expands consciousness, making us more attuned to the sensations, thoughts, and feelings. hence, we become more intuitive and insightful and able to enter the "flow state." this unveiling of full potential is termed as "up-leveling, " marked by extraordinary performance and fulfillment. the subsequent chapters explore the prospect of meditation as a tool for the development of collective consciousness. if one individual consciously learns to break the old habits by finding a gap between the trigger and impulsive reactions, this transformation in consciousness permeates other beings. keeping these in mind, we must make the z-technique a nonnegotiable daily practice. the author claims that, ultimately, this helps us become good in every sphere of life. for the contemporary fast-paced and stress-ridden society, the book serves as a foundational and practical guide for people who want to improve their physical and mental well-being but don't know where to begin. it is an amalgamation of ancient meditation practices, modern neuroscience, and pop psychology sans metaphysics or spirituality. it makes meditation accessible and understandable to all, not just high-achievers. the repetitive explanation of stress and lack of empirically investigated data to validate the technique is a bit unsettling. however, the book is simple, refreshing, and rewarding. in conclusion, the book addresses how meditation could remarkably improve productivity and efficiency in an overly stressed modern world. it emphasizes that meditation is much more than the austerity of a himalayan yogi. in meditation research, the issues of universality and secularity of a technique are not thoroughly addressed. such streamlined meditation practice could be easily adapted by anyone who has struggled with commencement, commitment, and consistency. the current need is to give equal emphasis to study such techniques of "meditation for the ordinary." the philosophy of "nishkam karma evolution of adaptation mechanisms: adaptation energy, stress, and oscillating death attention regulation and monitoring in meditation sleep drives metabolite clearance from the adult brain all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. 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.copyright © 2020 lodha and gupta. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-279214-7vna4uyo authors: goldfarb, elizabeth v. title: participant stress in the covid-19 era and beyond date: 2020-09-25 journal: nat rev neurosci doi: 10.1038/s41583-020-00388-7 sha: doc_id: 279214 cord_uid: 7vna4uyo the covid-19 pandemic represents a worldwide stressor. considering the influence of stress on research participants during this time and beyond may provide new insights and benefit the broader field of human neuroscience. as human neuroscience researchers, we invite people, with their disparate backgrounds, to participate in experi ments to help us uncover fundamental truths about the brain. we are now in the midst of a global pandemic that is changing our lives and the lives of our participants. for those of us who are currently able to work with human participants (in person or online), stress related to the pandemic may have farreaching and unanticipated effects on the data collected during this period. although the pandemic highlights the importance of stress as a factor in everyday life, stress and its neuro biological sequelae are not unique to the pandemic. indeed, stress can be routinely considered and measured in human neuroscience research. however, outside studies explicitly designed to measure stress or affec tive processes, stress is not typically considered. here, i discuss why and how stress should be considered in the design and interpretation of human neuroscience studies during the pandemic and moving forward. the stress of a pandemic stress is classically described as a response to something novel, unpredictable and uncontrollable -all features of the covid19 pandemic. the fear of illness for ourselves and our loved ones for an unknown period of time has been combined with, for example, prolonged disruptions to daily routine, education, child care, occupation and income that have resulted from efforts to limit the spread of the virus 1 . in the united states, these challenges have been disproportionately faced by black and indigenous people and other people of colour, who are also systema tically exposed to more stressful experiences 1,2 . isolation and lack of social support can also trigger maladap tive coping behaviours such as increased substance use that can potentiate risk for covid19 related complications 3 and interact with stress pathways to alter brain circuitry and cognitive function 4 . if having participants put their arm in a bucket of icy water for a few minutes (a common laboratory stress induction protocol) is sufficient to change participants' behaviour and brain responses 5 , one can imagine the scale of the impact that covid19 related stress may have on the data being collected now. stress leads to changes in neuronal structure and function throughout the brain. these effects are espe cially pronounced when a stressor is experienced repeat edly or over a long time period, known as chronic stress (which may characterize participants' experiences of the pandemic). research across different species has shown that chronic stress impairs prefrontal and hippocampal circuits (inducing dendritic atrophy and spine loss), and strengthens responses in the amygdala 6, 7 . these changes occur together with effects on myriad cognitive pro cesses, including attention, reward processing, learning, working memory, long term memory, decision making, strategy selection and top down regulation of emotion 8 . the clinical research community has emphasized the importance of considering the pandemic as a multi dimensional stressor to understand mental health consequences 1 . here, i suggest that this consideration extends beyond clinical science: all the data that are cur rently being collected from human participants are likely to be influenced by this sustained stress exposure. when designing studies to examine stress effects, a 'control group' is typically included to match a 'stress group' in as many ways as possible, short of the stress exposure. this enables us to draw conclusions about the specific con tributions of stress to the behavioural construct or neural process of interest. however, as practically all humans are now exposed to the pandemic, there is no temporally matched control group (although longitudinal studies may be able to consider pre pandemic versus post pandemic differences). crucially, experiences of and reactions to the challenges associated with the pandemic are highly vari able. for example, one participant may have been socially isolated yet had no changes in their employment; another may have grieved the loss of a loved one, taken on new caregiving roles, experienced income loss and increased their alcohol use; and yet another may have had fewer commitments and enjoyed more time with family. to better understand human data, and how they may be modulated by stressors like the pandemic, researchers can leverage tools developed by the stress field 9 . subjective measures. several well validated instruments have been developed to measure the number of stressors people experience and the extent of psychological dis tress they cause, typically including an assessment of when they occurred during the lifespan 10 . these can be implemented using self report or interviews by trained personnel. more recently, techniques such as ecolo gical momentary assessment (ema) use smartphone prompts to track daily fluctuations in experiences of and responses to stressful events. coping behaviours are also relevant to measure, as some responses, such as escala ting alcohol or drug use, can in turn influence behaviour and brain responses 4 . although self report measures rely on conscious insight and willingness to describe what may be stigmatized responses, they can help to predict later health outcomes 9 and can feasibly be incorporated into online and in person experiments. testing kits are widely used to measure stress related hormones in samples of saliva, plasma or hair. although salivary and plasma cortisol are frequently used to measure acute stress responses in the laboratory, they can also provide insight into more prolonged stress states. for example, basal cortisol levels at different times of day are altered with past stress exposure 10 , stress related psychopathology 8 and chronic drug use 4 . although obtaining saliva and plasma (poten tially infectious agents) may be challenging during the pandemic, these measures are routinely used in the stress field, providing a rich comparison data set against which to interpret new data. the body's stress response can also be assessed through measures of autonomic function. tools to measure autonomic responses include arm cuffs to quantify systolic and diastolic blood pressure, as well as electrocardiograms (ecgs) that can provide ambulatory indices of pulse and heart rate variability. these auto nomic functions also adapt with chronic stress and drug use 4, 9 . given the lack of need for bodily fluid samples to assess autonomic responses, such measures would prob ably be more practicable than neuroendocrine analyses during the pandemic. acute stress challenges. laboratory stressors are frequently used as experimental interventions and can also be used as assays for adaptations in acute stress responses result ing from chronic stress exposure. participants' reactivity profiles to acute stress can be heightened, blunted or show atypical recovery patterns as a function of their stress histories and biological backgrounds 9 . there are various techniques for inducing stress responses in the labora tory, including physical, psychological and cogni tive chal lenges 5 . by measuring subjective, neuroendocrine and/or autonomic responses before and after such procedures, researchers can quantify acute stress reactivity. beyond studies explicitly designed to examine effects of stress, obtaining stress measures provides opportunities for novel insights into brain and behavioural data. careful assessment of individuals' stress profiles may enable researchers to account for unanticipated sources of variance. this approach can also allow researchers to interpret indivi dual differences and divide participants into meaning ful cohorts, leading to the discovery of novel ways in which neural and behavioural processes are associated with stress. the need to consider the affective and physiological context in which people participate in research does not end with the end of covid19. we cannot assume that participants always complete our experiments in a neutral state, or that they have comparable histo ries of stressful experiences. individual differences in stress and coping might explain hitherto unknown boundary conditions for reproducing effects in human neuro science, and improve ecological validity by indicating how these processes may occur in the stress rich context of everyday life. going forward, participant level stress profiles can include assessment of remote stress experiences (including the pandemic) as well as in the experimental moment stress reacti vity. broadly consi dering potential stress effects provides opportunities to identify more brain functions that may be susce ptible to stress, elucidate neural factors that determine whether stress is associated with enhanced or impaired function 5 and uncover protective processes that promote resilience. knowing what we do about how stress alters fundamental neural processes, examining the effects of the pandemic and other stressors on data from human participants creates an opening to gain crucial insight into the mechanisms of the human brain. mental health and clinical psychological science in the time of covid-19: challenges, opportunities, and a call to action assessment of covid-19 hospitalizations by race/ethnicity in 12 states collision of the covid-19 and addiction epidemics drug-induced stress responses and addiction risk and relapse enhancing memory with stress: progress, challenges, and opportunities stress weakens prefrontal networks: molecular insults to higher cognition stress effects on neuronal structure: hippocampus, amygdala, and prefrontal cortex the effects of chronic stress on the human brain: from neurotoxicity, to vulnerability, to opportunity more than a feeling: a unified view of stress measurement for population science effects of stress throughout the lifespan on the brain, behaviour and cognition the author gratefully acknowledges m. d. rosenberg and d. v. clewett for helpful discussions. the authors declare no competing interests. key: cord-294422-hsqphc3t authors: sandesh, ram; shahid, wajeeha; dev, kapeel; mandhan, nikeeta; shankar, prem; shaikh, anam; rizwan, amber title: impact of covid-19 on the mental health of healthcare professionals in pakistan date: 2020-07-02 journal: cureus doi: 10.7759/cureus.8974 sha: doc_id: 294422 cord_uid: hsqphc3t introduction as a result of the ongoing covid-19 pandemic, health care professionals (hdps) are facing immense strain due to the heavy load of cases. in many cases, they work increasingly long hours, often with limited resources and a dubious infrastructure. thus, it is important to check on the mental health of caregivers. methods and materials this cross-sectional study was conducted in may 2020, at various hospitals in karachi, pakistan. all hcps posted in the covid-19 isolation wards were invited to participate and a total of 112 completed this study. a carefully structured form was created, which included the depression anxiety stress scale-21 (dass-21). results the overall mean score of anxiety was 19.01 ± 9.2, depression was 18.12 ± 10, and stress was 20.12 ± 12.0. there were 81 (72.3%) participants who suffered from moderate to extremely severe depression, 96 ( 85.7%) participants who suffered from moderate to extremely severe anxiety, and 101 (90.1%) participants who reported moderate to extreme stress levels conclusions it is evident that there are a high number of healthcare workers affected by various psychological ailments such as anxiety, stress, and depression. it is important that the government take steps to ensure that hcps' mental health is regularly checked and that efforts are made to reduce their burdens. , also known as coronavirus disease, was first identified in wuhan, china, in late 2019. it is a highly contagious disease with a rapidly rising incidence globally. the world health organisation (who) recognized it as a pandemic due to its widespread transmission [1] . as is the standard procedure in such pandemics, a lockdown is usually enforced to limit the spread of the disease and reduce potentially new cases by maintaining social distancing in all public places [2] . the general public undertakes such safety measures, but health care professionals (hcps) are unfortunately left exposed to deal with the many issues that arise due to this situation. firstly, due to a huge load of cases as a result of the pandemic, health care professionals face increasingly long work hours, often with limited resources and a dubious infrastructure [3] . secondly, they face physical discomfort and sometimes even breathing difficulties while wearing personal protective equipment (ppe), which is essential to keep them safe from exposure to [4] . another big problem faced by the hcps is that because little is known about the new virus/disease, and therefore, no established protocols or evidence-based clinical treatments are prevalent, many hcps feel unprepared to carry out their duties [4] . and then there is a very valid fear of autoinoculation and the risk of spreading the virus to their family and friends [5] [6] . this fear leads hcps to isolate themselves from their families, alter their daily routines, and even reduce their social support system, all in the hopes of keeping everybody potentially safe from themselves [4] . unsurprisingly, all these factors take a toll on the mental health of hcps. in 2003, during the outbreak of severe acute respiratory syndrome (sars), 18-57% of hcps suffered severe emotional problems and psychiatric symptoms both during and after the breakout [7] . in 2015, when the middle east respiratory syndrome (mers) emerged, also caused by a coronavirus, hcps suffered dysphoria and stress [7] . many other studies have reported that mental health implications for professionals involved in healthcare during epidemics and pandemics are long-lasting. even after some time had transpired after such events, high levels of stress, anxiety, depression, and even post-traumatic stress disorder (ptsd) were observed in many cases [8] [9] . therefore, it is extremely important to identify the hcps who are at high risk of burnout and are more likely to suffer from anxiety, depression and stress in this pandemic, so that help can be provided where and when needed. it is also equally important to identify and address the factors responsible for this stress. in this study, we will establish the frequency of hcps affected by anxiety, depression, and stress, as well as determine the causative factors behind them. this cross-sectional study was conducted in may 2020, at various hospitals of karachi, pakistan. all health care professionals (hcps) posted in the isolation wards for covid-19 were invited to participate. a total of 112 hcps completed this study. a carefully structured form was created, which included the english version of the depression anxiety stress scale -21 (dass-21). dass-21 is a 21-item self-report validated tool designed to measure the three related negative emotional states, which are: depression, anxiety, and stress [10] . the online form also included various reasons that hcps thought were predisposing factors for anxiety, stress, and depression. the participants could choose multiple reasons as applicable to themselves. the collected data were analyzed using spss version 21.0 (ibm corp, armonk, nj). mean and standard deviation (sd) were calculated for the scores of anxiety, stress, and depression. frequencies and percentages were calculated for the severity of dass-21 and reasons predisposing healthcare professionals to anxiety, stress, and depression. out of the 112 health care professionals (hcps) who participated in this study, 64 (57.1%) were male, and 48 (42.9%) were female. the overall mean score of anxiety was 19.01 ± 9.2, depression was 18.12 ± 10, and stress was 20.12 ± 12.0. there were 81 (72.3%) participants who suffered from moderate to extremely severe depression, 96 ( 85.7%) participants who suffered from moderate to extremely severe anxiety, and 101 (90.1%) participants who reported moderate to extreme stress levels ( table 1) . the study found that the most common reason for stress and anxiety among hcps treating covid-19 positive patients was the fear that they might infect their family members (89.2%), followed by the fear of getting infected themselves (80.3%). other reasons were: increased workload (64.2%), lack of ppe (62.5%), lack of security (62.5%), and lack of awareness among the general population about covid-19 (46.4%) ( the psychological response of hcps to an epidemic could be dependent on many factors which may cause anxiety and stress such as feeling vulnerable to infection, lack of control over the situation, the spread of the virus, health of their family, and being isolated [11] . other factors may also contribute to stress and anxiety among hcps such as shortage of ppes, medicines, etc. and an increasing number of actual and suspected cases [12] . these factors may contribute to various levels and severity of psychological pressure, which may lead to a feeling of loneliness and helplessness and may result in stress, anxiety, irritability, mental fatigue, and depression [4] . in this study, a significant number of pakistani participants had anxiety, stress, and depression. around 89% of hcps were afraid for their family, and 80% of hcps feared that they might get covid-19 themselves. these figures can be compared to wuhan, where among hcps looking after covid-19 [13] . factors such as improper infrastructure for patient care, lack of awareness among the masses, and poor compliance with safety measures can be accredited to this high prevalence of anxiety, depression, and stress in pakistan. another important thing to note is the persistence of mental health implications for healthcare workers. even after some time had passed after an epidemic, many studies reported that high levels of anxiety, depression, stress, and even ptsd were observed in many hcps [8] . in addition, the hcps who are directly involved in the treatment of a highly contagious disease such as the covid-19 may suffer a stigma whereas, at the other end of the spectrum, this pandemic has led to hcps being given the 'superhero' status. on the one hand, this adds value and gratification to the job, but on the other, it puts more pressure on the workers, leaving no margin for error. due to the sensational character of this worldwide pandemic, the 'superhero' status is being reinforced by the media, promoting the need for emotional support, encouragement, and appreciation [14] . in light of the results of this study, it is clear that an alarmingly high number of healthcare workers are the victims of mental health ill-effects caused by the covid-19 pandemic. it can be also seen that many of the factors due to which hcps suffer from these ill-effects can be potentially modified, for example, such as free provision of ppes to all healthcare workers, promoting general public awareness about covid-19, and building better infrastructure to encourage lighter work hours can all be carried out by the government and thus have a positive impact on the mental health of hcps. in our study, high levels of anxiety, stress and depression among health care professionals were noted, which is a cause for concern. both the government and health care agencies are responsible for protecting the psychological well-being of health care communities all over the world and ensuring a healthy work environment. since there is a high prevalence of anxiety, depression, and stress among hcps treating covid-19 patients, it is imperative to invest resources to promote the mental health welfare of frontline professionals. human subjects: consent was obtained by all participants in this study. civil hospital, karachi issued approval ch/20/04a. animal subjects: all authors have confirmed that this study did not involve animal subjects or tissue. 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. novel coronavirus (2019-ncov) situation reports isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus (2019-ncov) outbreak public responses to the novel 2019 coronavirus (2019-ncov) in japan: mental health consequences and target populations mental health survey of 230 medical staff in a tertiary infectious disease hospital for covid-19 the mental health of medical workers in wuhan, china dealing with the 2019 novel coronavirus timely mental health care for the 2019 novel coronavirus outbreak is urgently needed psychological impact of the 2015 mers outbreak on hospital workers and quarantined hemodialysis patients vicarious traumatisation in the general public, members, and non-members of medical teams aiding in covid-19 control pandemic fear" and covid-19: mental health burden and strategies safarizadeh mm: stress, anxiety and depression among medical university students and its relationship with their level of happiness the psychological impact of severe acute respiratory syndrome outbreak on healthcare workers in emergency departments and how they cope severe acute respiratory syndrome (sars) and healthcare workers factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019 recomendações para o bem-estar emocional da equipe multidisciplinar durante a pandemia pelo sars-cov-2 key: cord-316944-xt09q1z5 authors: preis, heidi; mahaffey, brittain; heiselman, cassandra; lobel, marci title: vulnerability and resilience to pandemic-related stress among u.s. women pregnant at the start of the covid-19 pandemic date: 2020-09-06 journal: soc sci med doi: 10.1016/j.socscimed.2020.113348 sha: doc_id: 316944 cord_uid: xt09q1z5 rationale. women pregnant during the covid-19 pandemic are experiencing moderate to high levels of emotional distress, which has previously been shown to be attributable to two types of pandemic-related pregnancy stress: stress associated with feeling unprepared for birth due to the pandemic (preparedness stress) and stress related to fears of perinatal covid-19 infection (perinatal infection stress). objective. given the well-documented harms associated with elevated prenatal stress and the critical importance of developing appropriately targeted interventions, we investigated factors predictive of pandemic-related pregnancy stress. method. between april 25 and may 15, 2020, 4,451 pregnant women in the u.s. were recruited via social media to complete an online questionnaire that included sociodemographic, medical, and covid-19 situational factors, as well as the pandemic-related pregnancy stress scale (preps). binary logistic regression was used to calculate odds ratios for high stress. results. nearly 30% of participants reported high preparedness stress; a similar proportion reported high perinatal infection stress. abuse history, chronic illness, income loss due to the pandemic, perceived risk of having had covid-19, alterations to prenatal appointments, high-risk pregnancy, and being a woman of color were associated with greater levels of one or both types of stress. access to outdoor space, older age, and engagement in healthy behaviors were protective against stress. conclusions. practices that may alleviate pandemic-related stress such as minimizing disruptions to prenatal care, ensuring access to outdoor space, and motivating engagement in health behaviors are of vital importance. particular attention is needed for more vulnerable populations including women of color, women with a history of abuse, and those with high-risk pregnancy. research focused on the short and longer-term impact of pandemic-related pregnancy stress on maternal mental and physical health, perinatal outcomes, and child development is critical to identify these effects and marshal appropriate resources to reduce them. experiencing a natural disaster or state of emergency during pregnancy is known to contribute to elevated emotional distress (brooks, previous research has identified several risk factors for elevated maternal stress, including primiparity, younger age, history of abuse, unplanned pregnancy, financial strain, being a woman of color, and pregnancy complications (see reviews by bayrampour, 2018; ibrahim & lobel, 2020). nonetheless, the specific factors contributing to women's elevated pandemic related stress have not been well-identified. recent research identified two major pandemic-related stress domains for pregnant women in the u.s., poland, israel, and germany: stress associated with feeling unprepared for birth due to the covid-19 pandemic and stress given the risks associated with elevated prenatal psychological stress and anxiety, and the critical importance of developing evidence-based, appropriately targeted interventions, we investigated which sociodemographic, medical, and situational factors are most associated with greater pandemic-related pregnancy stress among pregnant women in the u.s. and which appear to be protective. between april 25 and may 15, 2020 (six to nine weeks after the announcement of covid-19 as a global pandemic by the world health organization), 4,451 pregnant women in the u.s. 18+ years of age were recruited through social media to participate in the covid-19 pregnancy experiences (cope) study. facebook-paid advertisements targeted women in the u.s. with pregnancy-related interests. in addition, research assistants posted an identical advertisement for the study on various pregnancy-related social media groups and pages (i.e., facebook, instagram, and reddit). the advertisement included a request to share pregnancyrelated experiences during the covid-19 pandemic. participants completed the questionnaire through qualtrics, secure survey software, qualifying them for entry into a raffle for a $100 gift certificate awarded to one in every 100 participants. the study was approved by the institutional review board of stony brook university. the study questionnaire included sociodemographic factors (e.g., age, race/ethnicity), medical factors (e.g., parity, chronic illness), covid-19 situational factors (e.g., covid-19 related income loss, being diagnosed with covid-19), and the pandemic-related pregnancy we used binary logistic regression to calculate unadjusted and adjusted odds for high levels of preparedness stress and perinatal infection stress. cut-off scores (≥ 4 on the 1-5 response scale) were used to identify women most likely to suffer from moderate or severe levels of stress and to enhance the interpretability of study results. the unadjusted model reveals bivariate associations among predictors and stress outcomes, whereas the adjusted, multivariate models identify the independent association of each predictor after controlling for all other predictors. participants were on average 30.8 ± 4.7 years old, with an average gestational age of 27 weeks. participants were from all 50 states, and approximately half were primiparas (n = 2,270, 51.0%). approximately four-fifths of the sample (n = 3,651; 82%) identified themselves as non-hispanic/latino and white; one-fifth (n = 800; 18%) identified as hispanic/latino (n = 420, 9.4%), black/african american (n = 206; 4.6%), asian american (n = 128; 2.9%), native american (n = 105; 2.4%), or other (n = 134, 3.0%) (numbers and percentages exceed 4,451 and j o u r n a l p r e -p r o o f 100% because some women endorsed multiple racial/ethnic categories). fifty-three women (1.2%) reported being diagnosed with covid-19 during pregnancy, and one-third (n = 1,483, 33.3%) thought they might have contracted covid-19 during pregnancy but were not diagnosed. nearly half of the sample (n = 2,037, 45.8%,) lost income due to the pandemic, and 53.4% (n = 2,376) reported having prenatal appointments cancelled or rescheduled due to the pandemic. a large majority (n = 4,002, 89.9%) reported having access to outdoor space "whenever i want" or "sometimes" (versus rarely), and over half of the women (n = 2,492, 56.0%) reported that they were involved in healthy activities (e.g., "eat well, take vitamins, exercise, sleep enough"), scoring at least 4 on the 1 = very little to 5 = very much response scale for this item. nearly 30% of participants reported high levels of preparedness stress (27.2%) and perinatal infection stress (29.1%), with 17.9% of the sample reporting high levels of both. as can be seen in table 1 , in the unadjusted model, preparedness stress was predicted by an assortment of sociodemographic, medical, and covid-related situational factors and perinatal infection stress was predicted by a slightly different constellation of factors. common risk factors across the unadjusted models predicting preparedness stress and perinatal infection stress included sociodemographic vulnerability (e.g., experiencing previous abuse, below average financial status, being a woman of color, not having private insurance) and medical vulnerability (e.g., chronic illness, high-risk pregnancy status). having access to outdoor space and engaging in healthy prenatal behaviors were associated with lower odds of both types of stress in the unadjusted models. in the adjusted model, high preparedness stress was independently predicted by nine factors: previous abuse, primiparity, unplanned pregnancy, self-reported high-risk pregnancy j o u r n a l p r e -p r o o f status, multiple pregnancy, chronic illness, covid-19 related income loss, perceiving that one had been infected, and experiencing alterations of prenatal care (aors = 1.19-1.78). older age, healthy behaviors, and access to outdoor space were each independently protective against high levels of this type of stress (aors = 0.63-0.94). in the adjusted model, high perinatal infection stress was independently predicted by nine factors: being a woman of color, lack of a marital or cohabiting relationship, previous abuse, previous pregnancy loss, self-reported high-risk pregnancy status, chronic illness, covid-19 related income loss, perceiving that one had been infected, and experiencing alterations to prenatal care (aors = 1.19-1.53). older age and access to outdoor space were protective against perinatal infection stress (aors = 0.67-0.98). neither gestational age, fertility treatment (endorsed by 10.6% of the sample), psychiatric medication use (endorsed by 12.1% of the sample), nor diagnosis of covid-19 during pregnancy were significant predictors of high stress in the unadjusted or adjusted models, indicating that high stress was equally common for women irrespective of these characteristics. in the current study, nearly a third of women pregnant during the outbreak of the covid-19 pandemic experienced elevated levels of stress related to feeling unprepared for birth or being worried about perinatal infection. these high levels of stress appear to be especially common among particular groups of women, including those identified by prior research as vulnerable to high maternal stress: primiparas, women with high risk or unplanned pregnancy, and survivors of abuse (see review by ibrahim & lobel, 2020). although comprising a relatively small portion of the study sample, it is notable that woman of color also reported higher pandemic-related stress. this type of stress coupled with the stressful nature of racial j o u r n a l p r e -p r o o f discrimination is an especially detrimental combination for pregnant woman of color who are at significantly greater risk of adverse perinatal outcomes affected by stress (rosenthal & lobel, 2011; rosenthal & lobel, 2020) . apart from confirming the factors previously established as contributors to prenatal stress, we also identified risk factors that are unique to the pandemic, including experiencing pandemic-related income loss, believing one is infected with covid-19, and experiencing alterations of prenatal care. at the same time, more than two-thirds of the sample did not experience high levels of pandemic-related stress, and we were able to identify factors that appear to be protective against this type of stress, such as having access to the outdoors and practicing healthy behaviors (table 1) . these findings corroborate two recent studies that found that physical activity increases resilience among women in canada (lebel et al., 2020) prevention (e.g., hygiene, social distancing), may also be helpful in reducing stress related to concerns over infection. study findings, as well as other recent research (davenport et al., 2020) , also provide strong evidence to advocate for and promote women's access to the outdoors and to emphasize the value of practicing healthy behaviors, not merely for their physical health value, but also as a way to reduce stress. routine physical and emotional abuse screening is also more important than ever to protect women who may be sheltering in place with violent partners the present study contributes to our understanding of the experiences of pregnant women during the covid-19 pandemic and the overall literature on the experience of stress during pregnancy. it provides insight into which women are at greatest risk of elevated stress and how their stress might be alleviated. such work is critical to the welfare of women and their offspring, especially during periods of global challenges to health and well-being. j o u r n a l p r e -p r o o f stress and chronic illness: the inflammatory pathway psychological impact of infectious disease outbreaks on pregnant women: rapid evidence review. medrxiv risk factors for antenatal anxiety: a systematic review of the literature the covid-19 pandemic and perinatal mental health psychosocial vulnerabilities to upper respiratory infectious illness: implications for susceptibility to coronavirus disease 2019 (covid-19) uncertainty in times of medical emergency: knowledge gaps and structural ignorance during the brazilian zika crisis psychometric evaluation of the polish version of the pandemic-related pregnancy stress scale elevated depression and anxiety symptoms among pregnant individuals during the covid-19 pandemic intergenerational health consequences of in utero exposure to maternal stress: evidence from the 1980 kswangju uprising recommendations to the governor to promote increased choice and access to safe maternity care during the covid-19 pandemic patient-provider communication, maternal anxiety, and self-care in pregnancy examining inequities associated with changes in obstetric and gynecologic care delivery during the coronavirus disease 2019 (covid-19) pandemic. obstetrics & gynecology pandemic-related pregnancy stress and anxiety among women pregnant during the covid-19 pandemic psychometric properties of the pandemic-related pregnancy stress scale (preps) explaining racial disparities in adverse birth outcomes: unique sources of stress for black american women gendered racism and the sexual and reproductive health of black and latina women sociodemographic factors maternal age medicaid or uninsured adjusted odds ratios control for all other variables in the regression model † included all women who endorsed a racial or ethnic identity other than white-non-hispanic ¥ women who reported being high risk and those who were unsure were grouped together key: cord-332085-s58sd8e4 authors: wissmath, b.; mast, f. w.; kraus, f.; weibel, d. title: understanding the psychological impact of the covid-19 pandemic and containment measures: an empirical model of stress. date: 2020-05-16 journal: nan doi: 10.1101/2020.05.13.20100313 sha: doc_id: 332085 cord_uid: s58sd8e4 research suggests that epidemics and corresponding containment measures have negative consequences to the individual and cause stress. the psychological mechanisms that determine stress, caused by the covid-19 pandemic and containment measures, are not yet clear. in a survey during the lockdown in switzerland (n=1565), we found substantially increased levels of stress in the population. in particular, individuals who did not agree with the containment measures, as well as those who saw nothing positive in the crisis, experienced even higher levels of stress. in contrast, individuals who are part of a risk group or who are working in healthcare or in essential shops experienced similar stress levels as the general public. we conducted a path analysis to gain a deeper understanding of the psychological mechanisms during lockdown. experiencing fear of the disease is a key driver for being worried. our model further shows that worries about the individual, social, and economic consequences of the crisis, strongly boost stress. the infection rate in the canton (i.e. state) of residence also contributes to stress. positive thinking and perceived social, organizational, and governmental support mitigate worries and stress. to prevent stress, authorities should explain containment measures well, highlight positive aspects of the crisis, address worries, and facilitate support. 2 background covid-19 evokes stress in patients, healthcare professionals, and relatives (1) . the impact may not be limited to those directly affected; health worries and uncertainty are assumed to generate fear, anxiety, and severe stress in the general population (2) . hence, mental health practitioners anticipate a sharp rise in the need for mental health services (3) . some individuals may be more vulnerable to psychosocial consequences; risk factors include mental or physical preexisting conditions, working in healthcare, or social isolation (4, 5) . switzerland was among the countries first and most affected by covid-19 in europe. to curb the spread of the virus, the swiss government enacted a set of containment measures: encouraging hygiene, giving shelter-in-place orders, as well as closing borders, child-care facilities, schools, restaurants, bars, leisure facilities, and non-essential shops. events and gatherings of more than five individuals were banned. these measures directly affect the general population and may come with side effects. reduced social interactions are risk factors for mental disorders such as major depression (6) , and the containment measures may trigger an economic downturn, thus adding further stress (7) . early studies on covid-19 observed increased stress levels (8) . however, little is known about the psychological mechanisms that determine individual stress in this crisis. we assume that fear of covid-19 and the local infection rate trigger stress (9, 10) . individual, economic, and societal worries are also expected to increase stress levels. agreement with containment measures, perceived support, (9, 11) , and being optimistic about the crisis (12) , are likely to decrease stress. during the lockdown, we surveyed 1565 individuals in switzerland online. first, we compared the observed stress levels in our sample (m=15.58; sd=6.65) to a representative community sample under normal conditions (m=12.57; sd=6.24) (13) . a onesample gauss test showed higher stress levels during the lockdown period, z=19.08, p <.001. the effect (d=.48) is medium to strong. in a second step, we computed multiple comparisons (anova) of different subgroups (see table 1 ). women expressed higher stress levels than men, as has been found in non-epidemic situations (14) . young individuals reported the highest stress levels, whereas the levels were lowest for individuals older than 65 years (small to medium effect). the majority of respondents (66%) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint agreed with the measures of the swiss authorities. these individuals scored lower on stress than individuals who felt that the measures were either not sufficient or too extreme. individuals who strongly disagreed experienced even more stress (medium effect). individuals who reported that the lockdown situation also has positive aspects (89.4%) had substantially lower stress levels compared to those who did not see any positive aspects at all (strong effect). no other assessed variable influenced stress (cf. table 1 ). interestingly, individuals identifying as members of a risk group, as well as individuals working in healthcare or in essential shops with customer contact, did not experience more stress than the rest of the population. also, the expected duration of the pandemic and amount of news consumption had no influence on stress. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint in a third step, we conducted a path analysis with stress as the target variable. figure 1 depicts the resulting model, showing a good fit. in total, the measured variables explain 30% of variance in the endogenous variable, stress. worries contribute the most to stress, and worries mediate between fear of covid-19 and stress. seeing positive aspects of the crisis, as well as agreement with the government's containment strategy, are correlated, and both mitigate worries and boost perceived support. in addition, positive thinking directly minimizes stress. perceived support from family, friends, organizations, and authorities mitigates worries and reduces stress. our results show that the covid-19 pandemic and the mitigation strategies increase the population's stress level. in our model, fear of the virus is the most important booster of worries and the amount of worries is the most important driver of stress. agreeing with the authorities' containment strategy and seeing positive aspects of the crisis are important factors mitigating stress. correspondingly, feeling that the containment measures are not sufficient or too extreme is . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint associated with more stress. support provided by family, friends, organizations, and authorities are also important protective factors for stress. in our study, risk factors for high stress levels are a high amount of worries and perceived lack of support and, to a lesser extent, high infection rates. eleven percent of the respondents do not see anything positive about the crisis. this group experiences substantially more stress than individuals who see at least one positive aspect. although seeing something positive about the crisis may be associated with the individual situation, personality traits, and respondent's coping behavior (12) , our findings indicate that recognizing positive aspects alleviates stress. our model builds upon existing theories of stress and explains 30% of the variance in stress. surprisingly, members of the risk groups, old respondents and individuals working in healthcare or essential shops with customer contact, did not score higher on stress. one reason could be that extensive protection measures had been mandated. in addition, healthcare providers in switzerland have so far not been overwhelmed by cases of covid-19. our model could be useful in understanding and addressing the psychological impact of possible new waves of covid-19 cases and other epidemics. mitigation measures boost worries and stress, particularly for those individuals who feel that these measures are not sufficient or go too far. highlighting positive aspects about the crisis and convincing people of the effectiveness and the necessity of containment measures may not only boost compliance, but also decrease stress, since individuals feel protected by the authorities and experience less worries. this, in turn, will hopefully limit the impact on mental health as a consequence of the crisis. providing support is another important way to mitigate worries, enable coping, and reduce stress. from march 27 to april 26, 2020, a total of 1565 individuals completed the online survey. they were recruited via academic institutions and social media. all participants were swiss residents. information about the sample is provided in table 1 . individuals from geman-speaking (n=1206), french-speaking (n=247) and italian-speaking (n=112) regions, were surveyed. the study was reviewed and approved by the ethics committee of the faculty of human sciences, university of bern. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint measurements stress. we used the perceived stress scale (pps-10) (10-items) (13, 14) . the widely-used pss is based on the stress-related components of unpredictable, uncontrollable, and overloading life events (e.g. "in the last month, how often have you been angry because of things that were outside your control?"), (0=never; 4=very often). a pss score was calculated by summing up all items, with a high score indicating high stress levels. reliability was high (chronbach's alpha = .86). participants were asked to what extent they felt supported by the following individuals, organizations, or authorities: family, friends, neighbors, employers, authorities, schools, day-care centers, church, primary care physician, hospitals (0=little or no support; 3=strong support). to compute the score, we summed up the ratings. infection rate. the cases per 100,000 inhabitants in the participants' resident canton (state) were used (retrieved on april 30 2020: https://www.corona-data.ch/). agreement with containment measures. we asked the participants whether they thought that the measures were (a) not at all strict enough (b) not strict enough (c) just right (d) too strict (e) much too strict. for the analyses, the categories (a) and (e) were combined as "no agreement at all," and the categories (b) and (d) were combined as "no agreement." these combined categories were contrasted with (c) "agreement." seeing positive aspects. we asked the respondents whether there were any positive aspects for them in the current situation (1=no; 2=yes). participants' individual situation and demographics. we assessed the following: sex, age, education, type of household, having kids, employment, working in healthcare or essential shops with customer interaction, part of a risk group, media consumption, and estimated length of the crisis. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 16, 2020. . https://doi.org/10.1101/2020.05.13.20100313 doi: medrxiv preprint replication data are available on the open science framework (15) , https://osf.io/grvwa/ the psychological impact of quarantine and how to reduce it: rapid review of the evidence mental health and the covid-19 pandemic the consequences of the covid-19 pandemic on mental health and implications for clinical practice anxiety, worry and perceived stress in the world due to the covid-19 pandemic social consequences of ebola containment measures in liberia social and physical health risk factors for first-onset major depressive disorder in a community sample depression in the us population during the time periods surrounding the great recession immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china the stress (of an) epidemic is there a case for quarantine? perspectives from sars to ebola worries, perceived impact and preparedness in an avian influenza pandemic -a comparative study between healthcare workers in primary and tertiary care positive thinking in coping with stress and health outcomes: literature review the german version of the perceived stress scale -psychometric characteristics in a representative german community sample perceived stress in a probability sample of the united states understanding the psychological impact of the covid-19 pandemic and containment measures: an empirical model of stress key: cord-028176-wsveq2hk authors: alcibiade, alessandro; schlacht, irene lia; finazzi, francesco; di capua, massimiliano; ferrario, giacomo; musso, giorgio; foing, bernard title: reliability in extreme isolation: a natural language processing tool for stress self-assessment date: 2020-05-29 journal: advances in human factors and systems interaction doi: 10.1007/978-3-030-51369-6_47 sha: doc_id: 28176 cord_uid: wsveq2hk life in isolation is a condition that affects more people every day. from isolation of people in case of an epidemic (ebola, coronavirus, ..) to mineworkers or scientist in the antarctic environment, it is currently well established that extreme isolation is a stressor element which negatively affects human creating stress and social conflict. living in outer space is one of the most extreme forms of isolation that can be used as a test bench for isolation research. crews in space are expected to be able to manage independently stress and social conflict problems to be highly reliable and autonomous. human natural language (nl) has the potential of being a valuable source of information on crew-members stress and reliability in isolation. based on the use of nl, the psi (performance and stress in isolation) research group is developing an artificial intelligent tool for the autonomous monitoring of stress and reliability. in order to develop this tool, the first step (here presented) is to investigate the correlation of nl with stress and reliability within the psi experiment. the experiment consisted of three tasks to be performed periodically during the isolation: 1. analysis of free written communication (nl); 2. short self-reference questionnaire; and 3. detection of heart rate and blood pressure values. in this paper we present the preliminary data collected from 29 participants (17 males and 12 females) to 6 space analogue missions accomplished in europe, the usa and israel in the 2016 – 2019 time frames. that is, so far, the largest database ever analyzed of this kind. the results are coherent with what is described in the literature about nlp content and style analysis, with the novelty of the correlations found with the cardiovascular parameters, thus reinforcing the perspective of applying a nlp ai system for supporting stress management in extreme isolation contexts from space to earth spin off. abstract. life in isolation is a condition that affects more people every day. from isolation of people in case of an epidemic (ebola, coronavirus, ..) to mineworkers or scientist in the antarctic environment, it is currently well established that extreme isolation is a stressor element which negatively affects human creating stress and social conflict. living in outer space is one of the most extreme forms of isolation that can be used as a test bench for isolation research. crews in space are expected to be able to manage independently stress and social conflict problems to be highly reliable and autonomous. human natural language (nl) has the potential of being a valuable source of information on crew-members stress and reliability in isolation. based on the use of nl, the psi (performance and stress in isolation) research group is developing an artificial intelligent tool for the autonomous monitoring of stress and reliability. in order to develop this tool, the first step (here presented) is to investigate the correlation of nl with stress and reliability within the psi experiment. the experiment consisted of three tasks to be performed periodically during the isolation: 1. analysis of free written communication (nl); 2. short selfreference questionnaire; and 3. detection of heart rate and blood pressure values. in this paper we present the preliminary data collected from 29 participants (17 males and 12 females) to 6 space analogue missions accomplished in europe, the usa and israel in the 2016 -2019 time frames. that is, so far, the largest database ever analyzed of this kind. the results are coherent with what is described in the literature about nlp content and style analysis, with the novelty of the correlations found with the cardiovascular parameters, thus reinforcing the in every mission isolation is a stressor element which negatively affects human behaviour, mood and cognitive performances, creating stress and social conflict. living in outer space is one of the most extreme form of isolation that can be used as test bench for spin off of isolation research on earth. future crewed long lasting space missions beyond low earth orbit (beo) pose danger and challenges not previously faced by any human explorer. not only would the trip's length far exceed any previous mission but the crew members would also experience significant communication delays, endure lengthy periods of inactivity punctuated by crises and they would lose sight of the earth as they journey towards deep space. the effects of these unique circumstances on astronauts are still not completely described but they will likely test human psycho-physiological limits like never before and the importance of being able to self-monitoring the psychological and physiological factors will be crucial to their success [1] . in particular the relative lack of human factors data behond eath orbit (beo) creates a significant difficulty in understanding what the problems are and how to address them in the context of a crewed mission with associated constraints. what is already well described into the scientific literature is how "extreme" environmental stimuli give origin to an adaptive response, which comprehends systemic and behavioural changes aimed to develop the best homeostatic capability of the individual, thus increasing his survival chances. these adaptive changes are produced by chemical mediators, such as adrenalin, glucocorticoids, growth hormone and cytokines, that act on specific receptors localized in different organs. chronic stress can produce the so called allostatic state, characterized by an increased activity of the mediators on their target cells that leads to receptor desensitization and tissue damages, thus connoting the allostatic load [2] . this may cause dramatic consequences, all detrimental in the operating scenario of a crewed space mission that include insomnia, depression, cognitive impairment and various cardiovascular diseases. because of all those assumptions it is now well established that crewed missions beo are expected to be highly autonomous and automated in order to improve the human factor on the overall outcome of the mission and also, because of the communication delay with the earth station that imposes the spacecraft to be as self-reliant as possible in the accomplishment of its tasks. from these findings comes the need to design and implement more advanced systems of artificial intelligence (ai) and human-computer interfaces on board future spaceships. an effective and innovative human-computer interface cannot do without the implementation of the possibility of making astronauts communicate with ai in the most spontaneous way for humans: natural language. the branch of computer science that deals with "teaching" ai machines how to effectively communicate in the natural language of humans is called natural language processing (nlp). other than just potentially being a very effective human-machine interface, human natural language has the potential of being a valuable source of information regarding crew-members psycho-physiological well-being, stress and reliability. in fact, it has been well known for more than a century that psychological states and processes are encoded in verbal behavior and psychological constructs may be reliably explored by text analysis [3, 4] . a multitude of studies indicate the existence of a strong correlation between the variation of the word spectrum used in an individual's daily vocabulary in a given period and the variation in his level of physiological and psychological stress [5] [6] [7] . the aim of this study was to investigate the prospect of using nlp to monitor the psychological wellbeing and stressful conditions of personnel undergoing prolonged missions in isolated and confined environments (ices). nlp is a theorymotivated range of computational techniques for the automatic analysis and representation of human language [8] . the history of nlp began in the 1950s as the intersection of artificial intelligence and linguistics [8] . in recent years, increases in computing power have led to the development of sophisticated machine learning algorithms that allow researchers to analyze enormous quantities of biomedical data to detect covered patterns [9] . these developments have brought back the growing of the studies of human natural language from a psychometric perspective, consequently directing the progression of nlp algorithms as a diagnostic tool for psychiatric and psychological disorders based on the analysis of written and spoken language [10] . the studies of potential space applications of natural language processing (nlp) have been pioneered by the russian ibmp research group led by prof. vadim gushim, who analyzed texts extracted from space stations (mir and iss) and analogue missions (mars500 and mars105) crews communications with the respective ground controls. a computerized content analysis of written daily reports of the mars105 experiment crew (ibmp, moscow, 2009) revealed different tendencies in communicating behaviour. a stress-related strategy was "closing the communication channel" [11] , which was found to correlate with elevated urinary cortisol level. these results suggest as both language use and somatic markers can have a diagnostic value in identifying stressful conditions and changes thereof. current nlp analyses typically consist of both a content analysis assessing what a subject says and a style analysis evaluating how subjects express themselves. so far, the strongest associations according the scientific literature regarding content analysis have identified in the first-person singular personal pronouns the strongest nlp content indicator of depressive mood [12] . adjectives and adverbs expressing negative emotions are found to be strongly linked to depressive mood and suicidal thoughts [13, 14] . regarding style analysis, it has been found that individuals with suicidal thoughts, anxiety or depressive mood tend to use more absolutist words than the general population, thus expressing ideas of certainty [12] . this study explores the possibility to use the nlp to identify language markers that could be of diagnostic value in assessing crew-members psychological wellbeing and stress. moreover for the first time in literature, it looks for new correlations among: nlp-markers and known stress related cardio-vascular parameters such as heart rate, and nlp-markers and a self-reference psychometric short questionnaire. those findings could prospectively lead to the development of an ai learning tool to monitor remotely and non-invasively not only the astronauts' wellbeing and cognitive performance status in future missions beo but also on different spin off isolation context on earth. the psi (performance & stress in isolation) experiment aims to analyze stress levels during extreme missions without intrusive instruments. it has received the approval of the ethics committee of the pope giovanni xxiii hospital in bergamo on 11 august 2017. in this paper, the preliminary data collected from 29 participants (17 males and 12 females) to 6 space analogue missions (figs. 1 and 2) accomplished among europe, the usa and israel in the 2016-2019 time frames, are presented. that is, as yet, the largest database ever analyzed of this kind. the core of the psi experiment consisted of three tasks to be performed periodically during the isolation: 1. short text collection, to be analyzed through a nlp software. every crew member was asked to write in a maximum of 30 min a text with a maximum of 500 words in his mother tongue (e.g. diary collection or daily and official mission report if not affected by a strong imposed structure). a. short self reference questionnaire, to assess subjects' perceived psychological stress levels. it consisted of the following three questions: b. q1: "how stressed do you feel today?" (from low:1 to high:10); c. q2: "how well were you able to perform today?" (from low:1 to high:10); d. q3: "how comfortable do you feel today?" (from low:1 to high:10). 2. detection of heart rate (hr) and blood pressure (systolic, diastolic and mean arterial pressure) values, as markers of subjects physiological stress levels. the measurements were taken using an omron m3 device, provided to every crew, and crew members were instructed to measure the parameters according to the latest american heart association recommendations that at least two readings be taken, with a one-minute interval between them and the average of the measurements recorded. for each subject the written texts were categorized based on the editing date and time spent in isolation and according to the subject's gender and mother tongue as well. this research relied on the use of two content analysis software tools. one is an italian tool specifically tailored to this scope. the second tool is liwc (linguistic inquiry and word count), developed by the pennebaker lab [15] . the data was analyzed using standardized z-score output of linguistic markers from liwc software as well as using our own specifically tailored software which provides two variables not considered by the liwc software: the number of different words in a text (dw, assumed to be an index of verbal fluency) and the number of average characters per word in a text (cw, assumed to be an index of verbal and cognitive complexity). a pearson's correlation analysis was run to assess the strength of relationships between linguistic markers. statistical analysis correlations were searched between the nlp parameters and the answers to the self-reference psychological questionnaire and the cardiovascular parameters. numerous novel correlations were identified; those lead an interest for a confirmation with a larger number of subjects. here will be illustrated the most relevant correlations (fig. 3) . a strong direct correlation emerged at the person regression analysis (r = 0.490, p = 0.002) between the frequency of first person plural pronouns in a text and the scoring at the first questions of the self-reference psychological test ("how stressed do you feel today?"). perceived stress is thus directly correlated to the subject's tendency to express using the first person plural pronouns, which is linked to a feeling of detachment, selflessness and isolation [15] . a moderate negative correlation was found between the average number of words per sentence (wps) and the mean arterial pressure (map) values (r = −0.393, p = 0.013). wps is considered as an indicator of subjects' cognitive complexity [15] , while the map is an estimation of the average blood pressure in an individual during a single cardiac cycle. it is widely described in the scientific literature the link between higher blood pressure values and chronic [16, 17] , allowing us to assume that in individuals in extreme isolation higher map values are an physiological indicator of stress and that are negatively correlated to subjects' cognitive performances. the results confirm the literatures about nlp content and style analysis, with the important novelty of the correlation hypotheses between the nlp and cardiovascular parameters. being well assessed in the scientific literature [15, 16] how higher blood pressure values are linked to chronic stress and its complications, this finding open up the perspective of developing a nlp ai system to self-monitor not only astronauts' psychological and cognitive well being but also of their overall psycho-physiological status with spin off applications in different isolation contexts on earth. furthermore we know that the literature review is coherent in assessing that human nlp-psycho-physiological parameters correlations do not always follow the same trends within every subject, like on most parts of the human psycho-physiological phenomena [10] [11] [12] [13] [14] [15] . the reason is that nlp and stress-related parameters are highly influenced by many factors such as subjects' age, cultural background, education raining, gender and length of the isolation. the implementation of nlp in a software designed to let crew members to monitor their wellbeing in isolation, cannot be based on a single rigid algorithm but should be based on a machine learning tool trained to identify the peculiar pattern followed by each parameter in a given subject. finally, due to the restricted number of subjects enrolled in this study, the statistical power of this analysis is low and further research is needed to confirm specific linguistic indicators of functionally prohibitive stress responses. space habitability. tu-berlin sleep deprivation as a neurobiologic and physiologic stressor: allostasis and allostatic load motivation and language behavior: a content analysis of suicide notes the psychopathology of everyday life intermittency and scale-free networks: a dynamical model for human language complexity the variation of zipf's law in human language zipf's word frequency law in natural language: a critical review and future directions jumping nlp curves: a review of natural language processing research natural language processing: an introduction the psychological meaning of words: liwc and computerized text analysis methods content analysis as a source of information about personality during space flight linguistic markers of moderate and absolute natural language linguistic correlates of team performance: toward a tool for monitoring team functioning during space missions the psychology of word use in depression forums in english and in spanish: testing two text analytic approaches. association for the advancement of artificial intelligence the development and psychometric properties of liwc2015 psychological stress in pathogenesis of essential hypertension blood pressure and dementia: a comprehensive review key: cord-256132-ufs9kw3o authors: alateeq, deemah a.; aljhani, sumayah; aleesa, dalal title: perceived stress among students in virtual classrooms during the covid-19 outbreak in ksa date: 2020-08-01 journal: j taibah univ med sci doi: 10.1016/j.jtumed.2020.07.004 sha: doc_id: 256132 cord_uid: ufs9kw3o objectives: the coronavirus disease (covid-19) is a novel infection caused by the coronavirus, which rapidly became a global pandemic. as disease outbreaks can adversely affect mental health, in this study, we explored the perceived stress levels among students during the covid-19 outbreak that led to the adjournment of face-to-face teaching in ksa. methods: we conducted a cross-sectional survey of 367 students from ksa, which assessed their socio-demographic characteristics, perceived stress scale, emotions, and concerns during the covid-19 outbreak. results: most participants were female (74.7%) and secondary school (79.8%) students. more than half of the participants showed moderate levels of stress (55%), while 30.2% registered high levels. females and university students showed a significant association with stress level (p-value = 0.003 and 0.049, respectively). conclusions: there was a moderate to high level of stress among students in ksa at the start of the covid-19 outbreak. this study found a significant correlation between a high level of stress and female university students. the integration of online counselling and stress management programs would help mitigate the stress of students during distance learning. . females and university students showed a significant association with stress level (p-value ¼ 0.003 and 0.049, respectively). conclusions: there was a moderate to high level of stress among students in ksa at the start of the covid-19 outbreak. this study found a significant correlation between a high level of stress and female university students. the integration of online counselling q8 and stress management programs would help mitigate the stress of students during distance learning. the world health organization (who) defined pandemic as the spread of a certain disease worldwide or across international borders, affecting a large number of people. 1 covid-19 is a newly discovered infectious coronavirus that spreads from one person to another through droplets. 2 the virus emerged in december 2019 and was first discovered in wuhan, china. it was declared as a public health emergency of international concern in january 2020. 1, 2 among other countries, ksa implemented q10 strict precautions on its citizens in an attempt to control the spread. the country q11 transferred q12 its in-person educational system to virtual learning, closing public places of aggregation q13 , invoking travel bans and curfews, and even suspending the umrah pilgrimage. 3, 4 the literature on recent outbreaks, such as ebola, the severe acute respiratory syndrome (sars), and middle eastern respiratory syndrome (mers), showed unique consequences of mental health burden during pandemics. several factors were identified in the process of understanding how the public would respond during disease outbreaks, including disease course, media and misinformation, quarantine, neuropsychological sequelae of the infected individual, and the mental health burden among health care workers. during a quarantine, people face many consequences of physical and emotional social distancing, including isolation and future uncertainty. degrees of isolation vary between individuals, ranging from physical (i.e., contact) or symbolic (i.e., separation from loved ones), and affect the human psyche. 5 previous studies showed that disease outbreaks impacted individual mental health and well-being. multiple risk factors were addressed. it was found that women and those aged between 16 and 24 years exhibited a great risk of developing psychological distress. 6 similarly, recent studies showed a positive response to feeling panicked, depressed, or emotionally disturbed during the h1n1 pandemic. 7 during the sars outbreak, a population-based survey showed post-crisis mental distress. 8 comparably, during mers, the level of stress was high in medical students in ksa. 9 to our knowledge, there are currently no studies that have assessed stress among students during the quarantine related to the covid-19 pandemic. therefore, this study aimed to q14 explore the perceived stress level among students due to the coronavirus disease outbreak and the resulting suspension of in-person teaching in ksa. the data for this study were obtained q15 using a crosssectional survey, which was distributed from 18th until 28th of march 2020, during the coronavirus disease outbreak q16 . this study included both male and female arabic speaking students, over 12 years old, living in ksa q17 . an online survey was distributed using q18 non-probability convenient sampling to students from different levels living in ksa. google forms was used to create a link for the survey, which was posted in an announcement on the noon academy platform; a reminder was sent five days q19 later. noon academy is an online educational platform in the middle east and north africa (mena), where people q20 can study live with their facilitators and friends in an interactive and engaging manner. six million students are registered on the platform, from seven countries: ksa, kuwait, jordan, iraq, oman, egypt, and india. 10 the online survey consisted of three components: 1) socio-demographic characteristics that included age, gender, level of education, and region of residence. 2) perceived stress scale (pss) of sheldon cohen. 11 the scale consists of ten questions that are used to measure the perception of stress experienced by the participants over the past month. it includes a 5-point likert scale that capture responses ranging from never (0) to very often. 4 total mean scores of 0e13 are considered to be low stress, 14e26 indicate moderate stress, and 27e40 indicate high stress. the pss is an easily and widely used tool with acceptable psychometric properties. 12e14 an arabic-validated version was used. 15 3) a qualitative exploratory question was also included q21 : 'describe your emotions and concerns during the current outbreak'. a statistical analysis was performed using the statistical package for the social sciences (spss) version 23.0 software (spss inc., chicago, il, usa). categorical variables were expressed as percentages. the chi-square test was used to compare between perceived stress scales (low, moderate, and high perceived stress). mannewhitney u and kruskale wallis tests were also used. when p-values were less than 0.05, the differences were considered statistically significant. a total of 367 students responded to the survey. the demographic characteristics of the surveyed population are presented in table 1 table 2 displays the responses to the 10 items of the pss provided by the participants. through the month preceding the survey, the following trends were observed: 58 .1% of the students (fairly or very) often felt nervous and stressed; 58% were often angered due to things that happened outside of their control; 50.5% often felt that difficulties were piling up so high that they could not overcome them; 43% often found that they could not cope with all the things that they had to do; 42.2% often felt that they were unable to control the important things in their life; 34.9% were often upset because of something that happened unexpectedly. conversely , 49.3% of the students often felt confident in their ability to handle personal problems, 30.8% often were able to control irritations in their lives, 26.5% often felt that things were going their way, and 25.6% often felt that they were on top of things. the mean value of the total pss scores was 22.12 ae 7.33, with an absolute range of 1e38, median of 23, and inter-quartile range of 17e27. the cut off limits for the 25th, 50th q26 , and 75th percentiles were 17, 23, and 27, respectively. in general, 55% of the students had moderate stress, and 30.2% had high stress ( table 3 ). as shown in table 1 , there were significant associations between the level of stress and three sociodemographic variables: gender, age, and educational level. females had a significantly higher mean score of perceived stress level compared to males (22.75 versus 20.27; p q27 -value ¼ 0.003). however, more than half of them (52.9%) had moderate levels of stress, whereas 33.9% had high stress levels. university students had a significantly higher mean score of perceived stress compared to intermediate and secondary school students (25.30 versus 22.98 and 21.72, respectively; p-value ¼ 0.049). however, more than half of them had moderate levels of stress (60.9%), whereas 39.1% had high stress levels. the qualitative exploratory question regarding the emotions and concerns of the participants showed diversity. students reported feeling depressed, stressed, anxious, fearful, angry, and unmotivated. difficulties related to studying, problem solving, and time management were also reported. secondary school students who were aiming for high scores to get accepted in q28 universities were worried about their grades and their q29 future. one student reported that she was diagnosed with anxiety and was currently taking an antidepressant for her symptoms q30 , which helped her feel that she was able to control things, although she believed that she might panic in a given situation. this study is the first to explore the level of perceived stress during the covid-19 outbreak in ksa. students were included from all five regions of the country and various educational levels of both secondary and tertiary education. the mean score of the perceived stress scale in this study was 22.12 ae 7.33, and a high-moderate perceived stress was endorsed by 30.2e55% of the participating students. these scores are comparable to other national and international studies that were conducted in ksa, 16e18 india, 19, 20 iran, 21 and malaysia. 22 however, these studies were conducted among students from competitive specialties, such as medicine and dentistry. the result is not surprising as, at the time of conducting the survey, the total number of confirmed cases of covid-19 in ksa reached 900, with two coronavirus-related-deaths. the curfew measures have been extended in terms of the total amount of hours, and a prohibition was placed on both entering and exiting riyadh, makkah, and almadinah almunawwarah. 23 furthermore, three items on the 5-point likert scale of perceived stress (in table 2 ) were endorsed by more than half of the sample, which are often (fairly or very) felt nervous, stressed, and angered and unable to overcome piled up difficulties. similar results were recently found in other studies, which q31 evaluated the mental health of university students during covid-19 in china 24 and spain 25 and reported anxiety, stress, and depression. these psychological responses are more likely to occur and worsen due to the lack of interpersonal communication during the social distancing. 26, 27 in addition, distance learning was also found to be associated with stress, which is due to academic, financial, and social difficulties. 28 coping with the online mode might become a challenge for students. this includes students' ability to deal with technology, sufficient q32 home resources facilitating online learning, or stable internet connection. 29 previous studies regarding mental health during the pandemic have studied general populations in other regions of the world. 30e32 the outbreak of the virus and the implementation of sudden control measures may cause excessive fear and social isolation, while the lack of infectious disease knowledge can foster widespread panic. 30 the novelty of the virus itself and the unpredictability and uncertainty of when the situation will be entirely controlled has put people under excessive stress, especially when social face-to-face interactions are lost. 31 patients infected by the virus or who are suspected to have contracted the illness experience the fear of its potential fatality. 32 in our study, university students scored significantly higher in terms of levels of stress compared to students from intermediate and secondary schools. this is most q33 likely because university students are emerging adults who pursue identity exploration, work toward independence, and have different roles to fulfil. 33 other sources of stress include the frequency of and performance on examinations, broad curriculums, parental pressure, loneliness, and worrying about the future. 34 finally, female students reported significantly higher levels of stress, which may be due to female participants representing the majority of the sample. however, similar results were reported in previous related surveys. 18, 19, 21, 34 high levels of stress among females have been attributed to various factors, including hormonal changes and expression of emotions and thoughts regarding their social situation. 35, 36 limitations although our study represents the first survey of stress levels during the covid-19 outbreak in ksa, we acknowledge several limitations q34 . one of them is a convenient sample using an online platform, which limits the generalisability of the results. another possible limitation is reporting bias, as the study depends on self-reported information that may be affected by participants' interpretation of the items or their tendency to report their emotions in a certain manner. finally, the cross-sectional design precludes the ability to make causal q35 conclusions. this study showed high to moderate levels of stress among students in ksa during the covid-19 outbreak. this is most likely due to the mandatory curfew and distance learning. female and university students showed a higher level of stress. online stress management programs are recommended to improve stress and coping strategies, as well as prevent q36 further psychological consequences. further studies are necessary to conduct longitudinal assessments of psychiatric disorders, such as depression and anxiety, to produce evidence-based mental health interventions during crises. furthermore, as this is the first survey on the psychological impact of covid-19 on saudi students, these results could be used as a baseline to investigate the stressors and the extent of their impact. the author have no conflict of interest to declare. ethical approval was obtained from the institutional review board at princess nourah bint abdulrahman university (pnu) in riyadh, ksa. informed consent was obtained from the participants after the aims of the study were explained. daa conceptualised and designed the study. dda, saa, and dha drafted the questionnaire. daa and saa analysed q37 and interpreted the data. daa, saa, and dha wrote the manuscript. all authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 jtumed628_proof ■ 1 august 2020 ■ 5/6 please cite this article as: alateeq da et al., perceived stress among students in virtual classrooms during the covid-19 outbreak in ksa, journal of taibah university medical sciences, https://doi.org/10.1016/j.jtumed.2020.07.004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 the classical definition of a pandemic is not elusive kingdom's government sets preventive. precautionary measures to prevent covid-19 infection transmission, interior ministry says saudi arabia intensifies efforts to prevent outbreak of novel coronavirus saudi press agency psychiatry of 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reasons for and sources of stress among medical students at rabigh medical college academic distress, perceived stress and coping strategies among dental students in saudi arabia level of stress and its causes among 1st-year dental students -a cross-sectional study perceived stress and source of stress among undergraduate medical students of government medical college, mysore comparison of sources and severity of perceived stress between paramedical and medical students concurrent validity of the malay version of perceived stress scale (pss-10) saudi arabia records second coronavirus death j arab news the psychological impact of the covid-19 epidemic on college students in china psychological effects of the covid-19 outbreak and lockdown among students and workers of a spanish university novel approach of consultation on 2019 novel coronavirus (covid-19)-related psychological and mental problems: structured letter therapy rules on isolation rooms for suspected covid-19 cases in gp surgeries to be relaxed stress and coping strategies among distance education students at the university of cape coast, ghana closure of universities due to coronavirus disease 2019 (covid-19): impact on education and mental health of students and academic staff recommended psychological crisis intervention response to the 2019 novel coronavirus pneumonia outbreak in china: a model of west china hospital iranian mental health during the covid-19 epidemic timely mental health care for the 2019 novel coronavirus outbreak is urgently needed emerging adulthood: a theory of development from the late teen through the twenties perceived stress, sources and severity of stress among medical undergraduates in a pakistani medical school handbook of the sociology of mental health hormonal cycle modulates arousal circuitry in women using functional magnetic resonance imaging perceived stress among students in virtual classrooms during the covid-19 outbreak in ksa the authors would like to thank noon academy and noura alfulaij for their kind support in data collection. this research was funded by the deanship of scientific research at princess nourah bint abdulrahman university through the fast-track research funding program. key: cord-256504-odbaubqm authors: kuo, fang‐li; yang, pei‐hsuan; hsu, hsin‐tien; su, che‐yu; chen, chun‐hsien; yeh, i‐jeng; wu, yen‐hung; chen, li‐chin title: survey on perceived work stress and its influencing factors among hospital staff during the covid‐19 pandemic in taiwan date: 2020-08-19 journal: kaohsiung j med sci doi: 10.1002/kjm2.12294 sha: doc_id: 256504 cord_uid: odbaubqm this study aimed to investigate the perceived work stress and its influencing factors among hospital staff during the novel coronavirus (covid‐19) pandemic in taiwan. a web‐based survey was conducted at one medical center and two regional hospitals in southern taiwan, targeting physicians, nurses, medical examiners, and administrators. the questionnaire included items on the demographic characteristics of hospital staff and a scale to assess stress among healthcare workers caring for patients with a highly infectious disease. a total of 752 valid questionnaires were collected. the hospital staff reported a moderate level of stress and nurses had a highest level of stress compared to staff in the other three occupational categories. the five highest stress scores were observed for the items “rough and cracked hands due to frequent hand washing and disinfectant use,” “inconvenience in using the toilet at work,” “restrictions on eating and drinking at work,” “fear of transmitting the disease to relatives and friends,” and “fear of being infected with covid‐19.” discomfort caused by protective equipment was the major stressor for the participants, followed by burden of caring for patients. among participants who experienced severe stress (n = 129), work stress was higher among those with rather than without minor children. the present findings may serve as a reference for future monitoring of hospital staff's workload, and may aid the provision of support and interventions. the novel coronavirus pneumonia taiwan, to prevent disease spread, the government ordered the closure of a hospital due to severe cross infection. healthcare workers rushed out of the blockade line due to insufficient protective equipment and fear of the disease, which resulted in fear of the pandemic among medical staff and the public. as the covid-19 pandemic continues to grow, the collective memory of sars has been revived, and people have vowed not to repeat the same mistake. as a result, hospitals in taiwan have quickly implemented a containment campaign in response to the currently emerging pandemic by developing emergency response measures. staffs are required to wear protective equipment, screen patients and visitors entering and leaving the hospital, and set up an outdoor fever screening area in the emergency clinic. additionally, a scheduling roster has been set up, and all hospital staffs are deployed to fight against the pandemic by providing comprehensive care for patients in negative stress isolation wards or general inpatient wards, and by participating in rotational shifts to implement the quarantine measures at the hospital gate. the pandemic has disrupted the original schedule of hospital staff and has caused changes in their work or lives, resulting in substantial physical and mental stress. stress refers to individuals' reactions to different situations. it includes changes in their physiology or psychology in the face of events in the external environment that are evaluated as more than they can bear, such that they affect their physical and mental wellbeing. 3 stress at work is regarded as the process of interaction between the individual and the work environment, which represents an accumulation of negative emotions generated by the work. 4, 5 these negative reactions lead to the experience of high stress for a long period, which in turn affects physical and mental health. specifically, it causes multiple adverse symptoms, such as insomnia, headache, fatigue, anxiety, gastrointestinal discomfort, and immunity decline, as well as increased family conflicts, decreased work quality, interpersonal relationship disorders, and other negative effects. [5] [6] [7] [8] hospital staffs are under high stress during the care process. they have to tackle the effects of misunderstanding by the general public, face worries from family members, experience fear of the unknown disease and of becoming a transmitter of the disease, and experience challenges related to their professional skills of personal infection control. these were major stressors for healthcare workers during sars, 9 and as such, several of them experienced fear, exhaustion, and stress. 10 extensive research has been conducted to examine the correlation between personal demographics and work stress. studies have found that marital status, workplace, and educational level have significant effects on work stress. 12, 13 for example, wang 14 explored the relationship between work stress and social support among nurses, and found that the older they were and the higher years of work experience they had, the higher was their work stress. this finding suggests that, with the increase of age and accumulation of work experience, nurses are likely to have better attitudes toward and abilities to respond to problems, and accordingly, they would be more likely to be assigned the responsibility to handle unexpected situations at work. 12, 15, 16 unmarried, female, younger medical staff were found to be more prone to high work stress, 17 but work stress was also observed to decrease with the increase in age, years of work experience, and number of children. 16 another study found that healthcare workers are busy at work, and work stress could result from the conflict of clinical work with family or personal affairs. 18 however, mcgrath et al 18 found that marital status, work experience, but educational level had no significant impacts on work stress. 14 the contagion path of covid-19 is still unknown. therefore, since the outbreak of the pandemic, the taiwanese government has formulated an emergency response plan, and hospitals have adopted several preventive measures. moreover, hospital staffs have experienced the global impact of this highly communicable disease, whose sudden onset has caused a stress impact that is similar to that of sars. this leads to the question, "are hospital staff suffering from the same magnitude of stress as before?" accordingly, the objectives of the present study were a cross-sectional survey was conducted from the end of march to the beginning of april 2020, at the peak of the covid-19 pandemic in taiwan. participants were selected from a 1700-bed medical center and two 800-bed regional hospitals in southern taiwan. the survey was conducted using a web-based questionnaire and excluded new recruits, outsourced workers, research assistants, and other non-regular hospital employees. details of the survey website were provided to the survey participants through the mailboxes of the three hospitals, and the researchers compiled the responses from each hospital for analysis. this study recruited participants from the four main categories of hospital staff, namely physicians, nurses, medical technicians, and administrators. the questionnaire contained the following two sections 11 11 for sars. the scale comprises 4 subscales, namely, "worry and social isolation" with 10 items, "discomfort caused by protective equipment" with 8 items, "difficulties and anxieties related to infection control" with 7 items, and "burden of caring for patients" with 7 items, totaling 32 items. each item is rated on a 4-point likert scale (0: not at all, 1: about the same as usual, 2: slightly more severe than usual, 3: more severe than usual) to assess the degree of stress caused by various stressors. the total score ranges from 0 to 96, with a higher total score indicating a greater degree of stress. a total score of 46 to 96 indicates "severe stress," that from 33 to 46 indicates "moderate stress," that from 0 to 32 indicates "low stress," and 0 indicates "no stress." the content validity index of the scale was 0.92 in the original study by chuang and lou (2005) , 11 who tested it on healthcare workers (n = 543) from medical centers in taiwan. the cronbach's α values for the four subscales were 0.84 to 0.90 in the original study. in the present study, the cronbach's α value of the complete scale was 0.94, and that for the four subscales was 0.84 to 0.90. data were analyzed using jmp13.0 statistical package. regarding descriptive statistics, continuous variables related to demographic characteristics and perceived work stress were presented as mean ± sd; categorical variables were presented as counts and percentages. for inferential statistics, the pearson product moment correlation test was used to analyze the correlations between the study variables. since severe stress has a significant impact on the physical and mental health of employees 20 and willingness to care for patients. 11 with a projected power of 80%, an alpha of .05, and an effect size of 0. 30 one-way analysis of variance (anova) to identify if stress levels differed based on demographic characteristics, followed by a post hoc tukey-kramer comparison to identify groups with significant differences. this study was reviewed and approved by the institutional review the sample comprised 752 hospital staff from one medical center and two regional hospitals in southern taiwan. as shown in table 1 note: a score of 0 indicates "not at all," 1 "about the same as usual," 2 "slightly more severe than usual," and 3 "more severe than usual." note: rating scales: 4-point likert scale (0: not at all, 1: about the same as usual, 2: slightly more severe than usual, 3: more severe than usual). note: a score of 0 indicates "not at all," 1 "about the same as usual," 2 "slightly more severe than usual," and 3 "more severe than usual. stress could be attributed more to their fear of social isolation, discomfort due to protective equipment, and burden of patient care. in contrast, medical technicians had a higher degree of stress related to difficulties and anxieties related to infection control. the mean score for each of the 32 items on work stress was 1.5 ± 0.5 (table 3) , representing mild to moderate stress. the mean scores of each dimension and each item, and score rankings are presented in table 4 . on 19 items, more than 50% of the participants experienced moderate to severe work stress (with mean scores of over 2 points). the five highest stress scores were observed for the items "rough and cracked hands due to frequent hand washing and disinfectant use," "inconvenience in using the toilet at work," "restrictions on eating and drinking at work," "fear of transmitting the disease to relatives and friends," and "fear of being infected with covid-19". "hospital staff experienced discomfort caused by the use of protective equipment" and "the burden of caring patients" reported higher scores than other two dimensions of the scale. further, for hospital staff with severe stress (n = 129), we performed a t test or one-way anova to explore whether their stress levels differed based on their demographic characteristics of gender, marital status, years of work experience, educational level, staff type, and experience with caring for patients with sars. as shown in table 5 this study was the first one conducted in taiwan to investigate the perceived stress of hospital staff and to identify relevant influencing factors during the covid-19 pandemic, using a web-based structured questionnaire. the results showed that the total stress was moderate and discomfort caused by protective equipment emerged as the major stressor. nurses generally perceived higher stress as compared to the other types of hospital staff. difficulties and anxieties related to infection control were major stressors for medical technicians, while administrators were the least stressed among all types of staff. further, those with minor children experienced a higher degree of work stress. this study found that discomfort caused by protective equipment was the major stressor for the participants, followed by burden of caring for patients. the overall stress was slight to moderate. this result was consistent with the findings of yu et al, 21 but it was different from other similar studies conducted during the sars pandemic. specifically, those studies found that the main stressor among hospital staff was difficulties and anxieties related to infection control. 11, 22, 23 participants in the present study were more stressed by the discomfort caused by wearing protective equipment for a long period, probably because, to effectively prevent the source of infection from entering the hospital and causing cross infection, the hospital staff were required to wear masks throughout their stay in the hospital. additionally, they had to set up simple triage stations at the emergency and outpatient departments' entrances, while different types of staff took turns to participate in monitoring individuals entering the hospital. the staff on duty was required to wear basic protective gear, including goggles, protective clothing, surgical masks or n95 masks, and gloves, which made it inconvenient for them to eat and drink at work, as well as to use the toilet. they were required to wash their hands and use disinfectants frequently, and the fear that hands would become rough and cracked made them stressed. the covid-19 pandemic has changed the work style and environment of the healthcare system, leading to physical and mental imbalance, and thereby, an increased level of stress among hospital staff. 24 when the sars pandemic hit taiwan stressful events or stressors in the workplace can cause stress reactions, and an employee's personal characteristics and behaviors interact with the practice environment. 26 strong physical and mental demands lead to an increase in stress levels of hospital staff, which may in turn influence patient care. 27 a survey of 532 regional hospital staff examined the correlation between perceived overwork and musculoskeletal symptoms. findings revealed that nurses ranked the highest among hospital staff in terms of overwork, followed by administrative staff, medical technicians, and physicians, in order of decreasing overwork. 28 by nature, nursing is a highly stressful occupation. nurses reported highest stress level in this study. this could be attributed to physical discomfort caused by protective equipment, also, nurses often previous studies have shown that work stress is related to one's demographic characteristics, 13, 17 and that child care at home is a common problem for hospital staff, 28, 29 which is an important factor leading to work-family conflicts. 30 the present results showed that hospital staff with minor children was more stressed, confirming the viewpoint presented in prior research. however, these findings were different from those reported by wang et al 14 and lambert et al, 16 who found that employees with children were less stressed than those without. perhaps this is due to the parenting style difference between the east and the west world. 31 in the face of the covid-19 pandemic, since the end of january 2020, hospitals started hospital-wide staff mobilization to contain the pandemic, such as setting up triage stations at the entrances and exits, and medical tents and special wards at the emergency department. these measures changed the existing work pattern of staff and caused unprecedented stress on all types of healthcare workers. as the temperature rises in southern taiwan, wearing of protective equipment for prolonged periods causes discomfort in staff. such discomfort can be alleviated by shortening shift durations and installing more mobile air conditioners in emergency and outpatient departments. since healthcare workers are in a high-stress occupation, it is recommended that they should be assessed and monitored regularly, and appropriate interventions need to be implemented. it is necessary to provide psychological counselling and stress relief measures for this population. during the pandemic containment, some staff was concerned that they could be carries of the infection after caring for patients with covid-19, and that they would bring the source of infection home to their family and relatives. this led to higher work stress on hospital staff with minor children. to cope with this situation, it is recommended to assess the needs of hospital staff with minor children and provide appropriate arrangements for the transportation of employees' children to and from school or offer after-school programs. this would reduce the burden on frontline healthcare staff who are responsible for pandemic containment. in addition, hospitals may consider recruiting medical technicians as members in the emerging infectious diseases response drills in future, as this would familiarize them with various disposal processes. this would enable hospitals to commission these staff, as soon as necessary, for pandemic containment. such measures would in turn promote the response ability of medical technicians and reduce their stressors. due to workforce, financial, and time limitations, this study collected data only from one medical center and two regional hospitals in together, such findings could be used to provide appropriate support and response measures for hospital staff during a pandemic. the authors gratefully acknowledge the supports from kaohsiung medical university hospital, taiwan, grant number kmuh-c18. taiwan centers for disease control: coronavirus disease 2019 (covid-19) world health organization. global alert and response (gar): novel coronavirus infection in the united kingdom stress, 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among health care professionals during the covid-19 pandemic facing sars: psychological impacts on sars team nurses and psychiatric services in a taiwan general hospital occupational stress: toward a more integrated framework exploring the burden of emergency care: predictors of stress-health outcomes in emergency nurses analyses of burnout and musculoskeletal symptoms questionnaires and their relationships among employees in a regional hospital work-family conflict and job performance in nurses: the moderating effects of social support correlations of the "work-family conflict" with occupational stress-a cross-sectional study among university employees battle hymn of the tiger mom when middle-aged parents meet adolescent children: parent-child relationship patterns and parent's midlife perceptions survey on perceived work stress and its influencing factors among hospital staff during the covid-19 pandemic in taiwan the authors declare no potential conflict of interest. https://orcid.org/0000-0003-2699-6283hsin-tien hsu https://orcid.org/0000-0001-9638-1589che-yu su https://orcid.org/0000-0002-2946-3207 key: cord-011261-h1fzti0i authors: manning-geist, beryl; meyer, fremonta; chen, justin; pelletier, andrea; kosman, katherine; chen, xiaodong (phoenix); johnson, natasha r. title: pre-clinical stress management workshops increase medical students’ knowledge and self-awareness of coping with stress date: 2019-12-09 journal: med sci educ doi: 10.1007/s40670-019-00881-4 sha: doc_id: 11261 cord_uid: h1fzti0i objectives: to investigate the effects of a stress management workshop on medical students’ knowledge of stress and potential coping strategies. methods: a panel discussion with small group breakouts on stress in clinical medicine, learning challenges, competition with colleagues, handling stressful events, and recognizing burnout symptoms was conducted with medical students entering clerkships. a longitudinal survey design was utilized to measure pre-, post-, and long-term (3-month) changes in knowledge (impact of stress on personal health, learning, and patient care), confidence, perceived skills, and attitude (towards utilizing adaptive coping strategies) among participating students (n = 135). paired t test and multivariate analyses were performed to assess the differences between survey responses on a 5-point likert scale. results: survey response rates were pre-90.4%, post-77%, and long-term post-71.1%. compared to pre-workshop, students reported significant improvement in all four domains immediately post-workshop: knowledge (4.4 vs. 4.7, p < 0.05), confidence (3.6 vs. 3.9, p < 0.05), perceived skills (3.3 vs. 3.7, p < 0.05), and attitude (2.6 vs. 2.8, p < 0.05). compared to immediate post-workshop, students’ scores slightly decreased at 3 months but were overall significantly higher than the pre-workshop scores. conclusions: a stress management workshop can improve medical students’ knowledge of the impact of stress as well as the use of adaptive stress coping strategies. stress and burnout have been recognized as significant factors that influence medical trainees' aptitude [1] , learning [2] [3] [4] [5] , professionalism [6] [7] [8] [9] , wellness [9] [10] [11] [12] , and care of patients [8, 9, 13] . stress is a psychological and physical process that occurs when a person is faced with a demand that is perceived to exceed the resources available to respond to it effectively [14, 15] . burnout is a state of mental and/or physical exhaustion caused by excessive and prolonged stress, and can be measured through the validated maslach burnout inventory [14] [15] [16] [17] . a recent study reported that at least half of all medical students might be affected by stress and burnout during medical school [17] . dyrbye and colleagues revealed that the learning environment was significantly associated with medical student burnout; specifically, students rotating on hospital wards and those required to stay for overnight call were more likely to experience burnout [18] . psychological distress can lead to depression and thus medical students are at high risk [19] [20] [21] [22] . compared with us college graduates age 22-32, medical students were more likely to exhibit symptoms of depression and had higher levels of fatigue [23] . even more alarming is the recent finding that the prevalence of depression or depressive symptoms among medical students was 27.2% and that of suicidal ideation was 11.1% [24] . thus, more attention needs to be paid to the stress phenomenon during undergraduate medical education, including students' awareness of stress and how to improve students' stress coping skills. this is particularly important during the challenging transition from the mostly didactic-filled pre-clerkship curriculum to patient-care focused clerkships which requires medical students to adjust to new learning environments, unpredictable schedules, and different expectations. studies have investigated interventions for students to minimize the stress inherent to medical education and training and to improve wellness [25, 26] . specifically, the effects of mindfulness-based stress reduction (mbsr) training on preclinical and clinical medical students have been shown to reduce depression [27, 28] , anxiety [27] , psychological distress [27, 29, 30] , perceived stress [28, 31, 32] , and emotional exhaustion [28] and to increase empathy [27] , selfcompassion [28, 31] , mindfulness skills [28, 30] , positive mental health [30] , life satisfaction [30] , and spirituality [27] . medical students, however, use various coping mechanisms that vary by year in training and source of the stress [33] . the specific coping strategies that students use may determine the effect of stress on their academic performance and well-being [14] . however, little is understood regarding medical students' knowledge of the impact of stress on their own health, learning, and patient care. in addition, there is a paucity of literature on medical students' knowledge of coping strategies available and self-awareness of the need to utilize adapting coping strategies to prevent burnout and depression [9] . given the significant prevalence of psychological distress, burnout, and depression among medical students, it is critical for medical schools to equip students with the knowledge necessary to recognize stress, ability to determine when to seek assistance, and capacity to manage stress effectively to promote well-being and prevent burnout [9, 34, 35] . therefore, we conducted a longitudinal survey study in medical students poised to initiate clinical clerkships in order to investigate the impact of a stress management workshop on knowledge and self-awareness of stress symptoms, impact of stress, and potential adaptive positive coping strategies. a total of 135 medical students (representing one class year) were scheduled to participate in a stress management workshop at four academic hospitals affiliated with a single medical school prior to starting their clinical rotations as part of a larger transition to the principal clinical experience course (transition to clinical clerkships course). the goals of the 2-h workshop were for students to (1) demonstrate increased knowledge of the negative impact of stress on their learning, personal health, and patient care; (2) recognize symptoms of stress; (3) demonstrate increased selfawareness of adaptive coping mechanisms to reduce stress and reduce the risk of burnout; and (4) identify the value of seeking help and available resources. the standardized workshop format consisted of a 1 h didactic on stress and burnout with specific emphasis on manifestations in clinical medicine including definitions, epidemiology, time trends, hypothesized contributors, recognition in self and others, management, and approaches. in addition, the didactic highlighted the importance of seeking help and resources available to students. the second hour consisted of a panel discussion and small group breakouts on topics of role confusion, power in the hospital, benefits/risks of empathy, competition with colleagues, conflicts with other medical professionals/patients, dealing with disappointment, pimping, challenges of learning, self-care, handling stressful events, facade of perfection, and work/life balance, as well as recognizing and managing symptoms of burnout. the curriculum content was derived from the literature, a previously conducted needs assessment, and expert opinion. total population sampling was used to recruit student participants. a total of 135 medical students, representing one class year, were invited to attend a stress management workshop at their previously assigned academic hospital affiliated with a single medical school prior to starting clinical rotations. participation in the study associated with the workshop was voluntary. three surveys, including pre-workshop, immediate post-workshop, and a long-term (conducted 3 months after starting clinical rotations) post-workshop follow-up survey, were administered to the students. irb approval was obtained from partners and harvard medical school. we utilized paper and pencil surveys for the pre-and immediate post-workshop surveys. the long-term post-workshop survey was distributed with the qualtrics online survey tool, as students had started their clinical rotations in different hospitals. all surveys were anonymous and derived from validated instruments to measure students' knowledge of the negative impact of stress, symptoms of stress, utilization of positive coping strategies (derived from the coping strategies inventory [36] ), resources available, likelihood of utilizing resources, and current level of burnout, as assessed using the validated maslach burnout inventory [16, 37] . the maslach burnout inventory evaluates three aspects of perceived burnout including emotional exhaustion, depersonalization, and personal accomplishment [37] . the maslach burnout inventory is widely used to assess burnout in a variety of settings, and has been extensively validated as a tool to assess perceived burnout in medical professionals [38] . research team members reviewed and reached agreement on the final questions and construct of the survey instruments. cognitive pretesting was conducted with four medical students to ensure that students would interpret the survey questions in the intended manner. all survey instruments included identical questions to measure students' perceived changes in stress management in four domains: knowledge (impact of stress on their own health, learning, and patient care), confidence, perceived skills, and attitude (towards utilizing adaptive coping strategies to prevent burnout), which used a 5-point likert-type scale (1 = not at all, 5 = extremely). self-reported level of burnout (5point likert-type scale; 1 = completely burned out, 5 = no symptoms of burnout) was assessed in the pre-and long-term post-workshop surveys. ten questions (4-point likert-type scale; 1 = not at all, 4 = a lot) about workshop satisfaction and helpfulness were also included in the postworkshop survey. data were analyzed to determine the effects of the workshop on the study goals. statistical analyses were conducted using jmp pro statistical software (version 12) as well as stata (version 15.0). differences between pre-and immediate postworkshop responses were tested using paired t tests to assess differences between continuous variables. a p value of < 0.05 was considered significant. correlation coefficients were performed to measure the relationship between students' workshop satisfaction and their self-awareness of using positive stress coping techniques. we also calculated odds ratios to examine the association between students' reported level of burnout and other variables. power analysis was not applicable as total population sampling was used and the survey response rate was reported. the overall survey response rate was 79.5% (pre-90.4%, immediate post-77%, 3-month post-workshop 71.1%). in comparing combined ratings of 4 and 5 on the likert scale ("quite a lot" and "extremely") on pre-to immediate post-workshop responses, students' knowledge of the impact of stress on their own health, learning, and patient care increased. however, this was not completely sustained by the 3-month post-workshop survey (conducted 3 months after starting clinical clerkships). students' confidence and perceived skills towards utilizing adaptive coping strategies to handle stress (e.g., selfawareness) also improved from the pre-to immediate postworkshop, but also was not sustained by 3-month post-workshop. there was no change in attitude towards stress (seeking help is not a sign of weakness) over the 3 surveys. the trends examining impact of the workshop estimates over time (including pre-workshop, post-workshop, 3-month post-workshop) on the domains of knowledge, confidence, skills, and attitude are illustrated in fig. 1 . overall, students' mean survey responses demonstrated statistically significant improvement for knowledge about the impact of stress on their overall long-term success, health, learning and patient care, and confidence and skills in recognizing and utilizing adaptive strategies to handle stress and attitude towards seeking help increased from pre-to immediate post-workshop (t1 to t2) (see table 1 ). the difference between survey responses within the four domains from preworkshop and 3-month post-workshop (t1 to t3) was not statistically significant, except for scores on recognizing symptoms of burnout and stress within the confidence domain (see table 1 ). however, the workshop was effective in increasing students' knowledge of where to find supporting resources if they feel stressed during their clinical rotations both at the medical school (64.5% pre-workshop vs. 88.1% immediate post-workshop) and at their hospital site (18.2% pre-workshop vs. 70% post-workshop). the immediate post-workshop responses indicated that the majority of students enjoyed the workshop, learned the symptoms and impact of stress, and planned to use the knowledge of coping strategies gained from the workshop ( table 2 ). in addition, students found specific elements of the workshop to be overall helpful (improve wellness, know effects of stress, coping strategies, talking with peers). hearing classmates' concerns about the clinical year was reported to be the most helpful element of the workshop (mean score 3.21, sd ± 0.92) which occurred during the small group breakout section of the workshop. on long-term post-workshop surveying, students assigned a mean score of 2.21 (scale 1-5, sd ± 0.93) to the question "did the stress workshop help to improve selfawareness of using positive coping strategies during your clinical rotations?" we conducted spearman correlation coefficient analysis and found a moderate positive correlation (r ranged from 0.42 to 0.46, p < 0.05) between the following 5 items: satisfaction with the workshop, knowledge of stress symptoms, understanding of impact of stress, knowledge of coping strategies, and students' self-awareness of using adaptive stress coping strategies during clinical rotations in the future. confidence towards utilizing adaptive coping strategies in handling stress (e.g., self-awareness), perceived skills in addressing this stress, and attitude towards stress and seeking help (seeking help is not a sign of weakness) sd standard deviation; all with 1-5 scale; data have been re-coded; the higher rating, the better. the mean differences between pre-workshop and immediate post-workshop were compared using paired t tests. the mean differences between pre-workshop and 3-month post-workshop were also compared using paired t tests. a p value < 0.05 was considered statistically significant students were asked to rate their current level of burnout on the pre-workshop and 3-month post-workshop surveys (3 months after starting their clinical clerkships, scale 1-5; 1 = completely burned out, 5 = enjoy my work). there was a higher level of self-reported burnout at 3 months (mean 3.69 sd ± 0.84) compared to pre-workshop (mean 3.86 sd ± 0.83). we found no statistically significant relationship between students' self-reported burnout at 3 months and knowledge, confidence, perceived skill, or attitude scores. students who disagreed that asking for help was a sign of weakness were statistically more likely to have self-reported burnout at 3-month post-workshop (or 1.93, 95% ci 1.13-3.28) ( table 3 ). our study findings suggest that the 2-h stress management workshop had a positive impact on medical students' knowledge towards the symptoms and impact of stress as well as on their confidence, perceived skills, and attitude of using adaptive stress coping skills during their clinical rotations. students' self-reported knowledge, confidence, and perceived skills increased immediately after the workshop; however, this improvement was largely not durable at the 3-month postworkshop time point. it is possible that students' recall of the knowledge learned in the workshop was compromised by the high learning demands of clinical rotations. to prevent the knowledge decay, programs could incorporate a series of mindfulness-based stress training sessions [25] [26] [27] [28] [29] [30] [31] [32] and accessible support in addition to a one-time stress management workshop to enhance students' knowledge retention and skill acquisition. results of the pre-workshop survey illustrate that students generally demonstrated a high level of knowledge about stress symptoms and their impact, followed by confidence and perceived skills to cope with stress, and attitude towards seeking help. these are consistent with the immediate post-workshop survey results about workshop satisfaction and helpfulness: students rated hearing their peers' concerns and learning useful stress coping strategies as the two most helpful educational contents of the workshop. these findings suggest that future stress management workshops should focus on introducing stress coping skills and provide a safe environment for students to share concerns and experiences. students' self-reported burnout increased by 3 months compared to pre-workshop levels (3 months after starting clinical clerkships) and did not correlate with their knowledge about q questions, sd standard deviation; all with 1-4 scale, 1 = "not at all," 2 "slightly," 3 = "moderately," 4 = "very" or odds ratio; or = 1, exposure does not affect odds of outcome; or > 1, exposure associated with higher odds of outcome; or < 1, exposure associated with lower odds of outcome. sign of weakness was re-coded (5 = strongly disagree asking for help is a sign of weakness); *only compared students who were matched across 3 surveys (n = 70) stress, confidence, or perceived skill in coping with stress. burnout was correlated with students' attitude towards seeking help: students who were more open to seeking help were twice as likely report burnout at 3 months. this finding may be identifying premorbid depression, anxiety, or other mental health concerns; perhaps students who have already sought help in the past and are not opposed to reporting difficulty or seeking help in general. this highlights that it is critically important for students to know where to find resources should they need to seek help. further, this finding may also reveal students who are empathetic and thus more likely to talk about their feelings and seek help. one theory is that a high level of empathy may cause "compassion fatigue" which then leads to emotional exhaustion and burnout [39] . future studies are needed to further investigate the relationship between empathy and burnout among medical students, including whether increased screening for burnout should be used in students who are more likely to ask for help. our study has limitations, as previously described [40] . first, all participants are from a single medical school; results may therefore not be generalizable. second, all survey results represent students' self-reported knowledge, confidence, perceived skills, and attitude, and do not measure actual performance or changes in behaviors. as these data are self-reported, there is an unavoidable potential for social desirability bias. in conclusion, the stress management workshop had a positive impact on medical students' knowledge, confidence, perceived skills, and attitude of using adaptive stress coping strategies. programs are encouraged to empower students to effectively self-manage stress and prevent burnout during clinical training by providing additional interventions after a stress management workshop to increase students' knowledge retention. empathy in medical students as related to academic performance, clinical competence and gender medical student stress and clerkship 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high-stress clinical environment: stressors associated with medical students' clerkship training on labor and delivery publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that there is no conflict of interest. key: cord-302254-egt9qdib authors: amaral-prado, heloísa monteiro; borghi, filipy; mello, tânia maron vichi freire; grassi-kassisse, dora maria title: the impact of confinement in the psychosocial behaviour due covid-19 among members of a brazilian university date: 2020-11-08 journal: int j soc psychiatry doi: 10.1177/0020764020971318 sha: doc_id: 302254 cord_uid: egt9qdib background: the current situation due covid-19 may cause an eminent impact on mental health because the confinement restrictions. aims: the aim of this study was to analyze and compare perceived stress, resilience, depression symptoms and coping strategies on the members of university of campinas, in brazil, before and during the outbreak of the covid-19. methods: volunteers over 18 years of both sexes, members of the university of campinas (unicamp) in brazil answered instruments related to perceived stress, depression, resilience and coping strategies during final exams at the end of semester during 2018 to 2020. results: we obtained 1,135 responses (893 before covid-19 and 242 during covid-19). the volunteers did not show significant differences for perceived stress, depressive signs and resilience before and during the pandemic. in both periods, men exhibited lower scores for perceived stress and depression and higher scores for resilience when compared to women. undergraduate and graduate students exhibited higher perceived stress scores, more pronounced depressive signs and lower resilience, and employees and professors presented lower scores for perceived stress, depressive signs and greater resilience. conclusions: these first months of confinement did not directly affect the scores of perceived stress, depression and resilience, however, each subgroup adapted to the new routine by changing the coping strategy used. this study suggests the importance of monitoring the mental health of member in the university, especially in times of epidemic, in the search for policies that aim to improve the resilience of the population and seek positive and effective coping strategies within the university environment. the coronavirus disease 2019 was firstly detected in wuhan (hubei province, china) in december 2019 (odriozola-gonzález et al., 2020) . however, the pandemic reached latin america later than other continents (lancet, 2020) . until 29th october 2020, brazil has recorded 158,969 deaths and 5,494,376 confirmed cases of covid-19 since the beginning of the pandemic, according to the national council of health secretaries (conass). this panorama confirms brazil as the second country in the world with the highest number of cases and deaths in the pandemic of the new coronavirus. in the absence of a vaccine, several countries have implemented a series of interventions to reduce contagious and decelerate progression of the pandemic (aquino et al., 2020) . one of the containment measures was the total confinement of the population in their homes, also known as lockdown, which led to the disruption of most daily activities (ruiz-roso et al., 2020) . however, no national lockdown has been established in brazil, but some communities and urban areas did declare a mandatory quarantine at different times. confinement influences lifestyle and may induced many psychiatric individual and collective problems such as panic, anxiety, depression, post-traumatic stress disorders, among others symptoms (jakovljevic et al., 2020) . efforts to reduce the spread of the covid-19 virus among the younger and adult populations has prompted the suspensions of activities of schools, colleges, universities and other educational institutions in many countries (sahu, 2020) . the sudden change in the university's routine generated several new demands, such as the use of technological resources that had never been used before to carry out classes and administrative activities, as well as remote work and/or study. the university environment exposes its population to many situations linked to psychological distress (byrd & mckinney, 2012; pidgeon et al., 2014) . the enhancement of resilience and coping strategies become even more important in this pandemic moment for better management of daily adversities, preventing the emergence of physical and mental health problems in the future, such as depression (lupe et al., 2020; vinkers et al., 2020) . the current situation may cause an eminent impact on perceived stress, resilience, depression and coping strategies in this population due to the isolation restrictions, since all these topics are correlates (mahmoud et al., 2012; moreno-fernandez et al., 2018) . given the expected impact of the situation due the confinement and covid-19 crisis, the aim of this study was to analyze and compare perceived stress, resilience, depression symptoms and coping strategies on the members of university of campinas, in brazil, before and during the outbreak of the covid-19. volunteers over 18 years of both sexes, members of the university of campinas (unicamp) in brazil were invited through the communications channels of the university and social media. the volunteers signed the free and informed consent, declaring understanding on the procedures that would be performed during the protocol. the study was conducted according to the guidelines laid down in the declaration of helsinki and ethical approval was granted from research ethics committee of the school of medical sciences/university of campinas (caae: 97370018.0.0000.5404). the volunteers were divided into four categories: undergraduate, graduate students (master and phd), employees and professors. all data were collected during the final exam/test month at the end of the semester (june and december) in two different periods: before covid-19 (2018 covid-19 ( -2019 and during the outbreak of the covid-19 (2020). the participants answered five questionnaires that were validate for portuguese language. the first questionnaire was a sociodemographic questionnaire to identify the volunteer's sex, age, grade and period. the perceived stress scale from sheldon cohen is the most widely used psychological instrument for measuring the perception of stress (cohen et al., 1983) . it is a measure of the degree to which situations in one's life are appraised as stressful. items were designed to assess how unpredictable, uncontrollable, and overloaded respondents find their lives to be. we used the brazilian version with 14-question version, which total score is 56, with higher scores indicating a greater level of stress (luft et al., 2007) . the patient health questionnaire-9 (phq-9) is a nine item questionnaire designed to screen for depression in primary care and other medical settings (levis et al., 2019) . the total score ranges from 0 to 27, which the standard cut-off score for screening to identify possible major depression is 10 or above (arrieta et al., 2017) . the connor-davidson resilience scale (cd-risc10) is a questionnaire based on connor and davidson's operational definition of resilience, which is the ability to 'thrive in the face of adversity'. since its development in 2003, the cd-risc has been tested in a several contexts with a variety of populations. this work propose to work with the validated 10-item version of the measure validated for portuguese language, called the cd-risc10brasil, which total score is 40, with higher scores indicating a greater resilience (lopes & martins, 2011) . the folkman and lazarus inventory of coping strategies includes the thoughts and actions people used to handle the internal or external demands of a specific stressful event. it is a list of 66 items answered using a likert-type scale and the items are split into eight factors: confront, distance, self-control, social support, acceptance of responsibilities, escape-avoidance, problem solving and positive reappraisal (folkman et al., 1986; pompeo et al., 2016) . this scale is not associated with a total score as a sum for assessment, as the items should be assessed using relative scores within each factor (pompeo et al., 2016; vitaliano et al., 1987) . all data were collected and managed using redcap © electronic data capture tools hosted at unicamp. data are presented as means ± sem. the normality was confirmed by d'agostino-pearson test. for parametric intragroup and intergroups comparisons, we performed one-way anova followed by tukey. for non-parametric data, we performed friedman followed by dunn's for intragroup analysis and kruskal-wallis followed by dunn's for intergroups analysis. two-tailed pearson's partial correlation for parametric data and spearman's partial correlation for non-parametric data was performed for correlations. all statistical analysis was done with graph pad prism version 8.00 (graph pad software, san diego, california, usa). the acceptance level of significance was set at p < 0.05. this study obtained 1135 responses from volunteers' members from the university of campinas (unicamp). when separated by periods, 893 volunteers enrolled the surveys before the pandemic and 242 volunteers enrolled the surveys during the outbreak of the covid-19. demographic data showed a greater participation of women and individuals aged between 18 and 34 years. considering the university situation, undergraduates were the one that most adhered to participate in the research, followed by graduate students, employees and professors ( table 1) . the university members of this study did not show significant differences between the scores for perceived stress, depressive signs and resilience for the periods before and during the outbreak of the covid-19. in both periods, men exhibited lower scores for perceived stress and depression and higher scores for resilience when compared to women. volunteers who chose not to identify themselves as men or women were placed in a category named other. in the period before covid-19, this category showed higher scores of perceived stress and depressive signs when compared to men. compared to women, this category presented only higher scores for depressive signs. however, during the covid-19 period, these differences were not observed (table 2) . before covid-19 period, undergraduate exhibited higher scores for perceived stress and depressive signs and lower resilience scores when compared to employees and professors. this behaviour was also observed during the epidemic, but the difference in the resilience score when compared to employees ceased to exist. graduate students exhibited higher scores for perceived stress and depressive signs and lower resilience scores in the period before covid-19 when compared to employees and professors. however, during the covid-19 period, the difference between them and employees was no longer observed when the depressive signs and resilience scores were compared. employees and professors did not exhibit differences in perceived stress, depressive signs and resilience between them in any period (table 2) . for both periods analyzed, lower resilience scores lead to higher perceived stress and more accentuated depressive signs, as well as the greater resilience leads to an attenuation of the depressive signs, regardless of sex, university situation and period analyzed (table 3) . before the covid-19 scenario, the coping strategy most often used by undergraduates was acceptance of responsibilities, and the least used was confront. for graduate students, the coping strategy most often used was social support, and the least used was confront. for employees and professors, the coping strategy most often used was problem solving, however, the least used strategy for employees was confront and for professors was escape-avoidance. during the epidemic, almost all groups presented new coping strategies. in the confinement period, undergraduates and graduate students started to choose the self-control as the lead coping strategy. as the least used strategy, undergraduates opted for positive reappraisal and graduate students for confront. employees showed no changes in the most often and least used coping strategies in this new scenario. professors started to use problem solving as the most often coping strategy but kept escape-avoidance as the less used strategy (table 4) . as a next step, we performed correlations between the most used coping strategies in each group with the scores obtained in the other psychosocial instruments. in times before the pandemic, undergraduates exhibited a positive correlation with stress and depressive signs. thus, there is a trend towards higher scores of perceived stress and depressive signs with greater use of accepting responsibility as a coping strategy in this group. graduate students did not show any significant correlation with the psychosocial instruments used in this study. employees and professors showed a negative correlation for perceived stress and a positive correlation for resilience. however, only employees showed a negative correlation between coping strategy and phq-9. contrary to undergraduate students, the use of problem solving as a coping strategy leads to lower scores of perceived stress, depressive signs and greater resilience. during the pandemic, undergraduate and graduate students did not show significant correlations. employees and professors maintained the same coping behaviour observed before the epidemic (table 5 ). the ongoing covid-19 pandemic forced the world to take protective measures such as lockdown of cities, travel bans, confinement and social distancing (jakovljevic et al., 2020) . however, literature suggests that restrictive measures such as quarantine, isolation and social distancing, have an impact on psychological wellbeing of people as well as emotive reactions to pandemic itself (rubin & wessely, 2020; talevi et al., 2020) . in this fashion, it was expected that the population into the university would be negatively impacted, increasing the stress experienced at the end of the semester. however, the scores for perceived stress, depression and resilience were not affected. what has changed is the way that each group deals with the new situation caused by covid-19, highlighting the importance of the coping strategy as a quick response damper to keep the new routine without major losses. studies have documented differences between women and men with respect to symptom reporting, treatment seeking, coping strategies and several neurobiological variables pertinent to depression (nayak et al., 2019; wellman et al., 2018) . it has been shown that women are twice as likely as men to develop depression and suffer stress, in addition to presenting less resilience (nayak et al., 2019) . the unicamp community follows this same pattern described in the literature, with high scores for perceived stress, depressive signs and low score for resilience when compared to men. in our study, individuals who identified themselves with another option for sex or did not want to identify themselves with either sex exhibited a high score for perceived stress and depressive signs, and low scores for resilience. these data highlight the importance of further studies with this portion of the population for a better understanding of how to help deal with day-to-day problems. discrimination with this minority of the population is still present today, which can be one of the aggravators for the highest scores in these individuals. thus, the importance of inclusion measures becomes even more important. the correlations observed before and during the covid-19 pandemic in this study corroborate with studies from other countries, which also point to a positive correlation between perceived stress and depression and a negative correlation between stress and resilience (kermott et al., 2019; miceli et al., 2019) . positive coping strategies, which allow better coping with stress, enable the development of greater resilience, and consequently greater wellbeing for the individual. we observed that the average scores obtained in this study for perceived stress and depressive signs are higher the average of other populations studied (ali et al., 2019; polinski, 2020) . high perceived stress can be associated with physical and psychological health problems, as well as with emotional intelligence and coping strategies employed (enns et al., 2018) . in general, the unicamp community showed different profiles among classified subpopulations, with undergraduate and graduate students with the lowest performance: higher perceived stress scores, more pronounced depressive signs and lower resilience. in the normal routine of the university, the undergraduates used acceptance of responsibilities as the main coping strategy, while graduate students used social support. during the pandemic, both groups started to use selfcontrol as the main coping strategy. this change can be triggered by social isolation and by the transition from face-to-face teaching to online delivery. the use of self-control as the main coping strategy of this part of population becomes an important tool to face the new adversities, since the coping strategies previously used, in some way, have become limited due to the isolation predicted by the pandemic. however, regardless of the periods analyzed, these strategies were not shown to be effective enough for improvement or good management and coping with stressors. groups that have employment relationship with the university, such as employees and professors, showed the best performances: lower scores for perceived stress, depressive signs and greater resilience. these subgroups of the community used the problem solving as main coping strategy in both periods analyzed. the negative correlations between problem solving with perceived stress and depressive signs questionnaires, as well as the positive correlation between problem solving and resilience, indicate a better management of day-to-day stressors when these subgroups applied this strategy. therefore, the coping strategy focused on the problem, seeking to change and resolve the conflict, such as problem solving, proved to be effective. professors were the group that had the best performance among all. regardless of the period, they were the least to use escape-avoidance as a coping strategy when compared to other groups. this fact may indicate that this strategy may have a relationship with a possible increase in perceived stress and depressive signs, as well as less resilience, since the other subgroups showed greater use of this strategy compared to professors. the literature indicates escape-avoidance as a negative adaptation of human behaviour in dealing with stressful situations, which can lead to feelings of isolation and loneliness, and as a consequence the presence of apathy and demotivation to perform the activities that the individual needs to perform, such as example, academic or professional activities (pruessner et al., 2020) . studies in many populations have alarmed the impact of social isolation on mental health caused by covid-19. thus, the pandemic period stimulated many cross-sectional studies on mental health, mainly in groups without previous studies. these studies can often highlight findings due to the fact that there are no previous comparisons. cross-sectional studies with health-care workers from uk during covid-19 alarmed to the great incidence of mental health problems, however, longitudinal studies with prepandemic and postpandemic groups found no increase in mental distress among health-care workers due to covid-19 compared with the general population (lamb et al., 2020) . therefore, this type of information highlights the importance of longitudinal assessments that allows to track changes and better assess the situation without unnecessarily pathologizing common responses. despite the limitations, this is a cross-sectional study conducted at a brazilian university that started before an unprecedented situation, which allowed a screening of the new situation within the university. this study was performed in only university from brazil, which may contribute to some bias in the study results. the adherence from other universities in the same model of this study may extend and generalize the findings. moreover, the present study utilized self-report measures that considered the framework of an individual's subjective perception of perceived stress, depression and resilience. regardless of the emerging context of covid-19, the unicamp community exhibited high scores for perceived stress and depressive signs and low scores for resilience when compared to studies around the world. this population have been specially impacted by the covid-19 confinement, but, in these first months of confinement, it did not directly affect the scores of perceived stress, depression and resilience. on the other hand, each subgroup adapted to the new routine by changing the coping strategy used. groups with lower performances, such as undergraduate and graduate students, opted for new strategies to face adversity, while groups with the best performances, such as employees and professor, already presented efficient strategies and did not feel obliged to discover new ways to face the new situation imposed by covid-19. this study suggests the importance of monitoring the mental health of member in the university, especially in times of epidemic, in the search for policies that aim to improve the resilience of the population and seek positive and effective coping strategies within the university environment. heloísa monteiro amaral-prado 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neural substrates of emotion and motivation the authors wish to thank all the volunteers. part of these results were presented in the xxvii congresso de iniciação científica da unicamp (pibic) -2019 as a research reported poster. we state there are no competing financial interests in the work described. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported by coordenação de aperfeiçoamento de pessoal de nível superior -brasil (capes -finance code 001), conselho nacional de desenvolvimento científico e técnológico (pibic/cnpq) and fundação de amparo à pesquisa do estado de são paulo (fapesp). key: cord-349219-tl97bj42 authors: rayhan arusha, anowara; kishore biswas, raaj title: prevalence of stress, anxiety and depression due to examination in bangladeshi youths: a pilot study date: 2020-07-18 journal: child youth serv rev doi: 10.1016/j.childyouth.2020.105254 sha: doc_id: 349219 cord_uid: tl97bj42 an estimated 25% people suffer from mental health disorders worldwide. almost 7 million suffer from anxiety and depression in bangladesh. there are several factors that can cause stress among youths, both academic and non-academic, ranging from socioeconomic, environmental, cultural and psychological attributes. however, these are not widely researched in bangladesh. this study identified the factors that affect the mental health of students due to examinations in bangladesh, particularly the socio-demographic, lifestyle and psychological factors. an online cross-sectional survey was conducted on may 2020 with a sample size of 210 tertiary level students in dhaka. a modified dass-21 was used to measure stress, anxiety and depression scores related to examination. binary logistic model showed that those who lived with family, spent time with parents, had regular sufficient (self-assessed) sleeps and consumed balanced (self-assessed) diets had significantly lower stress, anxiety, and depression. balanced lifestyle with greater social bonding might help to better equip youths to reduce stress, anxiety, and depression during examination, which could be an avenue for future intervention studies. one in four people suffer from mental or neurological disorders worldwide (world health organisation, 2001) , including 264 million who suffer from depression (world health organization, 2020a) . a person with depression often functions poorly at work, school or in family environment, which, at its worst, can lead to suicide. due to depression, suicide is the second leading cause of death for 15-29 year-olds (world health organization, 2020a). the global prevalence of moderate to extreme levels of depression is 60.8%, anxiety 73%, and stress 62.4% (bayram and bilgel 2008; beiter et al. 2015; kulsoom and afsar 2015) . although not widely discussed, mental health issues are increasingly becoming a threat to low-and middle-income countries such as bangladesh. almost 7 million people in bangladesh experience depressive and anxiety disorders respectively (world health organization, 2020b ). an estimate of 10,167 committed suicide in 2012, and suicide attempts were considered by 4% of boys and 6% of girls by youths aged 13-17 years (world health organization, 2020b) . in bangladesh, the levels of depression, anxiety, and stress has been reported to be as high as 54.3%, 64.8%, and 59.0%, respectively (hossain et al., 2014; alim et al., 2017; saeed et al., 2018; mamun and griffiths, 2019; mamun et al., 2019) . there are myriads of factors that can cause stress among students, both academic and non-academic, ranging from socioeconomic, environmental, cultural to psychological attributes (brand and schoonheim-klein, 2009 ). stress levels vary across students depending on symptoms of anxiety, especially during examination periods. a prevalence rate of 10-35% of university students experience "functionally impairing levels of test anxiety" (chapell et al., 2005; neuderth, jabs and schmidtke, 2009) . students with test anxiety are more likely to delay and drop-out in university, which could lead to suicidal behaviors and high economic costs (schaefer et al. 2007) . it can be bidirectional as majority of students with stress display poor self-confidence and often poor academic performance (sohail, 2013; baste and gadkari, 2014) . several contributing factors of stress, anxiety, and depression among students were identified in literature including sex, strained relationships, family and peer pressure, high parental expectation, lack of financial support and hardships, sleep deprivation, future worries, loneliness, longer screen time, toxic psychological environment, academic pressure, workload, size of the academic curriculum, and heavy test schedules (brenneisen mayer et al., 2016; abdel wahed and hassan, 2017; saeed et al., 2018; silva and figueiredo-braga, 2018; ul haq et al., 2018; mamun and griffiths, 2019) . some of the major academic stressors for students include examinations, time demands, competition and class environment while the most common personal stressors have been intimate relationships, finance and parental conflicts (murphy and archer, 1996) . the stress of examinations and mental health of students are correlated. according to bayram and bilgel (2008) , exam stress and anxiety has negative impacts on students' academic achievement, physical health & development and standard of life. causes of exam anxiety can be associated with social stigma, where students from a rural background could feel incompetent compared to those who grew up in the metropolitan, coupled with the fear of not having competitive skills in english, the general medium for higher education in bangladesh, can demotivate them prior to and during exams which could lead to anxiety, stress, and frustration. higher education in recent times involves considerable financial burden (callender & kemp, 2000) . roberts et al.'s (1999) cross-sectional survey indicate that financial difficulties often lead to mental disorders such as depression for students. roberts et al.'s (1999) cross-sectional survey indicate that financial difficulties make students particularly vulnerable to depression. of all the adverse experiences assessed, financial hardship was the only one to show an independent relationship with depression when pre-entry anxiety and depression were controlled which indicates that the direction of causality is more likely to be from financial problems to depression than vice versa (andrews and wilding, 2004) . moreover, excessive parental control is assumed to inhibit the development of children's autonomy, which leads to perceptions of the environment as "uncontrollable and a limited sense of personal competence or mastery" (chorpita, albano and barlow, 1996; hudson and rapee, 2001; dadds, 2002; barlow, 2004; chorpita, brown and barlow, 2016) . in turn, these factors are postulated to contribute to the triggering anxiety in children. there is a knowledge gap concerning mental health problems in bangladesh. in 2019, four suicides and one suicide rescued among students attracted media and public attention nationwide, which suggested a need to examine the existing mental health issues among students in bangladesh (shamsuddin et al., 2013; arafat and al mamun, 2019) . to contribute to that gap, the present study investigated the prevalence of depression, anxiety, and stress among bangladeshi students and their associated risk factors with respect to socio-demographics and lifestyle measures. the objective of the study was to identify factors that affect the mental health of students due to examinations, particularly the socio-demographic and psychological factors using a survey conducted in dhaka, bangladesh in 2020. there are several theories that discuss risk factors associated with individual mental health. these include the social ecologic theory, the social cognitive theory, as well as the social stress model, among others (van praag et al., 2009; yen, michael and perdue, 2009 ). the present study was framed by the social ecologic theory and the social cognitive theory. the social ecologic theory suggests that individual and environment factors are interrelated and that good health can be achieved with behavioral, economic, and social factors promoting it (krieger, 1994; cohen, scribner and farley, 2000) . thus, the social ecologic theory suggested that a person living in a neighborhood with low socioeconomic status (ses) and low social support is expected to have worse health outcomes than someone from a better environment. the theory posits that environmental factors, socio-demographic factors in the current study context, influence the mental health of students. the most common socio-demographic factors discussed in the literature are the student's age, sex, place of residence, education, parents' education, family income, personal income, relationship status, frequency of contact with family and extra-curricular activities. social cognitive theory links these socio-demographic factors with the thought processes of students that contribute to their emotional, behavioral and psychological development such as seeking help, reading books, engaging in volunteering activities, getting sufficient sleep, maintaining a balanced diet, getting regular exercise and practicing relaxation techniques.. the theory also suggests that good health is linked to individuals having beliefs that they can achieve healthy outcomes, having goals to achieve these outcomes as well as having expectations of these outcomes. (bandura, 2004; fisher et al., 2004) . [ figure 1 . theoretical hypothesis for the study based on social ecologic and social cognitive theories.] an online survey was conducted among undergraduate and postgraduate students from tertiary institutions of dhaka, bangladesh. due to the covid-19 pandemic in bangladesh, the authors had to settle for online option and could only collect quantitative data. a total of 210 youths aged between 17 to 25 years participated in the survey using google online survey platform after receiving ethical clearance from university of dhaka, bangladesh. the data was collected during may 2020. the survey questionnaire included sociodemographic information as well as dass 21 scale which measured examination stress, anxiety and depression, and possible coping mechanisms. demographic information included sex, residence before joining university, current living status, relationship status, and contact with parents. the depression, anxiety and stress scale -21 items (dass-21) is a set of scales designed to measure the psychological states of depression, anxiety and stress. there are 7 items in each of the three dass-21 scales, divided into subscales with similar content. the depression scale assesses dysphoria, hopelessness, devaluation of life, self-deprecation, lack of interest/involvement, anhedonia and inertia (lovibond and lovibond, 1995) . the anxiety scale assesses autonomic arousal, skeletal muscle effects, situational anxiety, and subjective experience of anxious affect during examination. it examines the difficulty to relax, nervous arousal, and being easily upset/agitated, irritable/over-reactive and impatient (lovibond and lovibond, 1995) . scores for the three emotional states of mind are calculated by summing the scores for the relevant items. the stress/anxiety coping mechanism included questions regarding whether the participant ever sought help for depression/anxiety/stress, reading habits, engagement in volunteering activities, whether the participant made any effort to replace negative thoughts with positive ones, and self-assessment on quality of sleep and eating habits. bivariate distribution of the relevant variables across dass stress, dass anxiety and dass depression were quantified. the primary associations were evaluated using t-tests or analysis of variance (anova). to assess the relationships between the dass scores and socio-demographic variables, linear regression model was fitted. these models included only the variables that were found significant in the primary bivariate associations. p-value < 0.05 was considered as threshold for the level of significance. all statistical analysis was conducted with r (version 3.5.0). the online survey resulted to 210 responses. in the sample 169 were studying in undergraduate degrees, 40 in master's degrees and one in high school and aged between 17 and 25 years. the validity of the three dass scores were assessed using cronbach's alpha. the alpha values for stress, anxiety and depression were 0.84 (95% ci: 0.81 ~ 0.84), 0.80 (95% ci: 0.76 ~ 0.84) and 0.88 (95% ci: 0.85 ~ 0.90) respectively. these show that the scores were reliable. and balanced diet were fitted to linear regression model. the linear regression models observed a significant association of contact with parents with stress and depression (table 2) . those who kept contact with parents only once or twice a month were more likely to have higher examination stress and depression respectively compared to those who contacted daily with their parents. similarly, sleeping pattern was significantly (p < 0.05) associated with stress and depression during examinations. those who had sufficient sleep were less likely to have higher stress or depression compared to those who self-reported of insufficient sleep. only diet was associated with anxiety (table 2) . those who had balanced diet were likely to have lower anxiety during examination period compared to those with unbalanced diet. also, anxiety was marginally associated (p < 0.058) with living status, that is those living without family were more likely to experience anxiety than those living with it. for all the linear regression model, generalized varianceinflation factors (gvif) were computed and all scores were under 2, which means there were no multicollinearity in the models (fox and monette, 1992) the results showed that living with family, time spent with parents, sleeping patterns and diets were significantly associated with mental health factors. given the current study evaluated students who only had mild or moderate stress, anxiety or depression, it could be argued that a balanced lifestyle with sound social communication would help students deal with stress, anxiety and depression related to examination. talking with parents daily seemed to be a stress relieving mechanism among youths, as results indicate. students who talked to their parents daily over phone had the lower stress and depression scores resulting from examinations compared to those who talked 1-2 times a month. a pilot study of children with separation anxiety disorder (choate et al., 2005) found that a parent-child intervention designed to improve the attachment bond resulted in children no longer fitting criteria for separation anxiety disorder in most cases. that would work for examination stress as well, given, a student who shares the concerns are more likely to receive support and encouraging behaviors from their parents. social bonding has proved to be a good depression coping mechanism (karriker-jaffe, foshee and ennett, 2011). thus, improving the parent-youth attachment, particularly for academic examination related mental health issues, could be an avenue to alleviate excess anxiety and depression. students who generally had sufficient sleep regularly had lower examination related stress and depression compared to those who never had sufficient sleep. recent epidemiological studies suggest that insomnia is not just a typical symptom of depression, rather it could be an independent risk factor for depression in the long run (hohagen et al., 1993) . although this study did not evaluate a bidirectional relationship, depression is considered to be one of the most frequent and prominent causes of insomnia (hohagen et al., 1993) . however, academic performance might not necessarily be hindered due to insomnia (taylor, d.j., bramoweth, a.d., grieser, e.a., tatum, j.i. and roane, 2013) but lack of adequate sleep was found to be associated with a range of impairments in terms of academic functioning (hysing, m., pallesen, s., stormark, k.m., lundervold, a.j. and sivertsen, 2013) . a disruptive sleeping pattern or lack of it during examination period would suggest higher likelihood of stress, which would result in a deterioration in academic performance. many young adults face traumatic experiences and mental illnesses during university period. higher consumption of food due to anxiety is common. more often, anxiety leads to unbalanced diet and overconsumption of processed food, which hampers the bodily functions (kemp, bui, and grier 2013; weng et al. 2012) . in one study, m. hossain, naher, and shahabuddin 2005, found that participants who had healthy food habit had the lowest scores of anxiety, stress and depression respectively whereas students who did not have healthy food habit had the highest scores. food habit is also affiliated with students who live without family, more often they rely on campus canteens or substandard dormitory cafes. this could lead to less than adequate nutrient intake, particularly when studies have repeatedly questioned nutrition in food sources in bangladesh (hossain, naher and shahabuddin, 2005; khan et al., 2019) . all these are compounded by factors such as financial condition, sufficient resources and facilities which play a major role here. higher levels of examination anxiety were observed for students who lived without their family compared to those who lived with their family. results indicated that youths with higher anxiety level perceived their mothers and fathers as being more socially isolating, more concerned about others' opinions, more ashamed of the students' shyness and poor performance, and less socially active than youths with low anxiety level (caster et al., 1999) . staying with family reduces their stress, anxiety and depression, particularly during examination, as they could share some responsibilities with their families and focus more on academic workload. this, in turn, relieves some anxiety and improves their mental health. there were a few limitations to this study. first, there was no active identification of severely stressed individuals to be included in the survey, which might have observed stronger effect sizes in the models. second, this study was limited to only quantitative data, which means future studies could consider in-depth interviews to explore more focused examination related stress factors. also, as the sample size was small, the beta scores in the regression model might not be adjusted to reflect population estimates. thirdly, as the data was cross-sectional, there is hardly a scope to assess a causal link. based on the study findings, some intervention studies can explore the causal link between healthy lifestyle and mental health related factors during examination in bangladesh. moreover, future studies can consider calculating quality-adjusted life year (qaly) & disability-adjusted life year (daly) and assess their impacts. fourthly, the financial barrier between public universities and private institutions in bangladesh could act as a stressor, which was not collected in the current survey. finally, the sample was limited to only youths pursuing higher education in dhaka, bangladesh. for more generalized conclusion, wide scale surveys with funding would be necessary. examinations have a negative effect on the mental health of students in terms of stress, anxiety, and depression. this study explored some demographic factors associated with mental health for bangladeshi tertiary level students. balanced lifestyle with greater social bonding might help better equip fighting stress, anxiety, and depression during examination. future research could examine the relationship between academic results and its association with stresses and psychological disorders. amidst the high density of infections and non-communicable diseases, mental health is still not highly reorganized in bangladesh. implementation of stress management workshops, improvisation of academic environment, use of problem-focused and emotion focused strategies at the policy level should be targeted. moreover, students need assistance and co-curricular programming to help them develop "stress-related coping strategies" attuned to performance and the outcomes borne regarding the effects of stress, in particular regarding eustress, where a positive impact has been cited with respect to the effects of 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anxiety, and depression in pharmacy students stress and academic performance among medical students epidemiology of insomnia in college students: relationship with mental health, quality of life, and substance use difficulties psychometric study of depression, anxiety and stress among university students is there any relationship between dietary patterns and depression and anxiety in chinese adolescents? mental disorders affect one in four people, world health organization world health organization (2020a) depression, world health organization world health organization (2020b) mental health: current mental health situation in bangladesh neighborhood environment in studies of health of older adults. a systematic review key: cord-312362-i18rlo2r authors: yan, linlin; gan, yiqun; ding, xu; wu, jianhui; duan, hongxia title: the relationship between perceived stress and emotional distress during the covid-19 outbreak: effects of boredom proneness and coping style date: 2020-10-29 journal: j anxiety disord doi: 10.1016/j.janxdis.2020.102328 sha: doc_id: 312362 cord_uid: i18rlo2r the outbreak of a novel coronavirus (covid-19) pandemic was a great threat to the physical and mental health of the general population. our research aimed to investigate the relationship between perceived stress and emotional distress during the initial outbreak. furthermore, potential risk and protective factors, i.e., coping and boredom proneness, of stress-related emotional distress were also explored. data from 3233 participants in china were collected through an online survey platform during the initial outbreak of the covid-19 from january 31 to february 9 in 2020. the results showed that higher perceived stress was associated with more emotional distress including depression, fear, compulsion-anxiety, neurasthenia, and hypochondria. boredom proneness significantly and positively mediated the relationship between perceived stress and emotional distress. moreover, coping style moderated the stress-emotional distress relationship, i.e., individuals who mainly adopted positive coping strategies suffered fewer symptoms of depression, compulsion-anxiety, and neurasthenia under stress, while negative coping strategies aggravated emotional distress. the results from the present study provide practical value for mental health intervention during the emergent public health events. since pandemic (duan & zhu, 2020) . a recent online survey found that moderate-to-severe stress, anxiety, and depression were noted in 8.1%, 28.8%, and 16.5%, respectively, however, few studies explored the relationship between perceived stress related to the current pandemic and emotional distress. boredom was considered as one of the most relevant stressors in those who had experienced isolation during the pandemic (presti et al., 2020). the tendency to experience boredom or boredom proneness might be one of the potential variables that explain emotional distress during lockdown time. boredom was defined as a state that relates too low arousal with dissatisfaction due to perceived monotony and repetition (mikulas & vodanovich, 1993) . it was proposed that disengagement from the environment is a key contributor to the boredom feeling (eastwood et al., 2012) . boredom proneness refers to the propensity toward experiencing boredom, and it will vary across individuals (farmer & sundberg, 1986) . individuals with high scores on the boredom proneness scale (trait) were likely to report more frequent boredom experience (state) (mercer-lynn, bar, & eastwood, 2014) . previous studies indicated that boredom proneness was related to higher stress levels (wang, 2015; elhai et al., 2018; lee & zelman, 2019) . boredom proneness had also been demonstrated to be associated to emotional distress including depression, anxiety, and fear (farmer & sundberg, 1986 ; lepera, 2011; leong & schneller, 1993) . similarly, sommers and vodanovich (2000) found that individuals with a higher level of boredom proneness reported higher scores on the hopkins symptom checklist (derogatis et al., 1974) including obsessive-compulsive, somatization, j o u r n a l p r e -p r o o f anxiety, interpersonal sensitivity, and depression. people who scored higher on boredom proneness felt less control of themselves and thus persist disengagement from their environment (eastwood et al., 2012; isacescu, struk, & danckert, 2017) . in turn, uncontrollability and disengagement might lead to an emotional response to a demanding situation. therefore, boredom proneness might play a mediating role in an individual's perceived stress to the covid-19 pandemic and their emotional response to it. one of the factors which had been demonstrated in the effectiveness of mitigating the relationship between stress and mental health was coping style (coiro, bettis, & compas, 2017; wood & bhatnagar, 2015) . derived from the transactional model of stress, coping referred to "behavioral and cognitive efforts to reduce or tolerate the internal and external demands that were appraised as exceeding the person's resources" (lazarus & folkman, 1984) . in terms of its influence on physical and mental health, the coping style was divided into positive and negative (berman et al., 1996; xie, 1998) . positive coping is associated with better mental health outcomes while negative coping is related to worse mental problems (mark & smith, 2012a , 2012b . positive coping styles, such as problem-solving efforts, seeking information and social support, focus on active attempts to deal with stress and change the problematic situation. the characteristic items of negative coping styles include avoidance, wishful thinking, and substance use. negative coping focuses on stressorelicited emotion rather than the stressor itself (nowack, 1989) . adaptive emotion regulation, such as positive refocusing and appraisal, acted as a possible buffer between the subjective perception about the pandemic and the current virus anxiety (jungmann & witthöft, 2020) . differences in how individuals coped with events made some more susceptible to the negative consequences of stress (vollrath & torgersen, 2000) . for example, the influence of negative life events on depression was greater among those who took more negative coping strategies, such as worrying about problems instead of solving them (sawyer, pfeiffer, & spence, 2009 ). similarly, among females who adopted more negative coping strategies like passive acceptance and wishful thinking, high negative life events scores implicated significant increases in depressive and anxiety symptoms (blalock & joiner, 2000) . yu and colleagues (2016) also found that soldiers who tended to use more negative strategies and less positive strategies had higher levels of anxiety and depression during their recruit training. recently, a web-based survey of mental health during the covid-19 pandemic demonstrated that coping style was one of the influencing factors of psychological distress, i.e., participants with negative coping styles had higher levels of psychological distress, such as being nervous, hopeless and restless (wang et al., 2020c) . therefore, coping style might moderate the relationship between perceived stress and emotional distress during the outbreak of the covid-19 pandemic. j o u r n a l p r e -p r o o f this study aimed to explore (a) whether the level of perceived stress to the covid-19 pandemic would link with emotional distress (i.e., depression, fear, compulsion-anxiety, neurasthenia, and hypochondria), (b) whether boredom proneness would mediate the relationship between perceived stress and emotional distress, (c) and whether the direct path between perceived stress and emotional distress would be moderated by individuals' coping style. as an integrated model, the present study was guided by the following 3233 chinese respondents participated in this study and filled in the whole questionnaires. the sample had a moderately wide range in age, education, and monthly income (see table 1 ). information and responses would be anonymous and confidential. the pss10 is a 10-item scale to assess the respondents' perceived stress related to the covid-19 pandemic. this scale was originally compiled by cohen (1983) and the revised chinese version had been demonstrated to have good reliability and validity (yang et al., 2003) . items are rated from 1 (not at all) to 5 (very much). participants were asked to respond according to their feelings or thoughts of the covid-19 for the past month. the higher the score, the more stressful the respondents. the pss10 demonstrated good internal consistency (α = .801) in the current sample. j o u r n a l p r e -p r o o f the emotional distress to the covid-19 pandemic was measured by the pqeeph. the the sbps ( the scsq includes two dimensions, i.e., negative and positive coping styles (xie, 1998 ). the individual coping tendency is calculated by the standard score of positive coping minus the standard score of negative coping. if the value is greater than 0, indicates that the individuals' coping tendency is mostly positive (for example, "ask friends and family for advice"); if the value is less than 0, indicates that the individual mainly uses negative coping strategy (for example, "try to forget the whole thing"). in our study, the internal consistency coefficients of the positive and negative coping style subscale were .797 and .637, respectively. all the analyses were implemented by ibm spss statistics, version 24.0 (ibm corp., armonk, ny). the analyses of the hypothetical mediating role of boredom proneness and the moderating role of coping tendencies were conducted by hayes's (2017) process macro (model 4 and model 5). all continuous variables were standardized and the interaction effects were computed from these standardized scores. the bootstrapping method produces 99% bias-corrected confidence intervals of these effects from 5,000 resamples of the data. confidence intervals that do not include zero indicate significant effects (hayes, 2013) . before testing the models in j o u r n a l p r e -p r o o f process macro, the scatter plot of the standardized residuals showed that our data violated the homoscedasticity assumption as "the residuals roughly rectangularly distributed, with most of the scores concentrated in the center (along with the 0 points)" (pallant, 2011) . therefore, the hc3 heteroscedasticity-consistent standard error estimator proposed by davidson and mackinnon (1993) was applied for regressions in the current study, since hc3 was recommended (hayes & cai, 2007) and confirmed as the most reliable estimator (cribari-neto, ferrari, & oliveira, 2005). this estimator was also proposed to keep the test size at the nominal level regardless of the presence or absence of heteroscedasticity (long & ervin, 2000) . means and standard deviations (sd) for scores of all the questionnaires are presented in table 2 . pearson correlations between variables are shown in table 3 . the results demonstrated that individuals who experienced greater perceived stress about the covid-19 pandemic reported more emotional distress measured by pqeeph including depression, fear, compulsion-anxiety, neurasthenia, and hypochondria. likewise, individuals with higher boredom proneness had stronger emotional distress. additionally, individuals who mainly adopted negative coping would experience more emotional distress as well. note. * p < .05. ** p < .01. *** p < .001. correlation analysis showed significant and positive correlations among perceived stress, boredom proneness, and emotional distress, which provided a precondition for testing the mediating effect of boredom proneness. firstly, to examine hypothesis 1, a general linear model was built in which pss10 was treated as a predictor, emotional distress as outcome variables, boredom proneness as a mediator, and demographic variables (gender, age, education, and monthly income) as covariates. the indirect and direct path between perceived stress and emotional distress are presented in table 4 . as displayed in table 4 as the direct path of boredom proneness and distress was still significant. therefore, hypothesis 2 was supported that boredom proneness mediates the relationship between perceived stress and emotional distress. the process macro (model 5) by hayes (2013) was used to test the moderated mediation model. as demonstrated in table 6 , the interaction effect of pss10 and coping tendency negatively and significantly explained depression (β=-.047, p < .01), compulsion-anxiety (β=-.083, p < .001) and neurasthenia (β=-.100, p < .001). however, the interaction did not significantly associate with fear (β= .003, p > .05) and hypochondria (β= .013, p > .05). hypothesis 3 was largely supported that coping tendency moderates the relationship between perceived stress and emotional distress including depression, compulsion-anxiety, and neurasthenia. j o u r n a l p r e -p r o o f note. bootstrap sample size = 5000; pss10 = perceived stress; bp = boredom proneness. * p < .05, ** p < .01., *** p < .001. note. bootstrap sample size = 5000; pss10 = perceived stress; bp=boredom proneness; ct=coping tendency. * p < .05, ** p < .01., *** p < .001. to further explain the interaction effect, we plotted pss10 against emotional distress (i.e., depression, compulsion-anxiety, and neurasthenia), separated for low (m -sd) and high (m + sd) levels of coping tendency. the results showed that as the coping tendency moved from positive to negative, the interpretative effect of perceived stress on the depression was gradually strengthened, and β increased from .220 (p < .001) to .314 (p < .001) (see figure 2 . a); β increased from .333 (p < .001) to .498 (p < .001) for compulsion-anxiety (see figure 2 . b); β increased from .250 (p < .001) to .449 (p < .001) for neurasthenia (see figure 2 . c). these results indicated that, compared with the positive coping tendency, when participants tended to adopt more negative coping, perceived stress might lead to higher levels of depression, compulsion-anxiety, and neurasthenia. in summary, boredom proneness mediated the relationship between perceived stress and emotional distress (i.e., depression, compulsion-anxiety, and neurasthenia), and participants' coping tendency moderated the stress-emotional distress relationship. the current cross-sectional study explored the relationship between stress under the situation of lockdown during corona time, boredom proneness was discovered to play a partially mediating role in the relationship between perceived j o u r n a l p r e -p r o o f stress and emotional distress, i.e., individuals with a propensity to experience boredom were more likely to report greater emotional distress. it was proposed that individuals with higher boredom proneness might focus on themselves or their internal states, and further tend to have more awareness of existing psychological symptoms (sommers & vodanovich, 2000) . meanwhile, they were less able to regulate their emotion when experiencing a high level of stress (culp, 2006) . adopting positive coping strategies were more likely to experience less emotional distress, suggesting that positive coping might be a "resilient" factor. that is, positive and adaptive coping, such as training on stress management and encouragement of self-care, would contribute more to the improvement of emotional resilience (taylor, 2019) . in contrast, those who prefer to use more negative coping strategies are more likely to experience more emotional distress, therefore, are a "high-risk" population for mental illness under stress. there are several implications in the present study. firstly, psychological stress is related to emotional distress in the early stage of the pandemic during which the whole society is under panic. hotopf and wessely (1994) found that people with high levels of stress were more tend to fall ill with a viral infection and suffer more there are also some limitations to this study. first of all, it should be noted that this was a cross-sectional study without the temporal factor. it would be informative to follow up on the dynamic change of mental health along with the development of the covid-19 pandemic. for example, compared with the initial peak of the covid-19 pandemic, levels of stress and fear were decreased while depression levels were significantly increased during the remission phase when the number of cases declined . considering that the second wave of the covid-19 might be approaching, it remains to be investigated whether the rising number of cases and persistent social distancing are associated with a further deterioration of mental j o u r n a l p r e -p r o o f health. however, it is worthwhile to mention that data in our research was collected at the initial as well as the most serious stage of the outbreak, which may to a large extent reflect the most significant impact of pandemic-related stress on mental health. secondly, all questionnaires were self-reported, which might not be consistent with the objective assessment by mental health professionals. nevertheless, subjective measurements were widely used during the emergent public health events (lau et al., 2008; leder, pastukhov, & schütz, 2020) , and all questionnaires used here had been demonstrated high internal consistency. thirdly, though we aimed at the general population, our sample mainly consists of well-educated young people, who may have higher immunity to viruses as well as a better capability to manage stress. clinical characteristics of covid-19 showed that the mortality rate of the elderly patients was higher than that of young and middle-aged patients (liu et al., 2020), which implicated possibly higher psychological stress and more emotional distress in elderly individuals. last but not least, it should be noted that covid-19 has a greater negative impact on 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open rheumatol doi: 10.1002/acr2.11174 sha: doc_id: 326788 cord_uid: qzm3b3xy objective: response to the coronavirus disease 2019 (covid‐19) pandemic has resulted in shelter‐in‐place orders and major changes to individuals’ daily lives. the impact of such stressors on disease activity in individuals with axial spondyloarthritis (axspa) is unclear. the aim of this study is to examine whether stress, anxiety, and depression are associated with patient‐reported disease activity, after accounting for important factors. methods: we administered a survey to an axspa cohort from a single center with well‐defined demographic and disease characteristics. we included questions about job status changes, exercise, medication use, disease activity (by the bath ankylosing spondylitis disease activity index [basdai]), and psychological factors (stress, depressive symptoms, and anxiety). separate multivariable linear models examined the associations between perceived stress, anxiety, and depression with the basdai. results: after adjustment for potential confounders, those with higher levels of stress had a statistically significant 0.54‐point higher basdai, on average, compared with those with lower levels of stress (95% confidence interval [ci]: 0.11, 0.97). those with higher levels of anxiety also had a statistically significant higher basdai, on average, compared with those with lower levels of anxiety (β: 0.95, 95% ci: 0.18, 0.99). the association between depression and basdai was not statistically significant. we did not find differences in these associations among subgroups of age, job status, or county of residence. conclusion: individuals with axspa with higher levels of stress and anxiety had significantly higher disease activity levels, although with a difference below clinical importance. further planned studies will evaluate the trajectory of disease activity. severe acute respiratory syndrome coronavirus 2 is a novel coronavirus that causes coronavirus disease 2019 (covid19) , which the world health organization declared a pandemic in 2020 (1) . by early april 2020, there were over 100 000 confirmed cases in the united states, with global cases climbing past a million (2) . in response to rising case numbers, san francisco, california, was placed under a shelter-in-place order on march 17, 2020, with many cities, counties, and states subsequently doing the same (3) . prior literature in other rheumatic diseases has been conflicting in terms of how major natural disasters, such as hurricanes and earthquakes, impact disease activity (4) (5) (6) (7) (8) (9) . stress is implicated as an important factor that affects disease activity, as has been demonstrated in rheumatoid arthritis (ra) (10) (11) (12) . however, there have been few studies of axial spondyloarthritis (axspa) that have directly examined the association of stress with disease activity (13) , and none them have evaluated the association of major stressors such as natural disasters or pandemics with disease activity. as a shelter-in-place order impacts the entire community and may result in occupational changes (eg, working from home or loss of income), limitations on physical activity (eg, closure of nonessential businesses including gyms), and medication changes (eg, in light of concerns of increased infection risk with immunosuppression), covid-19 represents a unique situation to study the impact of stress on disease activity in axspa. the purpose of this study is to study the impact of this major event, particularly in light of local shelter-in-place ordinances and major economic changes, on disease activity in an existing natural history cohort of individuals with axspa. the primary aim is to examine whether stress, anxiety, and depression are associated with a change in disease activity, after accounting for important factors. study population. the natural history of axspa study is an ongoing prospective cohort of 473 subjects at the university of california, san francisco (ucsf). adults who met either the modified new york criteria for ankylosing spondylitis (as) or the assessment of spondyloarthritis international society (asas) criteria for nonradiographic axspa were enrolled from 2007. the purpose of the cohort study is to explore potential mechanisms responsible for disease, risk factors for the development and progression, associated comorbidities, treatments and treatment complications, and the natural history of spondyloarthritis. patients are followed at cohort study visits every 6 months. data collection and variables. for the current study, subjects were emailed a link to a survey hosted online through the hipaa-compliant platform qualtrics that was specifically focused on issues surrounding the past 2 months during the covid-19 pandemic. survey objectives, risks, benefits, and study team contact information were provided to patients before questions were initiated. this study received internal review board (irb) approval from ucsf following a modification to the cohort irb. the university of washington determined that further irb review was not needed for the analysis of this study. medication use. patients were asked about baseline nonsteroidal anti-inflammatory drug (nsaid) and biologics use, and whether there was any change over the prior two months (increased, decreased, stopped, stayed the same, switched, or did not use at baseline). the interval of 2 months (february to april 2020) was chosen to capture potential changes that patients may have instituted on account of the pandemic. exercise. patients were asked about baseline ("six months ago") exercise frequency, duration, and specific type. then they were asked about change over the prior 2 months (increased, decreased, stopped, stayed the same, or did not exercise at baseline). job status. patients were asked for details about their current employment status, including whether they were unemployed, and their current work location if employed. covid-19. patients were asked whether they had received a nasal swab or other form of testing for the diagnosis of covid-19 and whether the results were positive. if testing had been performed, they were asked about symptoms and any treatment received. they were also given the opportunity to provide any concerns they had regarding covid19 in an open-ended question. disease activity. the bath ankylosing spondylitis disease activity index (basdai) was administered as an assessment of disease activity over the prior week. the basdai is a composite measure of as disease activity and consists of six questions that address five major symptoms in as: fatigue, spinal pain, peripheral joint pain and swelling, localized tenderness, and morning stiffness (14) . scores range from 0 to 10, with higher scores reflecting greater disease activity. psychological factors were measured by the following instruments: the perceived stress scale is a 10-item, self-reported instrument that measures perceived stress in relation to life events over the past month (15) . scores range from 0 to 40, with higher scores indicating greater stress. the center for epidemiologic studies -depression (ces-d) scale is a 20-item, self-reported measure of depression symptoms with an emphasis on depressed mood in the past week (16) . scores range from 0 to 60, with higher scores indicating more severe symptoms. a score of 16 or greater is suggestive of possible depression and a score of 20 or greater of probable depression. the patient reported outcomes measurement information system (promis) anxiety short form is a six-item, selfreported measure of anxiety over the past 7 days (17) . scores are converted to a standardized t-score, with a population mean of 50 and standard deviation of 10. cutoff scores to define levels • few studies have evaluated the relationship of major stressors and disease activity in axial spondyloarthritis (axspa), and none have examined this association in the setting of a natural disaster or pandemic. • in april 2020, during the peak of the coronavirus disease 2019 (covid-19) pandemic in the united states, we surveyed patients with axspa from a single center cohort regarding changes in job status, exercise, medication use, disease activity, and psychological factors including perceived stress and anxiety. • after adjustment for important confounders, including baseline disease activity, those with higher levels of perceived stress had significantly higher disease activity compared with those with lower levels of stress. the same association was seen for anxiety levels and disease activity. • these findings highlight stress and anxiety as independent predictors of patient-reported disease activity among individuals with axspa during the covid-19 pandemic, particularly while shelterin-place measures were in effect. | 535 of anxiety have been suggested as none/slight (<55), mild (55.0-59.9), moderate (60.0-69.9), and severe (≥70) levels of anxiety (18) . baseline measures, including demographics, axspa disease characteristics, medication use, and comorbidities were obtained from either the cohort database (in the case of time-invariant characteristics) or from the most recent study visit prior to march 2020. disease activity was recorded using the basdai. nsaid use was recorded as none, low dose, or high dose (19) . biologic use, including tumor necrosis factor inhibitors and interleukin-17 inhibitors, was recorded as a binary variable (using, yes/no). comorbidities were extracted using information available in the electronic medical record problem list and medication list, as appropriate. analysis. we performed descriptive statistics for baseline characteristics and survey responses, which included calculation of pairwise correlation coefficients for stress, anxiety, and depression scales. we used multiple imputation with chained equations with 10 iterations to impute missing values for the basdai at baseline (20) (21) (22) . we included the following variables as predictors in the imputation model: age, gender, race, survey basdai, baseline basdai, change in nsaid use, change in biologic use, change in exercise, change in job status, total promis anxiety score, and total ces-d score. complete case analyses were performed as a secondary analysis. because stress, anxiety, and depression may be closely related, we examined correlations among these scores. for the first primary analysis, we compared the survey basdai score between those with higher versus lower levels of stress (perceived stress scale dichotomized at the median). we used multivariable linear regression and adjusted for the following potential confounders identified a priori: age (years), gender, race (white versus other), baseline basdai, decrease or cessation of nsaid use, decrease or cessation of biologic use, decrease or cessation of exercise, and whether there was a change in job status. for the second analysis, we used multivariable linear regression to compare the survey basdai between those with higher versus lower levels of anxiety (promis anxiety score dichotomized at the median) after adjusting for the same confounders as in the first analysis. for the third analysis, we compared the survey basdai between those with higher versus lower scores on the ces-d scale (dichotomized at the cutoff of 16) using multivariable linear regression with adjustment for the same confounders as in the previous two models. in exploratory analyses, we tested interactions (α = .10 for statistical significance) in the primary analysis model between perceived stress level and 1) age greater than 65 years, 2) job change, and 3) residence outside of san francisco. all analyses were conducted in stata software version 15 (statacorp) using robust standard error estimates and an α level of .05 (with the exception of tests of interaction). as of april 2020, there were 473 patients in the cohort. the survey was initially sent on april 2, 2020. several reminders, including emails, messages through the electronic medical record, and telephone calls, were sent. as of april 28, 2020, the final response rate was 43% with 203 complete responses. overall, survey respondents had a mean age of 46.4 ± 12.5 years, 66% were male, and 77% were white. baseline data are described, by stress level based on the perceived stress scale, in table 1 . groups by stress level were similar, with the exception of a lower proportion of males in the higher stress group (54% versus 79%) and a higher proportion with diagnoses of depression in the higher stress group (46% versus 17%). table 2 . groups were similar in terms of gender, as classification, and treatment for axspa. nonrespondents were older and a higher proportion were white as compared with respondents. nonrespondents had a numerically higher basdai at baseline but also a higher proportion of missing data for the basdai. survey outcomes are shown in table 3 , stratified by stress level. the majority of respondents did not alter their nsaid or biologic dosing (81% and 88%, respectively). however, 56% reported decreasing or stopping their exercise as compared with 6 months prior, and 13% reported a change in their job. nine received testing for covid-19, but none reported a positive test. there were more people who reduced or stopped their nsaids (but not biologics) in the high-versus low-stress group; and more people who had job changes and were not currently working in the high-versus low-stress group. the scores for ces-d, anxiety, and basdai were higher in the high-stress group compared with the low-stress group. stress, anxiety, and depression scores were highly correlated (pairwise correlation coefficients ranged from 0.70 to 0.74, table 4 ), so separate multivariate regression analyses were conducted for each factor. there were missing data for the baseline basdai for 43 respondents, which were imputed using multiple imputation with chained equations. after adjustment for potential confounders and baseline disease activity, those with higher levels of stress had a statistically significant 0.54-point higher basdai, on average, compared with those with lower levels of stress (95% confidence interval [ci]: 0.11, 0.97). those with higher levels of anxiety had a statistically significant 0.59-point higher basdai, on average, .08 note. continuous variables are reported as mean ± sd and median (interquartile range) and categorical variables as n (%). to assess differences between high-and low-stress level groups, we used χ 2 tests for categorical variables and mann-whitney u tests for continuous variables. values missing for the following: as classification (n = 7 nonrespondents), biologic use (n = 7 nonrespondents), nsaid use (n = 7 nonrespondents), baseline basdai (n = 43 respondents, n = 102 nonrespondents). abbreviations: as, ankylosing spondylitis; basdai, bath ankylosing spondylitis disease activity index; nsaid, nonsteroidal antiinflammatory drug. a total cohort of 473. not included in this table: 26 declined survey participation and 7 who were deceased. | 537 compared with those with lower levels of anxiety (95% ci: 0.18, 0.99), after adjustment for potential confounders and baseline disease activity. the average difference in the basdai was not significantly different compared with those with higher versus lower scores on the ces-d scale (β: .38, 95% ci: −0.10, 0.85). results were similar using complete case analysis (data not shown). in exploratory analyses, the association of stress with disease activity did not differ across subgroups of age, job status, or county of residence (p = 0.44, 0.90, and 0.99, respectively), nor did the association of anxiety with disease activity differ by these subgroups (p = 0.69, 0.22, and 0.62, respectively). in this study, higher levels of perceived stress and anxiety, but not depression, were significantly associated with higher disease activity levels among individuals with axspa. we did not find differences in this association among subgroups of age, job status, or county of residence. to our knowledge, this is the first study to evaluate patient-reported disease activity in relation to psychological factors among individuals with axspa during a major pandemic. although the estimated average difference in the basdai comparing between stress and anxiety levels was less than the minimal clinically important difference (mcid) of 1.0 reported by pavy et al (23) , the differences between groups may become more apparent upon longitudinal follow-up of this cohort as the pandemic evolves. stress is related to disease activity in rheumatic disease (10) (11) (12) . however, whether stress is an independent predictor of elevated disease activity has not been definitively demonstrated in axspa. jiang et al examined psychological status, sleep quality, and stress due to life events over the prior 12 months in patients with as and found an association between anxiety/depression and disease activity (13) . previously published studies have evaluated patients with ra, systemic lupus erythematosus (sle), and inflammatory bowel disease (ibd) following natural disasters (4) (5) (6) (7) (8) (9) , with inconclusive findings. wallace et al followed 13 patients with ra and 10 with sle for 6 months after the 1994 northridge earthquake, but no flares were documented (4) . a study in japan surveyed 192 women with ra who had experienced a natural disaster (predominantly typhoons, tornados, and torrential rain) between 2004 and 2006. surveys were administered 1 and 6 months following the event, with the finding that 14% experienced deterioration of functional status, whereas 22% experienced a worsening of self-rated health status (6) . in contrast, a study in taiwan compared patients with sle inside and outside of the disaster zone following the major earthquake of september 1999. the researchers found that 6 months following the disaster, neither the exposed nor the comparator group had experienced a significant change in the clinical symptoms of sle (7) . factors affecting short-term and long-term impact can vary. two studies examined the frequency of disease relapse in ibd immediately following the great east japan earthquake of march 2011, and then with follow-up data 1 and 2 years postdisaster. the authors found that the factors that influenced long-term relapse were different from those that influenced short-term relapse (8, 9) . however, many of these studies were small, some did not have adequate comparators, and there was high potential for unmeasured confounding. the covid-19 pandemic represents a unique universal stressor on our patient population, as the shelter-in-place orders affected multiple cities, counties, and entire states in the united states, with impacts on job status, the ability to exercise, and access to health care. information on covid-19 risk for people living with rheumatic disease was also scarce in the early days of the pandemic, as these comorbidities were not reported in the initial large case series published from china, europe, or the united states (24) (25) (26) (27) (28) (29) . larger reports from a global rheumatology registry are forthcoming (30) . at the end of march 2020, michaud et al surveyed a large us registry of patients with rheumatic disease (for-ward) and found that common themes relating to covid-19 were concerns over risk of infection and how best to manage immunosuppressive medications (31) . the strengths of this study include the use of a well-defined axspa cohort with detailed baseline data on important confounders as well as baseline data on disease activity. we were able to administer a survey with disease-specific questions, capturing a fairly homogenous population of rheumatic disease. the survey had a moderate response rate of 43%. additionally, we used validated measures of anxiety, stress, and depression with mean levels in our cohort that were similar to those in other rheumatic disease cohorts (32, 33) . there are limitations of this study that we must acknowledge. first, this is an observational study limited to two time intervals: before and after the pandemic. there is potential selection bias regarding survey responses, as those with lower stress levels and disease activity may be more inclined to respond to the survey. generalizability may be limited, as all of the patients in this study were under the care of one rheumatologist. during the survey period, most of the patients were living in the bay area of california, which had shelter-in-place implementation that differed from other areas of the country. unmeasured confounding and measurement error are also possible, as we were limited in the granularity of detail that we could include in our survey. we did not have stress or anxiety measures in prior surveys, so we could not measure changes in these variables. finally, our use of the basdai as a patient-reported measure of disease activity is limited by its use of subjective, rather than objective, questions. our survey-based study of an axspa cohort at a single us center found that stress and anxiety were significantly associated with patient-reported disease activity, independent of confounding factors. although the average difference in basdai was below the mcid, these findings suggest that the covid-19 pandemic may have had an impact on axspa disease activity through increased stress and anxiety. we will continue this study with further iterations of the survey as a repeated measure. this will allow us to look at both population-and subject-level trajectories over time. world health organization. coronavirus disease (covid-19) pandemic san francisco department of public health. order of the health officer no. c19-07b can an earthquake cause flares of rheumatoid arthritis or lupus nephritis? the impact of hurricane hugo and the san francisco earthquake on a sample of people with rheumatoid arthritis impact of natural disasters on the functional and health status of patients with rheumatoid arthritis changes in the clinical and laboratory features of lupus patients after the big earthquake in taiwan long-term course of inflammatory bowel disease after the great east japan earthquake life-event stress induced by the great east japan earthquake was associated with relapse in ulcerative colitis but not crohn's disease: a retrospective cohort study does stress affect the joints? daily stressors, stress vulnerability, immune and hpa axis activity, and shortterm disease and symptom fluctuations in rheumatoid arthritis the role of stress in rheumatic diseases experimental stress in inflammatory rheumatic diseases: a review of psychophysiological stress responses the relationship between disease activity measured by the basdai and psychological status, stressful life events, and sleep quality in ankylosing spondylitis a new approach to defining disease status in ankylosing spondylitis: the bath ankylosing spondylitis disease activity index perceived stress scale the ces-d scale: a self-report depression scale for research in the general population patient reported outcomes measurement information system (promis) promis anxiety scoring manual asas recommendations for collecting, analysing and reporting nsaid intake in clinical trials/epidemiological studies in axial spondyloarthritis introduction in multiple imputation for nonresponse in surveys flexible imputation of missing data using the outcome for imputation of missing predictor values was preferred establishment of the minimum clinically important difference for the bath ankylosing spondylitis indices: a prospective study clinical features of patients infected with 2019 novel coronavirus in wuhan, china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention clinical characteristics of coronavirus disease 2019 in china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region clinical characteristics of covid-19 in new york city presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the rheumatic disease and covid-19: initial data from the covid-19 global rheumatology alliance provider registries experiences of patients with rheumatic diseases in the us during early days of the covid-19 pandemic association between pain sensitization and disease activity in patients with rheumatoid arthritis: a cross-sectional study determinants of sleep problems in patients with spondyloarthropathy all authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. dr. liew had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. study conception and design. liew, castillo, katz, haroon, gensler. acquisition of data. castillo, gensler. analysis and interpretation of data. liew, zaccagnino, katz, haroon, gensler. key: cord-302616-1uwrcvjx authors: steenblock, charlotte; todorov, vladimir; kanczkowski, waldemar; eisenhofer, graeme; schedl, andreas; wong, ma-li; licinio, julio; bauer, michael; young, allan h.; gainetdinov, raul r.; bornstein, stefan r. title: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the neuroendocrine stress axis date: 2020-05-07 journal: mol psychiatry doi: 10.1038/s41380-020-0758-9 sha: doc_id: 302616 cord_uid: 1uwrcvjx nan and interactions between these two systems play a central role in adaptation. patients with stress-related disorders may exhibit increased vulnerability to mental illness for several years after the stress experience [12] . on a systemic level, stressors activate the sympathetic branch of the autonomic nervous system followed by activation of the hpa axis. splanchnic nerve activation triggers chromaffin cells in the adrenal medulla to release the catecholamines, epinephrine and norepinephrine, for mediating a rapid response. when the hpa axis is activated, corticotropin-releasing hormone (crh) and arginine vasopressin (avp) are secreted from the paraventricular nucleus of the hypothalamus. crh and avp then activate the anterior pituitary to secrete adrenocorticotropic hormone (acth), which in turn stimulates the adrenal cortex to produce corticosteroids [13] . glucocorticoid receptors are expressed in several areas of the adult brain such as the hypothalamus, pituitary, hippocampus, and amygdala, which are all involved in glucocorticoid feedback. an appropriate response during acute stress is imperative for survival, but frequent or prolonged activation can change the functional tone of stress systems with ensuing deleterious effects. excessive or chronic activation of the endocrine stress axis, which also leads to a disrupted feedback loop, triggers and contributes to numerous human disorders and stress conditions in humans, including anxiety, post-traumatic stress disorder, major depressive disorder, and occupational burnout. in addition, gonadal steroids can differentially affect the hpa axis resulting in sex differences in responses to stress [14] . animal models and human studies support an increased risk in males for behavioral or neurodevelopmental disorders in response to prenatal and early life stress, whereas females appear to have an increased risk in adolescence and throughout adulthood, especially if they have experienced stress-related early life adversity or trauma. during the perimenopause and menopausal transition, women are at greater risk for presenting with affective disorders including depression as well as schizophrenia [15] . entry of the corona viruses sars-cov and sars-cov-2 into cells depends on binding of the viral spike (s) glycoproteins to cellular ace2, which the virus uses as its entry receptor. furthermore, s proteins are primed by host cell proteases [16, 17] . this subsequently leads to fusion of the viral envelope and the plasma membrane or the endosome membrane of the host. it has been indicated that the sars-cov-2 s protein binds to ace2 with 10-20-fold higher affinity than the s protein of sars-cov. this might underlie the high spread of sars-cov-2 [18] . ace2 is a carboxypeptidase, which controls the generation of the vasodilating angiotensin 1-7 from angiotensin ii. ace2 can also cleave angiotensin i to angiotensin 1-9, which can then be further converted to angiotensin 1-7 by ace. ace2, angiotensin 1-7 and its mas receptor constitute the vasoprotective arm of the raas. excessive activation of the classical raas leads to arterial hypertension, accompanied by immuno-metabolic disorders and obligatory culminates in end-organ damage in the absence of adequate therapy (for recent reviews see [19] [20] [21] [22] [23] ). the raas and in particular ace2 appear to be attractive targets to fight covid-19. therein, the role of ace inhibitors and at1r blockers is currently highly controversial. on the one hand, these drugs could increase ace2 expression and potentiate sars-cov-2 cell entry [24] . on the other hand, increased ace2 production would result in a higher circulating level of soluble ace2, which neutralizes sars-cov-2 [25] . yet, the precise role of the available raastargeting drugs in covid-19 disease waits to be unraveled. ace2 has been shown to have an effect on the stress response and on anxiety. mice overexpressing ace2 are less anxious and have reduced plasma corticosterone and pituitary expression of proopiomelanocortin [26] . the same effect was observed when ace2 overexpression was targeted to the crh-synthesizing cells of the hypothalamus suggesting that especially the hpa axis is affected [27] . in mice transgenic for ace2, the olfactory bulb of the brain is a major target organ for sars infection leading to transneural spread [28] [29] [30] . expression of ace2 has been noted not only in the respiratory tract, lung, ileum, bladder, esophagus, heart, and kidney [31] , but also in the the hypothalamus, pituitary and adrenal [32] . these results fit with the organ distribution of sars-cov observed in patients who died of sars [33] . intriguingly, in these patients, following the lung, the adrenal belonged to the organs with the highest concentration of virus particles. in addition, the pituitary was affected [33] making it clear that the hpa axis is targeted during a coronavirus infection. furthermore, the gastrointestinal tract is targeted [33] suggesting associations with appearance of gastrointestinal infection symptoms in some patients [34] . as ace2 is also expressed in the human brain this might explain why infection of the central nervous system is regularly observed in sars patients [35, 36] . although an equal number of covid-19 cases between men and women has been reported, there seem to be sex differences in mortality and vulnerability to the disease [37] . emerging evidence suggests that more men than women are dying [38, 39] . it has been speculated that this is due to sexbased immunological [40] or other differences, such as patterns and prevalence of smoking [41] . an alternative hypothetical explanation could involve sex-dependent differences in responsiveness of hpa and other hormonal/stress systems. further research is needed to confirm this hypothesis. in conclusion, the strong interaction and reciprocal communication between the organs of the endocrine stress system in a coronavirus infection require a deeper understanding of the adaptation of all components to the infection. furthermore, it has to be considered that certain therapies impacting expression of ace2 might also influence the raas and the neuroendocrine stress axis, which may lead to long-term consequences, as prolonged exposure to stressors increases the risk to develop major depressive, anxiety and post-traumatic stress disorders [42] . maintenance of homeostasis during immune challenges, such as a virus infection, involves activation of the immune system, resolution of the challenge, and protection of the host against potentially toxic inflammatory processes. upon immune challenge, the immune system is activated to produce a number of cytokines. one functional group of cytokines mediates the innate immune response and includes several proinflammatory cytokines: tumor necrosis factor-α (tnf-α), interleukin-1 (il-1), and il-6, and the type i interferons (ifn-α/β). these cytokines are released in the early stages of an immune response. another group of cytokines, such as the t cell cytokines il-2 and ifn-γ (type ii interferons), which are especially important in mediating antiviral defenses, mediates the later adaptive immunity. in addition to contributing to the progression of the immune response against viral infection, cytokines activate the hpa axis, resulting in the release of adrenal glucocorticoids [43] . in turn, glucocorticoids exert negative feedback influences on immune cells to suppress further synthesis and release of cytokines, thereby protecting the host from the detrimental consequences of an overactive immune response (e.g., tissue damage, autoimmunity, or septic shock) [44, 45] . a number of cytokines have been measured in serum of covid-19 patients [46] . initial plasma il-1b, il-1ra, il-7, il-8, il-9, il-10, bfgf, gcsf, gmcsf, ifn-γ, ip-10, mcp1, mip-1a, mip-1b, pdgf, tnf-α, and vegf concentrations were higher in patients than in healthy adults, whereas plasma levels of il-5, il-12p70, il-15, eotaxin, and rantes were similar in patients and controls. ifn-α and β were not measured. these results suggest that an overproduction of cytokines might be responsible for the damage of the lungs in covid-19 patients. however, also host factors such as expression of ace2 and underlying diseases such as hypertension, chronic obstructive pulmonary disease, diabetes, and cardiovascular disorders might influence susceptibility to infection and progression of the disease [34, 47] . both sars-cov and middle east respiratory syndrome (mers)-cov were shown to induce very little type i ifn in most cell types. this might be due to different reasons such as storage of coronaviral dsrna in double-membrane vesicles and activation of numerous mechanisms dedicated to suppress dsrna-dependent ifn induction [48] . this explains how these viruses employ mechanisms to escape, dampen, or block the antiviral interferon response in human cells. furthermore, the ability of certain pathogens to escape from host immune response has been reported to be accomplished by host protein mimicry [49] . thereby, no or a limited amount of antibodies are created, which also explains why certain vaccines may be ineffective. on the other hand, the similarity between pathogenic antigens and host proteins may lead to immune cross reactivity, whereby the reaction of the immune system toward the pathogenic antigens may harm similar human proteins, eventually causing autoimmune disease [50] . it has been proposed that an escape mechanism of sars-cov could be accomplished by viral expression of amino acid sequences mimicking acth. when the host produces antibodies against these viral antigens, the antibodies also bind to acth thereby limiting hpa activity and secretion of corticosteroids, which could lead to adrenal insufficiency [51] . this suggests that corticosteroids may be used to treat covid-19 patients thereby helping the immune system to fight the infection. indeed, corticosteroid supplements have improved the clinical conditions of many sars patients [52] . on the other hand, a number of studies reported corticosteroid-treatment of sars patients to be rather harmful (reviewed in [53] ). for example, early steroid treatment has been associated with delayed viral clearance [54] , and it has been found that patients with psychosis received higher cumulative doses of steroids than patients without psychosis [55] . in a study with 138 covid-19 patients, where 45% received methylprednisolone, no effective outcome was observed after the treatment [56] . thereby, the use of corticosteroids for the treatment of covid-19 is not recommended [34] . as mentioned above an infection with sars-cov-2 can be expected to lead to activation of the endocrine stress axis in association with mental stress. in addition, to the actual infection and treatment, psychological stress is likely to occur due to pandemic restrictions. isolation during a pandemic is somewhat similar to physical immobilization stress, which leads to increased expression of enzymes involved in steroidogenesis [57] and elevated plasma corticosteroids [58] . chronic restraint stress in rats also alters the raas by reduction of plasma aldosterone levels despite significant increases in plasma renin activity. public anxiety of being infected is high; health care workers, who have higher vulnerability of acquiring and spreading the virus, are in particular those most likely to be distressed. in a study about the impact of mers in health care workers in saudi arabia, a significant proportion expressed anxiety about the risk of acquiring mers-cov infection and for transmitting it to family members [59, 60] . in addition, during quarantine or curfew, people might experience loneliness because of the lack of communication and engagement with others. loneliness, defined as the discrepancy between a person's desired and actual social relationships, is an emotional response to social isolation, while social isolation is an objective measure of the lack of social connections or interactions [61, 62] . social isolation and loneliness are common sources of chronic stress in adults. they are correlated to a higher risk of mortality and cardiovascular disease, which have been associated with activation of the hpa axis and the sympathetic nervous system. repeated and chronic social stress leads to glucocorticoid release, enhanced myelopoiesis, upregulated proinflammatory gene expression, and oxidative stress [63] . furthermore, psychological stress due to social isolation is known to cause abnormal feeding behaviors. in addition to psychological reactions, such as depression and anxiety, stress triggers various physiological reactions including an increase in respiration and blood pressure. an activated stress axis also influences the body's metabolism [64, 65] . in particular, changes in feeding behavior are a well-known phenotype related to stress [66] . people who survived an infection with sars-cov have shown elevated stress levels long after the outbreak [67] . leow et al. observed a direct effect on the hpa axis in sars survivors 3 months after their recovery, where hypocortisolism was diagnosed. one year after recovery, hpa dysfunction had largely recovered [68] . however, even though physical conditions continuously improved, mental health did not. psychiatric morbidities and chronic fatigue persisted and continued to be clinically significant up to at least 4 years after the sars outbreak [69] . this has also been shown by lee et al., where health care workers during the outbreak showed an elevated stress level when compared to non-health care workers. one year later, stress levels of survivors remained persistently elevated and higher than in control subjects. specifically, health care workers showed significantly higher stress levels and had even higher depression, anxiety, and post-traumatic symptom scores than other survivors [70, 71] . progenitor and stem cell populations are required for the successful homeostasis and adaptation of most tissues. stem cells are dynamically regulated by signals originating from their niches, helping to regulate appropriate proliferation and differentiation. external and internal stressors influence the process of cell differentiation of stem and progenitor cells in the hpa axis in order to form the fully functional endocrine stress system [72] . in relation to coronavirus infections, it was shown that pulmonary stem/progenitor cells that express ace2 are targeted by sars-cov in primary cultures [73] . the infected cells support active virus replication, which leads to their own destruction [73] . another study by mallick et al. has further revealed that a subset of these stem cells, the bronchoalveolar stem cells, are the prime target of the sars-cov infection of stem cells [74] . these observations suggest that, in addition to pneumocytes, lung stem cells/ progenitors are involved in coronavirus infection. this might also explain the long course of illness, in the context of continued deterioration of lung tissues and apparent loss of capacity of lung repair observed in covid-19 patients. as, after the lung, the adrenal is one of the most prominent organs infected by coronavirus. it would be reasonable to hypothesize that in a manner analogous to what happens in the lung, stem cells of the hpa axis could be affected by sars-cov-2. we and others have shown that stem cells play an important role in stress [75, 76] . our work on the adrenal suggests that stem cell populations in both the adrenal cortex and medulla are highly susceptible to stress [77, 78] . under normal conditions, they seem to be quiescent or only slowly contribute to organ homeostasis. however, in stressful situations they enter the cell cycle leading to differentiation into mature lineages. furthermore, we have shown that differentiation of adrenocortical stem cells is faster in females than in males [79] . in a recent paper, de laval et al. have shown that a transient immune challenge with lipopolysaccharide not only induced an acute response in hematopoietic stem cells but also established persisting epigenetic modifications in myeloid lineage and innate immunity genes [80] . these results prove that permanent epigenetic changes are induced not only in the adaptive immune system but also in stem cells of the innate immune system after an infection. a pandemic such as the ongoing sars-cov-2 outbreak is extremely stressful for all members of society, with longterm outcomes, not only for those who have been actually infected, but also for other people, who are not necessarily infected themselves, but who may suffer lasting consequences as a result of loss, grief, isolation, deprivation, and other psychological outcomes of ongoing quarantine or shelter in place measures. this includes health care workers, and others whose lives were severely affected. the impact of multiple supportive and experimental treatments also needs to be considered. many of these affect the endocrine stress axis, which again might lead to longterm consequences. from earlier coronavirus outbreaks, it is evident that survivors and health care workers are at increased risk of developing mental illness years 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stress-inducible-stem cells: a new view on endocrine, metabolic and mental disease? beyond the paradigm: novel functions of reninproducing cells isolation and characterization of adrenocortical progenitors involved in the adaptation to stress multipotent glia-like stem cells mediate stress adaptation the adult adrenal cortex undergoes rapid tissue renewal in a sex-specific manner c/ebpbeta-dependent epigenetic memory induces trained immunity in hematopoietic stem cells conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-314014-d9jwy5b6 authors: duan, hongxia; yan, linlin; ding, xu; gan, yiqun; kohn, nils; wu, jianhui title: impact of the covid-19 pandemic on mental health in the general chinese population: changes, predictors and psychosocial correlates date: 2020-08-18 journal: psychiatry res doi: 10.1016/j.psychres.2020.113396 sha: doc_id: 314014 cord_uid: d9jwy5b6 the current covid-19 pandemic is not only a threat to physical health, but also brings a burden to mental health in the general chinese population. however, the temporal change of mental health status due to pandemic-related stress in relation to protective and risk factors to hostility is less known. this study was implemented at two timepoints, i.e., during the peak and the remission of the covid-19 pandemic. 3233 chinese individuals participated in the first wave, and among them 1390 participants were followed in a second wave. the result showed that fear significantly decreased over time, while depression level significantly increased during the second wave compared to the first wave of the survey. younger age, lower-income, increased level of perceived stress, and current quarantine experience were significant predictors of depression escalation. younger people and individuals who had a higher initial stress response tended to show more hostility. furthermore, the use of negative coping strategy plays a potential intermediating role in the stress-related increase in hostility, while social support acts as a buffer in hostility in the general population under high stress. as the whole world is facing the same pandemic, this research provides several implications for public mental health intervention. since december of 2019, china has experienced a dramatic outbreak of a novel coronavirus disease 2019 (covid19) , which rapidly spread in china and abroad. wuhan, the center city of the epidemic, was locked down for 76 days from january 23 to april 8, and many other regions in china initiated first-level responses to major public health emergencies to curtail further disease transmission. since the announcement of global pandemic threat by the world health organization (who) on march 11, 2020, many other countries initiated their lockdowns on their own. only one study explored the change of mental health problem from the initial outbreak phase (end of january) to the epidemic's peak or acute phase (end of february) in the general public (n = 333) (wang et al., 2020b) . however, they could not investigate the temporal change of mental health problems at the individual level because they were not able to identify repeated respondents at the second time interval. a commonly reported phenomenon in the media during this pandemic is an increase in hostility against people from such as medical profession, other nations or domestic ethnic minorities around the world (wikipedia: list of incidents of xenophobia and racism related to the covid-19 pandemic). in laboratory studies, a wide variety of stressors, like physical pain or minor daily hassles have been demonstrated to increase harmful social reactions such as aggression in both animal models and human participants (for reviews, see sprague et al., 2011; takahashi et al., 2018) . however, the role of stress caused by social quarantine and constant low-level threat due to uncertainty during the pandemic and how this might relate to increased hostility is rarely studied. in the present study, hostility is defined as comprising feelings and/or behaviors characterized by anger expression such as aggression, irritability, rage, and resentment (holi, 2003) . in addition, it was proposed that stress per se is less crucial to mental health than coping strategies a person uses in response to stress (compas et al., 2001) . the construct of coping, the cognitive and behavioral strategies to meet the demands of stressful situations, has been considered an important mediator of the stress-emotion relationship (folkman & lazarus, 1988; main et al., 2011) . coping can be described as negative/avoidant (e.g., distraction, withdrawal, wishful thinking, substance use) or positive/approach (e.g., problem-solving efforts, seeking information) (day & livingstone, 2001). moreover, researchers have found that positive/approach coping is generally related with less psychological symptoms, while negative/avoidant coping is associated with more symptoms in the chinese population (ding et al., 2015; zheng et al., 2012) . furthermore, it is important to examine the social factors that might protect an affected population from developing hostility. social support of individuals by family, friends and even institutions have been widely demonstrated to have positive effects on both physical and psychological well-being, and also to have a moderating effect of stress on health, the so-called "stress-buffering hypothesis" (cohen & wills, 1985) . according to this hypothesis, the moderation effect of social support on stress may arise through both processes depending on the amount of social support (main effect) and processes depending on the interaction of the stress level and the amount of social support (buffering effect). the buffering effect of social support, therefore, becomes more crucial as the stress level individuals perceived increases. consistent with stress-buffering hypothesis, we predicted that social support might act as a buffer between stress and hostility. therefore, the first aim of the current study was to explore the effect of the covid-19 pandemic on mental health of a general chinese population sample during the course of acute pandemic activity the peak of the outbreak (january 31 st to february 9 th ) to the epidemic's remission phase (15 th to 28 th of march) at the individual level. the second aim was to examine whether perceived stress level to the covid-19 pandemic would predict an increase in hostility and whether coping strategies would play a potential intermediating role in the relationship between stress and hostility. it was expected that the underlying mechanism between stress and hostility would function through coping strategies, such that higher perceived covid-19 stress would be related with an increased use of negative coping, which, in turn, would lead to more general hostility. the third aim was to identify protective psychosocial factor from the perspective of perceived social support in the relationship between perceived stress and hostility in the general population sample. according to the stress-buffering effect, individuals with high stress level benefit more from social support than individuals with low stress level (cohen & wills, 1985) . therefore, it was expected that social support would act as a protective factor between stress and hostility. this study was conducted from january 31 st to february 9 th (first wave of the survey, t1) and march 15 th to march 28 th (second wave of the survey, t2), which covered the time from the peak of the outbreak to the remission of covid-19 epidemic in central china. as is shown in fig. 1 , at the first timepoint of this study, china was undergoing a difficult period during which the confirmed and suspected cases reached its peak, and pressure on the healthcare system was intensified. there was little information about the causal agents and available treatment methods for the disease. were asked to fill in the questionnaire according to their current situation in a relatively quiet environment to avoid interference as much as possible. they were also informed that their personal information and responses will be kept anonymous and confidential. this study was approved by the ethics committee of peking university. all participants provided electronic informed consent before the commencement of the two waves of survey. the lines indicate case counts per day of confirmed cases in yellow (note the spike of confirmed case on 14 th of february was due to a change in diagnostic criteria from only by test kits to clinical (radiological) diagnosis of patients), suspected cases in blue, recovered cases in green and casualties in red. to assess the perceived stress to the covid-19 epidemic, perceived stress scale during the second wave of the survey. all the questionnaires were described in detail in the following part. the pss10 includes 10 items assessing stress for the past one month during the covid-19 epidemic. this scale was originally compiled by cohen (1983) and the revised chinese version has been demonstrated to have good reliability and validity (yang et al., 2003 ). an example item is "during the outbreak, how often have you felt that you were unable to control the important things in your life". each item is rated from 1 (not at all) to 5 (very much). the average score is calculated by the sum of each item score divided by the total number of items (range: 1~5). the higher the score, the more stressed the respondent is. the pss10 demonstrated good internal consistency (α = .801) in the current sample. the mental health status during the covid-19 pandemic was measured by the the hostility subscale of the scl-90 (derogatis & savitz, 1999 ) was used to measure hostility levels in the second wave. this hostility subscale comprises 6 items such as temper outbursts that you could not control in the past one week, and each item is assessed by a 5-point likert scale (0 = "not at all" to 4 = "all the time"). the average score is calculated by the sum of each item score divided by the total number of items (range: 1~5). an example item is "how often did you get into frequent arguments within the last week". it reflects the respondents' hostility from three aspects: thoughts, emotion and behavior. in the current sample, the internal consistency coefficient is α = .858. coping style has been widely investigated around the world. however, the difference in research aims contributed to various evaluation tools for coping style. in china, one of the main understandings of coping style is defined it as the tendency an individual is used to adopting. based on this, xie (1998) considering the characteristic of "collectivism" in chinese culture, the chinese psss includes not only supports from family and friends, but also supports from others government. for example, "when we need help, our nation's medical forces can be the first to provide help". this scale was used to measure the subjective and perceived social support of participants in the second wave. the degree of support is rated and average score is calculated (range: 1~7), with higher score indicating more perceived social support. the internal consistency of our research is α = .828. all the statistical analysis was conducted by ibm spss statistics, version 24.0 (ibm for the second and third aim, the intermediating role of coping styles and the moderating role of perceived social support on hostility were analyzed by process macro (model 1 and model 4). in these models, we controlled for sex, age, education, monthly income, and quarantine experience (t1 and t2). all continuous variables were standardized and the interaction terms were computed from these standardized scores. the bootstrapping method produces 95% bias-corrected confidence intervals of these effects from 5,000 resamples of the data. confidence intervals that do not include zero indicate effects that are significant (hayes, 2013) . the standard error of the linear regression model is estimated using hc3 as proposed by davidson and mackinnon (1993). demographic description of the respondents who participated in the two waves of the survey (n = 1390) are shown in the to explore predictors of depression level at t2, a hierarchical regression model was used. specifically, for independent variables, depression level and quarantine experience measured in wave 1 were placed in the first step as the controlled variables; demographic variables (i.e., gender, age, education and monthly income), which were all measured in wave 1, were entered in the second regression step; and increase in perceived stress as well as quarantine experience measured in wave 2 were placed in the third step. as table 3 shows, after controlling initial depression level (depression_t1) and initial quarantine experience (quarantine_t1), δpss10 (β from t1 to t2 is associated with higher depression level at t2. quarantine experience at t2 (β = -.063, p < .05) significantly predicted depression at t2, such that respondents who have the recent experience of quarantine reported higher depression level at t2. furthermore, age (β = -0.056, p < .05) and monthly income (β = -0.054, p < .05) were also significant predictors, indicating that younger people and individuals with lower income experience worsening depression symptoms. there was a significantly positive correlation between pss10 at t1 and hostility at t2 (r = .382, p < .001). in the hierarchical model, initial perceived stress and quarantine experience measured in wave 1 were placed in the first regression step; demographic variables (i.e., gender, age, education and monthly income), which were all measured in wave 1, were entered in the second regression step; and quarantine experience measured in wave 2 was placed in the third step. the result (see table 4 ) showed that the level of perceived stress at t1 (β = .366, p < .001) significantly predicts the higher level of hostility at t2. additionally, age (β = -.082, p < .001) negatively predicts the hostility such that younger people show higher hostility in general. the level of hostility at t2 was positively correlated with negative coping (r = .287, p < .001) and negatively correlated with positive coping (r = -.123, p < .001), which provided a precondition for testing the intermediating effect of coping styles of stress-hostility relationship. a mediation model was built in which perceived stress at t1 was treated as a predictor, the level of hostility at t2 as the outcome variable, negative and positive coping as mediators, and demographic variables (gender, age, education, and monthly income) as covariates. this mediation model was tested using the process macro (model 4) developed by hayes (2013) . as reported in table 5 , perceived stress at t2 is negatively correlated with positive coping (β = -.167, p < .001) and positively correlated with negative coping (β = .055, p < .05). furthermore, positive coping at t2 negatively predicted the level of hostility at t2 (β = -.161, p < .001), while negative coping at t2 positively predicted the level of hostility at t2. furthermore, positive coping and negative coping at t2 independently intermediates the relationship between perceived stress at t1 and the level of hostility at t2 (see result in supplementary material). the results of mediation effect are summarized in fig. 3 . to summarize, positive coping negatively intermediates the effect of stress on hostility, while negative coping positively intermediates the effect of stress on hostility. there was a significantly positive correlation between pss10 at t1 and hostility at t2 (r = .382, p < .001). meanwhile, perceived social support at t2 was negatively correlated with the level of hostility at t2 (r = -.208, p < .001). therefore, the process macro (model 1) by hayes (2013) was used to test whether perceived social support at t2 could moderate the relationship of perceived stress at t1 and the level of hostility at t2. as displayed in table 6 , the interaction effect of perceived stress at t1 and perceived social support at t2 could negatively predict the level of hostility at t2 (β = -.081, p < .001). to further explain the interaction effect, the relationship between perceived stress at t1 and hostility at t2 was plotted. as the moderator, the levels of perceived social support at t2 was divided into low (m -sd) and high (m + sd), respectively. the results showed that as the level of perceived social support at t2 reduced from high to low, the predictive effect of perceived stress at t1 on the level of hostility at t2 was gradually strengthened, and β increased from .241 (p < .001) to .402 (p < .001) (see fig. 4) . therefore, perceived social support buffered the relationship between elevated perceived stress and elevated hostility. in the current study, we investigated the temporal change of mental health status in a sample of the chinese population from the peak to the remission phase of the covid-19 pandemic. we found that compared to the peak phase, levels of perceived stress and fear decreased, while depression levels were significantly increased during the remission phase. regression analysis showed that younger age, lower income, higher perceived stress to the covid-19 pandemic as well as current quarantine experience (measured at t2) are risk factors of depression deterioration. furthermore, we found that the younger age and initial perceived stress during the peak phase predict more hostility in its remission phase. our mediation model showed that however, due to the cross-sectional design, these two studies could not clarify whether these symptoms were elevated above the level as when the pandemic occurred. an assessment of hospital employees in china found that compared to a non-segregated sample, the experience of being segregated during sars was associated with high depressive symptoms three years after the epidemic . with a prospective design, our study provided insights for depression development under the epidemic in the general population. individuals who are young, economically disadvantaged, who have been socially isolated or quarantined and who show a strong initial stress response might be particularly at risk for elevated depression in the aftermath of such a pandemic. we found that younger people expressed more hostility in this covid-19 epidemiological situation, which might be due that younger adults adopt ineffective emotion regulation supported by cognitive control (jackson & finney, 2002) . after reviewing data from structural and functional brain imaging, nashiro and colleagues (2012) found that with preserved amygdala in older adults, they show greater prefrontal cortex activity than younger adults while engaging in emotion-processing tasks. furthermore, individuals perceived higher stress of the covid-19 pandemic at the peak of the outbreak predicted more hostility at the remission phase of the epidemic. this is consistent with berkowitz's (1990) however, our mediation model showed that relations between perceived stress and hostility could be accounted for by the coping strategies individuals selected. this is consistent with stress and trauma literature that the association between life stress and psychological adjustment is strongly mediated by coping strategies (runtz & schallow, 1997; tremblay et al., 1999) . positive coping, such as problem-solving efforts, seeking information and cognitive reappraisal, involves focusing on the cause of the stress and attempting to actively do something to mitigate the stress (carver et al., 1989) . individuals with positive coping strategies believe they have more control over the situation and might in turn develop fewer hostile responses to a stressful situation. negative coping, such as denial, withdrawal, wishful thinking and substance use, involves emotion-focused passive coping strategies in an attempt to reduce the emotional stress elicited by a stressful situation. individuals with passive coping believe they have little control over the situation (folkman & lazarus, 1980; , and therefore display more hostility to the environment. furthermore, the relationship between perceived stress and hostility was moderated by social support, which was consistent with the stress-buffering effect that social support has. compared to individuals with lower levels of stress, individuals with higher levels of stress show a more substantial and beneficial influence of social support (cohen & wills, 1985) . our study provides some implications for public mental health. first, it is crucial to develop and implement effective screening procedures at the institutional level to identify risk and resilience factors to provide precise intervention (yang et al., 2010) . in this context, we identified that younger people are at risk of both depression and hostility in the aftermath of the covid-19 pandemic. second, the effect of pandemic-related stress on emotional disturbance (especially depression) suggests the importance of early individualized psychological intervention in the general population, with a focus on individuals who are economically disadvantaged, individuals who have been quarantined and individuals that exhibit high levels of stress. third, positive coping strategies as well as social support should be encouraged even in the context of social distancing. there are some limitations to our study. first, we investigated a chinese sample only to assess the temporal dynamics of mental health during this pandemic, which limits generalizability to other countries. in the same vein, the questionnaires we used here are not for clinical diagnosis and include constructs that capture culturally-dependent phenomenon such as neurasthenia, social support from the government level. second, all the constructs in the current study were assessed by self-report. however, more and more studies reach the consensus that it is the subjective appraisal rather than the stressor itself that has large impacts on mental and physical health (mathur et al., 2016; pascoe & smart richman, 2009 ). third, the retention rate from the first wave to the second wave in our study was low (approximately 43%). the respondents who participated in the two waves were mainly young and well-educated who might also be more physically and psychologically resilient. older and socio-economically disadvantaged people might constitute a population that is more at risk for severe trajectories of the disease and also consequently might be more prone to perceived stress. despite these limitations, our study tracked the psychological change at the individual level during the peak and remission phase of the covid-19 epidemic in china. fear significantly decreased while depression level significantly increased over the course of the pandemic. we identified that younger age, lower income, higher perceived stress, and current quarantine experience are risk factors for depression deterioration. younger age and higher initial stress predicted higher hostility, which was mediated by negative coping style. furthermore, social support can buffer the effect of stress on hostility. our results might provide implications for public mental health intervention. declaration of competing interest: the authors declare no competing interests. conceptualizing the prospective relationship between social support, stress, and depressive symptoms among adolescents examining an affective aggression framework weapon and temperature effects on aggressive thoughts, affect, and attitudes on the formation and regulation of anger and aggression: a cognitive-neoassociationistic analysis pain and aggression: some findings and implications tracing "fearbola'': psychological 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epidemiological study on stress among urban residents in social transition period life events, coping, and posttraumatic stress symptoms among chinese adolescents exposed to 2008 wenchuan earthquake the multidimensional scale of perceived social support key: cord-297673-rh8o4eu9 authors: vahedian-azimi, amir; moayed, malihe sadat; rahimibashar, farshid; shojaei, sajad; ashtari, sara; pourhoseingholi, mohamad amin title: comparison of the severity of psychological distress among four groups of an iranian population regarding covid-19 pandemic date: 2020-08-08 journal: bmc psychiatry doi: 10.1186/s12888-020-02804-9 sha: doc_id: 297673 cord_uid: rh8o4eu9 background: coronavirus disease 2019 (covid-19) pandemic has caused serious psychological problems, including panic attack, anxiety, stress, and depression. the main objective of this study was to measure the prevalence and compare the severity of this psychological distress among four groups of an iranian population. method: in a cross-sectional survey, the mental health status of four groups of an iranian society including community population, patients with covid-19, medical staff, and medical students were investigated by the self-report questionnaire of depression, anxiety, and stress scale (dass). dass-21 questionnaire and the demographic data sheet were filled out by the participants. all statistical analyses were done using r version 3.6.1 software. p-values less than 0.05 were considered as statistically significant. anova test was used to compare the severity of stress, anxiety, and depression between the four study groups. results: of the 886 participants in this survey, 554 (62.5%) were men and 332 (37.5%) were women, and the mean ± standard division of age was 40.91 ± 10.7 years. among these participants, 241 (27.2%) were selected from community population, 221 (24.9%) were patients with covid-19, 217 (24.5%) were medical staff, and 207 (23.4%) were medical students. the mean score of stress, anxiety, and depression in medical students and patients with covid-19 was significantly higher than in medical staff and community population (p < 0.05). overall, the anxiety score in men was higher than that in women (27.4 ± 4.6 vs. 26.48 ± 4.8, p = 0.006), and unmarried participants had a significantly higher depression score compared with the married group (27.5 ± 4.8 vs. 26.7 ± 4.6, p = 0.023). in addition, the score of depression was higher in female medical staff (27.08 ± 4.6 vs. 25.33 ± 4.3, p = 0.011) and community population (26.6 ± 4.3 vs. 25.3 ± 4.3, p = 0.02) than in male. conclusion: covid-19 patients and medical students in contact with these patients were at a high risk for mental illness due to lower experience compared with professional medical staff and community population. continuous surveillance and monitoring of psychological distress for outbreaks should become a routine part of preparedness efforts worldwide. the novel coronavirus disease 2019 (covid19) , formerly known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first detected in december 2019 in wuhan city in china [1] . as of 4 april 2020, more than 1, 250,000 cases of covid-19 were reported by more than 209 countries, hence characterized as a pandemic [2] . as a result of the rapid increase in the number of confirmed cases and deaths, both medical staff and the public have been experiencing psychological pressure and other health-related issues [3] . these concerns arose with all infections, including influenza, severe acute respiratory syndrome (sars), and middle-east respiratory syndrome (mers) that took place years ago. during the outbreak of these infections, several psychiatric comorbidities such as depression, panic attack, anxiety, psychomotor excitement, suicide, and stress symptoms were reported [4] [5] [6] . however, covid-19 has been highlighted as a unique threat that has added to panic, stress, anxiety, and the potential for depression due to its fast transmission pattern, inadequate preparedness of health officials, and the absence of a comprehensive and definitive treatment protocol or vaccination program [7] . pandemics can psychologically affect everyone in the community. however, healthcare workers and medical students, particularly those involved in the treatment of covid-19 patients, are highly susceptible to both infection and mental health problems. during previous sars outbreak, research on healthcare workers showed that many presented high levels of psychological distress and frequent concerns regarding their health and functional ability and their families' health [8] [9] [10] [11] [12] [13] . previous studies also revealed psychological disorders and chronic fatigue in the survivors of diseases such as sars and mers [14, 15] . moreover, depression and post-traumatic stress disorders (ptsd) have been reported to last for as long as a year after illness [16] . there is a paucity of research on the psychological impact and mental health of the iranian population during the covid-19 pandemic. therefore, we conducted this cross-sectional survey for the first time to measure the prevalence and compare the severity of the psychological distress (stress, anxiety, and depression) among community population, patients with covid-19, medical staff, and medical students in an iranian population. we hope our study findings will provide data support for the target intervention on psychological health in iran and different parts of the world during the pandemic. this cross-sectional survey was carried out from february to march 2020 in tehran, iran; the aim was to measure the prevalence and compare the severity of psychological distress (stress, anxiety, and depression) among community population, patients with covid-19, medical staff, and medical students in an iranian population. the present study was approved by the ethics committee of baqiyatallah university of medical sciences, tehran, iran, with code ir.bmsu.rec.1398.442. cochran's sample size estimation formula was used in the epidemiologic study. the first and second type errors were considered 0.05 and 0.02, respectively. a 50% satisfaction probability was assumed to estimate the maximum sample size for each group (196 participants). given the nature of the study and the probability of dropout, a 10% drop was considered, and the final sample size was estimated to include a minimum of 216 participants in each group. of the 886 participants in this cross-sectional survey, 241 (27.2%) were selected from community population, 221 (24.9%) were patients with covid-19, 217 (24.5%) were medical staff, and 207 (23.4%) were medical students. adult subjects aged 18 years or older, interested in participation, able to read and write, and having no physical disability or mental disorder (based on selfreports) were included in the community population group. patients with covid-19 were selected from those referred to baqiyatallah hospital, one of the main referral centers for specialized diagnosis and treatment of covid-19, between february and march 2020 in tehran, iran. all patients with covid-19 enrolled in this study were diagnosed according to world health organization interim guidance [17] . we included the medical staff members of baqiyatallah hospital who treated patients with covid-19 infection for at least a week in february and march 2020 and volunteered to participate. medical students included in the study were randomly selected from the faculty of nursing and medicine (baqiyatallah university of medical sciences, tehran, iran). all the medical students in the study were interns working under the supervision of residents and fully licensed staff physicians at university hospitals. therefore, the included medical students had at least a week experience of working with covid-19 patients. dass-21 questionnaire and the demographic data sheet were filled out by all participants. demographic variables included age, gender, and marital status. in addition, work experience (years) and experience working with covid-19 patients (weeks) were recorded for medical staff and medical students. the study participants were all informed about the objectives of the study and written informed consent was received from each participant. they were also assured of confidentiality. mental health status was measured using the depression, anxiety, and stress scale (dass-21). this questionnaire was designed and validated by lovibond in 1995 [18] to measure the psychological distress in a community with 21 items. dass-21 is a unique, simple, and approved instrument for assessing depression, anxiety, and stress both in clinical settings and communities [19] . dass is a short screening tool that measures depression, anxiety, and stress by a 21-item self-report questionnaire. for each disorder, seven questions are considered, and the final score is obtained by the total score of the questions related to it. each question was scored using a likert-scale ranging from 0 (did not apply to me at all/never) to 3 (applied to me very much, or most of the time/almost always). higher scores indicated a higher level of disorder based on a specific classification scoring system. individuals were categorized into normal, mild, moderate, severe, and extremely severe based on their responses. comparison of dass-21 results with psychiatric interviews showed that this tool had a sensitivity and specificity of 75 and 89% and was capable of accurately screening depression, anxiety, and stress [20, 21] . the reliability and validity of the translated version of the persian questionnaire was confirmed for an iranian population. in a study by sahebi et al. [22] on 970 students and military men, the translated questionnaire was reported to be comparable with the original one with a high internal correlation; cronbach's alpha of depression, anxiety, and stress subscales were 0.77, 0.79, and 0.78, respectively. in addition, the study by moradipanah et al. [23] in iran reported a cronbach's alpha of 0.94 for depression, 0.92 for anxiety, and 0.82 for stress. categorical variables were described as frequency rates and percentages, and continuous variables were described using mean ± standard deviation (sd) values. the scores of the dass subscales for each group were expressed as mean and standard deviation. anova test was used to compare the severity of stress, anxiety, and depression between the four study groups. moreover, the mean scores of stress, anxiety, and depression were compared between the two groups via (tukey) post hoc test. all tests were two-tailed with a significance level of p < 0.05. statistical analysis was performed using r version 3.6.1 software. a total of 886 participants responded to the questionnaire. the results showed that the majority of participants had extremely severe anxiety 862/886 (97.3%), and the mean score of anxiety level was higher in men than in women (27.4 ± 4.6 vs. 26.48 ± 4.8, p = 0.006, 95% ci: 0.27-1.56). meanwhile, in terms of stress (p = 0.446) and depression (p = 0.774), there was no statistically significant difference between men and women. in addition, unmarried participants had a significantly higher average depression score compared with the married group (27.5 ± 4.8 vs. 26.7 ± 4.6, p = 0.023, 95% ci: 0.11-1.38). however, the mean score of stress and anxiety levels did not differ between married and unmarried participants (p > 0.05). among these participants, 241 (27.2%) were selected from community population, 217 (24.5%) belonged to the medical staff, 207 (23.4%) were medical students, and 221 (24.9%) were patients with covid-19. table 1 shows the demographic characteristics and severity of psychological distress in the participants of the four study groups. the occupational status for community population and patients with covid-19 was divided into five subgroups: employed, self-employed, looking for work or retired, student, and homemaker. the highest frequency in both groups was related to selfemployed subgroup (more than 35%). however, this factor did not affect the level of stress, anxiety, and depression in these two groups. anova test was used to compare the severity of stress, anxiety, and depression between the four groups under study (table 2) including sex, marital status and age group (lower or upper 40 years); the results showed significant differences among four groups of study mean score of stress (f: 13.9, p < 0.001), anxiety (f: 14.8, p < 0.001), and depression (f: 23.9, p < 0.001) in. in addition, the mean scores of stress, anxiety, and depression were compared between the groups two by two with (tukey) post hoc test. results showed that the mean scores of stress, anxiety, and depression in medical students and patients with covid-19 were significantly higher than the medical staff and community population (p < 0.05). moreover, medical students had a significantly higher average depression score compared with covid-19 patients (29.36 ± 4.4 vs. 28.07 ± 5.06, p = 0.031, 95% ci: 0.08-2.5). in terms of stress and anxiety, on the other hand, no statistically significant differences were detected between medical students and covid-19 patients (p > 0.05). according to the main effect model analysis, the depression score was significantly different between the groups according to gender (p = 0.015). the results showed that the average depression score in female medical staff (f/m: 27.08 ± 4.6 vs. 25.33 ± 4.3, p = 0.011, 95% ci: 0.39-3.08) and female community population (f/m: 26.6 ± 4.3 vs. 25.3 ± 4.3, p = 0.02, 95% ci: 0.21-2.47) was higher than in males. as far as age groups and marital status are concerned, no significant differences were observed between the groups according to stress, anxiety, and depression levels. to our knowledge, this is the first study to report the prevalence and compare the severity of the stress, anxiety and depression among four groups of iranian society at the initial stage of the covid-19 outbreak. the main results of the present study showed that covid-19 patients and medical students with at least 1 week experience of working with them had significantly higher scores of stress, anxiety, and depression compared to the medical staff and community population; this suggests that they are the main targets of psychiatric assessment and care. in total, the anxiety score was higher in men than in women, and unmarried participants had a significantly higher depression score compared with the married group. in addition, the score of depression in female medical staff and community population was higher in comparison with the males. previous studies have revealed an association between epidemics and mental disorders during the spread of [24, 25] . hawryluck et al. [4] reported that isolation and quarantine during the outbreak were stressful; they observed that certain subjects, due to quarantine for sars in canada, displayed symptoms of ptsd (28.9%) and depression (31.2%). al-najjar et al. [26] examined the psychological reactions of adults to the mers epidemic in western saudi arabia; they found that anxiety was significantly increased by the increasing susceptibility to infection and social behaviors associated with travel and being in public places. lee et al. [27] assessed the psychological impacts of the mers outbreak and found that ptsd symptoms were very high among hospital staff even many years after the outbreak. there are not many studies on the psychological impacts of covid-19 pandemic on society, except for a few investigations conducted in china [28] [29] [30] . in a cross-sectional study by wang et al. [31] , psychological impacts, depression, stress, and anxiety were evaluated in 1210 participants from 194 cities in china at the beginning of the covid-19 outbreak; their results showed that 53.8% of these individuals experienced severe psychological impacts of the outbreak. moreover, 16.5, 28.8, and 8.1% of the respondents reported moderate to severe levels of depression, anxiety, and stress, respectively. in a cross-sectional observational study, xiao et al. [32] measured the levels of anxiety, self-efficacy, stress, sleep quality, and social support in 180 medical staff; their findings showed that medical staff in china who were treating patients with covid-19 infection had high levels of anxiety, stress, and self-efficacy that were dependent on sleep quality and social support. the results of the present study and all previous studies show that the psychological trauma caused by the prevalence of infectious diseases is highly common in societies. infectious pandemics can cause disruptions in societies and individuals on many levels [33] . covid-19 imposes irreversible psychological impacts on all groups of community members, such as general population, healthcare workers, and students due to the commuting restrictions, fear of contracting the virus, anxiety about the closure of schools and businesses, the depression following the loss of friends and family, and fear of death [34] [35] [36] . furthermore, according to the results of our study, the score of anxiety level in men was significantly higher than that in women. this could be attributed to the economic pressure caused by the pandemic because in most iranian families, men are responsible for daily expenses. the economic impact of covid-19 and its effects on community behavior, such as hoarding and stockpiling of resources and financial predicaments can cause psychological problems for the householder. furthermore, the mean depression score in female medical staff and the female in the community population was higher than in men. in line with previous studies from china [31, 37] , women seemed to experience elevated psychological symptoms related to this pandemic as compared to men. in this exceptional situation, women are faced with additional responsibilities, such as family care and child support in learning due to school closures. female medical staffs are more likely to suffer from depression due to loneliness and separation from families and children. hence, supporting women in this situation might be especially important. the results of this study revealed that patients with covid-19 and medical students in contact with these patients are the main targets of psychiatric assessment and care. perhaps the main reason for the higher psychological distress among medical students compared with fully licensed physicians as medical staff is the former group's lack of experience in controlling infectious diseases in high-risk environments and the fear of medical errors in the face of new cases. other reasons are physical and emotional exhaustion due to a high-pressure health care system, rapid changes in medical information and procedures, self-perception of risk to themselves, the impact of pandemic on lifestyle, fear of inadequate medical equipment, such as masks and gloves, long working hours, and separation from families. to reduce the psychological distress among medical students, health policymakers are to introduce new policies that attract more medical students to the healthcare system with more awareness and readiness and also increase the flexibility of supervisors. health services that employ interns must continue to supervise them and provide them, as much as possible, with meaningful educational clinical experiences. these interns should also be trained in caring forcovid-19 patients, informed about protective measures, and supported during challenging times. unpredictability, uncertainty, seriousness of the disease, misinformation, social isolation, and the overwhelming news may cause anxiety and fear in the public. the general public may also experience boredom, frustration, and irritability under isolation measures [29] . in patients with covid-19, these can be related to the fear of severe disease consequences, contagion, isolation treatment, loss of trust in health services, and fear of death. consequently, they may experience loneliness, denial, anxiety, depression, insomnia, and despair, which may lower the treatment adherence. some of these cases may even run increased risks of aggression and suicide. due to the sudden shock of the outbreak and the lack of information on the disease, interventions to promote mental well-being may not be possible at the beginning of global pandemics. as a possible solution for this challenge, public health decision makers need to perform appropriate psychosocial interventions and incorporate mental health management plans in next few months. other steps to lowering the psychological distress in society can be the assessment of the accuracy of information, enhancing social support, reducing the stigma associated with the disease, maintaining a normal life while adhering to safety measures, and using available psychosocial services. the main strengths of the present investigation were the comparison of the severity of this psychological distress among the four groups of society and the large sample size in each group. however, this study is not without its limitations. first, this is a single-center crosssectional survey which limits the generalizability of our findings. our participants in three groups of study were recruited in the same hospital, which cannot represent the iranian population, hence the need for further research. second, we were unable to investigate the history of participants' mental disorders. thus, participants with a history of mental disorders based on self-report were excluded from the study. this study showed the high severity of anxiety, stress, and depression among iranian subpopulations during covid-19 pandemic. covid-19 patients and the medical students in contact with them were the main targets of psychiatric assessment and care. increasing public awareness, building trust in the media, and providing information on patients' recovery can reduce the psychological burden of this pandemic in the society. in addition, based on the previous experiences with sars outbreak, some patients and health professionals are traumatized by the covid-2019 outbreak and still suffer from persistent psychiatric symptoms even after the outbreak. therefore, we should expect long-term negative psychological outcomes as post-traumatic stress disorder (ptsd) among covid-19 survivors and healthcare workers. this indicates the necessity of designing psychological interventions so as to improve mental health during and after the pandemic. ethics approval and consent to participation the present study was approved by the ethics committee of baqiyatallah university of medical sciences, tehran, iran, with code ir.bmsu.rec.1398.442. the study participants were all informed about the objectives of the study and written informed consent was received from each participant. a novel coronavirus outbreak of global health concern worldometer: covid-19 coronavirus outbreak timely mental health care for the 2019 novel coronavirus outbreak is urgently needed sars control and psychological effects of quarantine 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epidemic among the general population in china the effects of social support on sleep quality of medical staff treating patients with coronavirus disease 2019 (covid-19) in january and february 2020 in china the psychological impact of quarantine and how to reduce it: rapid review of the evidence covid-19 and anxiety: a review of psychological impacts of infectious disease outbreaks psychosocial effects of an ebola outbreak at individual, community and international levels the psychological effects of quarantining a city psychological impact and predisposing factors of the coronavirus disease 2019 (covid-19) pandemic on general public in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are grateful to the guidance and advice from the clinical research development unit of baqiyatallah hospital of medical sciences, tehran, iran. authors' contributions av-a developed the study concept. all authors contributed to the study design. testing and data collection were performed by av-a, msm, and mr data analysis and interpretation were performed by sa and ssh under the supervision of m-ap. sa drafted the manuscript, and m-ap and av-a provided critical revisions. all authors approved the final version of the manuscript for submission. no funding was received for this study.availability of data and materials all data collected and analyzed during the current study can be provided by the corresponding author on reasonable request. not applicable. none to declare. key: cord-302185-pnw3xiun authors: bodecka, marta; nowakowska, iwona; zajenkowska, anna; rajchert, joanna; kaźmierczak, izabela; jelonkiewicz, irena title: gender as a moderator between present-hedonistic time perspective and depressive symptoms or stress during covid-19 lock-down date: 2021-01-01 journal: pers individ dif doi: 10.1016/j.paid.2020.110395 sha: doc_id: 302185 cord_uid: pnw3xiun although numerous studies have addressed the impact of the covid-19 lock-downs on psychological distress, scarce data is available relating to the role of present-hedonistic (ph) time perspective and gender differences in the development of depressive symptoms and stress during the period of strict social distancing. we hypothesized that gender would moderate the relationship between ph and depressiveness or stress levels, such that ph would negatively correlate with psychological distress in women but correlate positively in men. the present study was online and questionnaire-based. n = 230 participants aged 15–73 from the general population took part in the study. the results of moderation analysis allowed for full acceptance of the hypothesis for depression as a factor, but for stress the hypothesis was only partially confirmed, since the relationship between ph time perspective and stress was not significant for men (although it was positive, as expected). the findings are pioneering in terms of including ph time perspective in predicting psychological distress during the covid-19 lock-down and have potentially significant implications for practicing clinicians, who could include the development of more adaptive time perspectives and balance them in their therapeutic work with people experiencing lock-down-related distress. in january 2020, the world health organization announced that covid-19 constituted a global pandemic (mahase, 2020) . the virus then proliferated worldwide and government actions to mitigate spread have significantly affected various areas of life, such as healthcare, transportation, freedom of movement and daily activity (simpson & katsanis, 2020; zajenkowski, jonason, leniarska, & kozakiewicz, 2020) . in poland, public health safety measures were initiated in january 2020, followed by declaration of a state of epidemic emergency and imposition of lock-down measures on march 14th and the declaration of a state of epidemic from march 20th (pinkas et al., 2020) . lock-down and social isolation, although quite effective in slowing down the pace of the epidemic, have been shown to impact emotional and mental health (de quervain et al., 2020; li et al., 2020; shigemura & kurosawa, 2020) . according to these reports, one of the most significant adverse consequences of the changes in everyday life due to epidemic is an elevation of stress and depressive symptoms in the population. for instance, initial results of the swiss corona stress study (de quervain et al., 2020) suggested that there was a 50% increase in stress levels during the lock-down compared to the period preceding it. changes in stress levels were strongly associated with changes in depressive symptoms as 57% of participants reported an increase in depressive symptoms, which is not unexpected considering the strong link between stressful life events and depression (hammen, 2005) . interestingly, approximately 25% of the participants reported lower stress levels during lock-down than before. the authors of the report suggest that, in this group, the decrease might have been due to a reduction of stressors or having more time for recovery from stress during lock-down than under non-lock-down circumstances. accordingly, the level of experienced stress during lock-down and the impact it may have on mental health may be an individual matter. one promising avenue for investigation is found in gender differences and their potential associations with perceived stress and depressive symptoms during lockdown. it is worth noting that a greater number of depression diagnoses are observed in women than men (essau, lewinsohn, seeley, & sasagawa, 2010; van de velde, huijts, bracke, & bambra, 2013) . women have higher incidence rates of clinical diagnosis of dysthymia, recurrent brief depression and minor depression (for a review see angst et al., 2002) , as well as major depressive disorder and its chronic course (essau et al., 2010) . women were also found to report twice as many depressive symptoms as men (girgus & yang, 2015) , were more likely to admit being under stress and were more likely to develop depressive symptoms after a stressful event (sherrill et al., 1997) . ruminative tendencies, chronic strain and low mastery were also found to be more common in women and mediate the gender difference in depressive symptoms (nolen-hoeksema, larson, & grayson, 1999) . this gender difference might also stem from hormonal fluctuations (for a review of psychosocial factors in depression across genders see leach, christensen, mackinnon, windsor, & butterworth, 2008) . additionally, social roles, among other determinants, have been acknowledged as potential risk factors for developing depression in both genders (piccinelli & wilkinson, 2000) . gender schemas (martin & halverson jr, 1981) may be connected to how women and men attribute the causes of their depression onset. for instance, physical illnesses or problems were the most important precipitants of depression for both genders but especially for men (angst et al., 2002) . for women, problems in relationships and illness or death in the family were identified as other significant causes, whereas, for men, additional causes included problems at work and unemployment. furthermore, the question of whether elevated levels of depressive symptoms in women might be a consequence of gender inequality has been a topic of wide discussion (salk et al., 2017) . such an idea is supported by the association of female social roles with lower role overload and lack of choice (szpitalak & prochwicz, 2013; van de velde et al., 2013) and the well-established linkage between feelings of powerlessness, lack of control in one's own life and depression (mirowsky & ross, 2003) . despite a climate of social change in gender roles (eagly, nater, miller, kaufmann, & sczesny, 2020) , a number of cross-cultural similarities in the gender division of labor has been observed in advanced industrial societies (pérez & tavits, 2019) . women were found to typically invest more time in raising children, preparing food and caring for home. in contrast, men were found to typically invest more time in extra-domestic tasks. the context of lock-down creates the situation of needing to remain at home, the constant presence of all family members at the home, an increased importance of female gender schema-related activities, and either a shifting of extra-domestic activities to the home space or reduction of these activities. therefore, typically, women during lock-down might be encouraged to play more gender schemacongruent roles in the course of everyday lock-down life, in contrast to men. the remote work lifestyle, as well as fear of job loss due to the economic crisis resulting from the epidemic, might be especially gender schema-threatening for men and contribute to depressive symptoms. additionally, a lock-down situation shifts attentions to everyday activities and the uncertainty of the present moment (versluis, van asselt, & kim, 2019) . as no one could predict the duration of lock-down and the covid-19 epidemic, time perspective (at an individual consideration) may be a particularly noteworthy factor in explaining adaptations to the adverse situation. time perspective is generally defined as an "often unconscious process whereby the continual flows of personal and social experiences are assigned to temporal categories or time frames that help to give order, coherence and meaning to those events" (zimbardo & boyd, 1999 , p. 1271 . a habitual bias to process time in a certain manner might become a relatively stable individual difference, formed through learning processes and cultural influences (jochemczyk, pietrzak, buczkowski, stolarski, & markiewicz, 2017) . boyd (1999, 2008) in their seminal works distinguished five time perspectives: past-negative, past-positive, present-hedonistic (ph), present-fatalistic and future. a tendency to focus on particular time perspectives, especially past-negative and present-fatalistic might be predictive of a higher level of depressive symptoms, whereas past-positive (anagnostopoulos & griva, 2012; zimbardo & boyd, 1999) appeared to protect individuals from elevated levels of depressive symptoms. in general, people rating high on past-positive and ph time perspectives also exhibit increased well-being and life satisfaction (stolarski, bitner, & zimbardo, 2011; zhang & howell, 2011) . additionally, they are happier, in contrast with those scoring higher in the past-negative time perspective, who experienced less happiness (drake, duncan, sutherland, abernethy, & henry, 2008) . however, compared to other time perspectives, ph time perspective was the most robust predictor of current emotional states (stolarski, matthews, postek, zimbardo, & bitner, 2014) . hedonism, from which the name for the ph time perspective is taken, is defined as openness to pleasurable experience (veenhoven, 2003) , and is associated with lower levels of depressive symptoms (disabato, kashdan, short, & jarden, 2017) , as well as with mania in bipolar disorder (gruber, cunningham, kirkland, & hay, 2012) . therefore, the ph time perspective is especially interesting for investigating depressive and stress symptoms during covid-19 lockdown. the main aim of the current study is to contribute to the knowledge about potential gender differences in the linkages between ph time perspective and depressive symptoms or perceived stress during covid-19 lock-down. personal characteristics, including time perspectives, are related to how people experience social events. phs are habitually oriented to pleasures of the present and excitement with little consideration of future consequences (zimbardo & boyd, 1999) . strong social situations "providing salient cues to guide behavior and having a high degree of structure and definition" (snyder & ickes, 1985; p. 904) can be more important in predicting certain behaviors or experiences than personality traits (sherman, nave, & funder, 2012 ). an epidemic, considered to be a strong social situation, can increase psychological distress, especially depressiveness and stress level. it is possible that, due to strict social distancing, the impossibility of realizing most needs outside of home and, hence, the blockage of pleasant stimuli could predict depressiveness and stress experience. moreover, lock-down compels the discounting of immediate rewards for the sake of the one's own health and that of others, which might be difficult for ph-oriented people in general (jochemczyk et al., 2017; stolarski et al., 2011) . therefore, one might suppose that people who tend to fulfill their hedonistic needs outside of their homes might experience greater lock-down distress than people who tend to take pleasure from homeand family-oriented activities. considering the gender schema theories, it is possible that the lockdown situation could prove more depressing for men. according to such theories, men might be inclined toward valuing hedonistic extra-domestic activities (compared to typical domestic activities), which were significantly limited due to the lock-down. moreover, although in general women tend to present higher levels of depression, the factors leading to this discrepancy are distinct for women and men. for instance, men more frequently attributed the onset of their depression to current life events, such as unemployment or problems at work, than females did (angst et al., 2002) . the lock-down was not only linked to shifting work life to homes but sometimes caused employment uncertainty and financial insecurity. based on the above-mentioned theoretical assumptions, our hypothesis is that gender would moderate the relationship between ph and depressiveness or stress levels, such that ph would be negatively related with psychological distress in women m. bodecka, et al. personality and individual differences 168 (2021) 110395 but positively correlated with psychological distress in men. we recruited 230 participants (141 women, 89 men) online. power analysis conducted in g*power 3.1 (faul, erdfelder, buchner, & lang, 2009; faul, erdfelder, lang, & buchner, 2007) indicated that this sample size would allow for the detection of a small effect of partial r 2 increase of 0.05 (alpha = 0.05) with a power of 0.81. the participants were not reimbursed. all participants were between the ages of 15 and 73 years (m = 30.37, sd = 10.21). only 4 participants had not graduated high school, 31 participants (27.4%) declared secondary education, 63 participants (27.4%) were students and 130 individuals reported higher education (56.5%). the majority of participants lived in cities with either less than 100,000 inhabitants (n = 45, 19.6%) or more than 100,000 (n = 124, 53.9%) while the other participants lived in the countryside (n = 61, 26.5%). participants were married, (n = 71, 30.9%), in a partnership (n = 53, 23%), single (n = 97, 42.2%), divorced (n = 8) or widowed (n = 1). participants mostly lived with other people (n = 204, 88.7%), including with family (children, spouse, parents and other family members), with romantic partners or with friends. 24 individuals (10%) declared that they were currently in psychotherapy. participants were recruited through social media, primarily facebook, through paid advertisement, a post about the study on the lab profile and on private profiles using the snowball method. the study conformed to the declaration of helsinki (world medical association, 2001) , and all participants provided informed consent to take part in the study. the respondents were informed that the purpose of the study is to examine "how people deal with the current situation, how they feel, what they think", that the survey is fully anonymous and that they could discontinue at any time. the average time for survey completion was approximately 15 min. depressive symptoms. a 9-item patient health questionnaire (phq-9) was used to assess severity of depressive symptoms. its items correspond to criteria for diagnosis of dsm-iv and dsm-v depression symptoms (kroenke & spitzer, 2002; mitchell, frayne, wyatt, goller, & mccord, 2019) and enabled grading of depressive symptom severity. it contains questions about psychological well-being within the last two weeks (e.g., how often have you been bothered by little interest or pleasure in doing things?), including a question related to hurting oneself (i.e., how often have you been bothered by thoughts that you would be better off dead or of hurting yourself in some way?). in the current study, the phq-9 provided a severity measure with scores ranging from 0 to 27-each of the nine items can be scored from 0 ("not at all") to 3 ("nearly every day"). depression severity was defined by the scale's authors as: 1-4 none, 5-9 mild, 10-14 moderate, 15-19 moderately severe and 20-27 severe. the phq-9 was found to be a reliable measure in our study (α = 0.86). perceived stress. the perceived stress scale (pss) was applied to measure levels of stress (cohen et al., 1983) and it measures the degree to which situations in one's life are considered stressful. the scale consists of 10 items (four positively stated, e.g., in the last month, how often have you felt that things were going your way? and six negatively stated, e.g., in the last month, how often have you been upset because of something that happened unexpectedly?) that can be scored from 0 ("never") to 4 ("very often"). the pss scores are obtained by reversing the responses to the positively stated items and then summing across all the scale items. the cronbach's alpha coefficient in the present research was α = 0.91. time perspectives. the zimbardo time perspective inventory (ztpi) was used to measure the ph time perspective (zimbardo & boyd, 1999 and future (13 items e.g., when i want to achieve something, i set goals and consider specific means for reaching those goals). the participants were asked to score on a five-point likert scale the degree to which each statement referred to him/her (1 = very untrue, 5 = very true), and some items were reverse coded. the level of a specific time perspective was obtained by summing the items results for each scale. in the current study, the cronbach alpha for ztpi ph subscale was α = 0.77. reliability of ztpi subscales which were not of the interest of the current study is presented in appendix table a .1. all analyses were conducted using ibm spss 25.0.0.2 for windows. our main hypotheses were tested employing regression analysis with bootstrapping methods using andrew f. hayes process 3.2.01 macro (hayes, 2018) . frequency analysis of the results from the phq-9 indicated that 40 (17.4%) participants had no depressive symptoms, whereas the rest of the sample displayed mild (n = 85, 37.0%); moderate (n = 53, 23.0%); moderately severe (n = 37, 16.1%) or severe (n = 15, 6.5%) depressive symptoms. next, we investigated descriptive statistics, performed correlation analysis and tested for gender differences in time perspectives, stress and depression scores. the results of these analyses for the variables of interest of the current study are presented in table 1 . correlations and descriptive statistics for all study variables including ztpi subscales other than ph are presented in the appendix table a.2. it should also be noted that depression scores were not significantly associated with ph. stress was negatively, although weakly, correlated with ph. women and men did not differ in ph. results also indicated that women declared higher perceived stress and more intensive depression symptoms. mean depression scores for women fell into the interval for m. bodecka, et al. personality and individual differences 168 (2021) 110395 moderate level of depressive symptoms, while the mean for men fell within the mild depressive symptoms level. next, we tested our main hypothesis using regression models with a bootstrapping method for depressive symptoms and stress as dependent variables in two separate models. ph was included as the predictor and gender was included as a moderator in both models. coefficients with 95% ci for both models are presented in table 2 . data from table 2 suggests that both models predicted a significant amount of variance in the dependent variables. the results also showed that ph was negatively related with depression and with stress. women were coded 1 and men were coded 2; thus, a negative relationship indicated that women were higher on stress and depression scores. in both models, the interactions were also significant. the interpretation of ph and gender interaction with simple slopes showed that the relationship between ph perspective and depression scores was also significant and negative for women, whereas for men it was significant and positive. the relationship between ph and stress was significant and negative in women, while this relationship was not significant in men. the results allow us to accept the hypothesis in the case of depression but, in the case of stress, the hypothesis was only partially confirmed, since the relationship between ph and stress was not significant for men (although it was positive, as expected). the relationship between ph and depression scores in men and women is presented in fig. 1 and relationship between ph and stress in men and women is presented in fig. 2. the aim of the study was to explore gender differences in the relationships between ph and depressive symptoms or perceived stress during the specific context of lock-down due to the covid-19 epidemic. we tested for two independent models predicting depression and stress. both of these variables were strongly related, which is in line with previous studies (see hammen, 2005) . the majority of participants displayed at least mild depressive symptoms (82.6%). the study was performed during the strict social distancing period in poland and the risk of distress connected to being apart from other people might have been heightened. a study conducted on a representative sample during covid lock-down suggested that depressive symptoms were twice as high as before the measure was introduced (gambin et al., 2020) in our study, gender moderated the relationship between ph and depressiveness, such that women that scored higher for ph presented with fewer depressive symptoms than women scoring lower on this time perspective. for men, the relationship was inverse-men scoring higher for ph displayed more depressive symptoms than men with lower ph scores. interestingly, this was observed even though men and women did not differ in their levels of ph. although a hedonistic view of the present was found to be related to a high positive affect (desmyter & de raedt, 2012) , other research suggests a significant positive association between ph and depression and anxiety (davies & filippopoulos, 2015) . based on these inconsistencies we can assume that in stress, ph might lead to the development of both adaptive or maladaptive forms of coping, especially emotion-focused forms (blomgren, svahn, åström, & rönnlund, 2016) . our results suggest that the way in which men and women actualize this time perspective may be different. as a consequence, ph-oriented women were able to succeed in the lock-down circumstances, while ph-oriented men were not (blomgren et al., 2016) . vandello and cohen (2008) conducted five studies to show that masculinity as opposed to femininity is a much more uncertain and vulnerable state, dependent on constant external stimulation and social acknowledgement in interactions with others. it m. bodecka, et al. personality and individual differences 168 (2021) 110395 is possible that ph-oriented men are likely to meet their hedonistic needs in contact with other people outside of their homes, and lockdown might have been a circumstance that restricted opportunities to maintain such contacts. ph was also found to be negatively related to stress levels only in women. in men, the relationship between these variables was not significant. one of the crucial stressors during lock-down might have been a fear of viral infection. women, although generally found to be more concerned about their health than men (thompson et al., 2016) , when high on ph, might have been concentrated on the present and oriented at pleasure so that they found pathways for reducing their stress levels. it should be noted that negative life events, such as an epidemic, may not always result in a decrease in well-being or deterioration in mental health but can lead to effective coping with the adversities and to sustained health (luhmann & eid, 2009 ). despite inconsistent findings (see eisenbarth, 2019) , some data has shown that men use avoidance (e.g., sigmon, stanton, & snyder, 1995) , and drugs or alcohol to cope (e.g., kieffer et al., 2006) more often than women. women are more likely than men to seek emotional support across a range of stressors (tamres, janicki, & helgeson, 2002) . it is possible that men high on ph are particularly willing to distract themselves from thinking about the danger of viral infection, in contrast to women, who might seek more social contact and support from close-others. however, these are just speculations and further studies are needed to investigate coping strategies during the recent pandemic in both men and women with high ph. these findings have potentially significant implications for practicing clinicians. given that time perspectives are based on learning processes, clinicians can utilize them to enhance the development of adaptive time perspectives and balance them-in order to enhance well-being and reduce lock-down related distress. several limitations necessitate a degree of care when interpreting these findings. the sample consisted mainly of caucasian participants from a developed country. it is possible that in more embedded cultures, where people live with several generations of relatives in the same household, our result would not be valid. it should also be noted that the forms that displayed depressive symptoms take on can differ between genders (martin et al., 2013) . including a wider variety of measures of distress might result in a more accurate estimation of depression prevalence, especially in men. the study was cross-sectional, which makes it impossible to form causality statements about the linkages between variables. furthermore, it is advisable to continue searching for other indicators of depression and stress during lock-down, such as feelings of loneliness or perceived social support. the involvement of mb and az in preparation of the manuscript was supported by national science centre of poland, grant no. umo-2017/ 26/d/hs6/00258 awarded to az. 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putting time in perspective: a valid reliable individual differences metric the time para-dox key: cord-263518-6puccigu authors: maarefvand, masoomeh; hosseinzadeh, samaneh; farmani, ozra; safarabadi farahani, atefeh; khubchandani, jagdish title: coronavirus outbreak and stress in iranians date: 2020-06-20 journal: int j environ res public health doi: 10.3390/ijerph17124441 sha: doc_id: 263518 cord_uid: 6puccigu iran has faced one of the worst covid-19 outbreaks in the world, and no studies to date have examined covid-19-related stress in the general iranian population. in this first population-based study, a web-based survey was conducted during the peak of the outbreak to assess stress and its correlates in the iranian population. a 54-item, valid, and reliable questionnaire, including items on demographic characteristics and past medical history, stress levels, awareness about signs and symptoms of covid-19, knowledge about at-risk groups and prevention methods, knowledge about transmission methods, trust in sources of information, and availability of facemasks and sanitizers, was deployed via social and mass media networks. a total of 3787 iranians participated in the study where the majority of the participants were females (67.4%), employed (56.1%), from developed provinces (81.6%), without chronic diseases (66.6%), and with ≥13 years of formal education (87.9%). the mean age of study participants was 34.9 years (range = 12–73), and the average stress score was 3.33 (sd = ±1.02). stress score was significantly higher for females, those who were 30–39 years old, housewives, those with chronic diseases, individuals who were aware that there is no vaccine to prevent covid-19, those who could not get facemasks or sanitizers, and individuals with higher knowledge about at-risk groups (p < 0.05). there was a significant correlation of stress scores with knowledge about prevention methods for covid-19 (r = 0.21, p = 0.01) and trust in sources of information about covid-19 (r = −0.18, p = 0.01). all of the predictors, except knowledge of two important at-risk groups and education, had a significant effect on stress scores based on a multivariate regression model. the covid-19 outbreak could increase stress among all population groups, with certain groups at higher risk. in the high-risk groups and based on experience with previous pandemics, interventions are needed to prevent long-term psychological effects. professional support and family-centered programs should be a part of pandemic mitigation-related policymaking and public health practices. since the emergence of the covid-19 outbreak in china in december 2019, the disease has rapidly spread across the world [1] [2] [3] [4] [5] [6] [7] . by june 2020, covid-19 had affected more than 8 million people who tested positive, and almost half a million people died worldwide [8] . iran reported its first confirmed case of covid-19 infection in february 2020 in qom [9] . soon after, other provinces in iran reported covid-19 cases, and as a result, schools and universities were closed in the affected provinces, and several cultural, sports, and religious gatherings were canceled as well. in early april 2020, there were 8522 covid-19-associated deaths worldwide, with a large proportion of deaths being reported from iran [10] . travel and other types of warnings and advisories have been issued by the iranian government regularly since the first case was found [11] . despite growing attempts to increase public awareness on prevention, people around the world were suffering from widespread fear, stress, and anxiety. this could be more prominent in areas of peak transmission and spread, with people feeling stressed and anxious about the transmission of the disease. for example, a major psychological burden on the public was identified during the peak of the covid-19 outbreak in china [12] [13] [14] . young people, people who spent too much time searching for information or working on the frontlines, healthcare workers with exposure to confirmed or suspected cases, and survivors of covid-19 had the highest levels of anxiety, depression, and mental distress [13] [14] [15] . the outbreak itself and the control measures may lead to widespread fear and panic, especially stigmatization and social exclusion of confirmed patients, survivors, and family members, which may escalate into further negative psychological reactions, including adjustment disorder and depression [16] . during the covid-19 pandemic, chinese individuals, especially those who were quarantined and had limited access to face-to-face communication, experienced serious psychological problems (e.g., anxiety, psychosis, depression). [16] . in the published literature, post-traumatic stress disorder (ptsd) and depressive disorders have been reported as prevalent long-term psychological consequences of epidemics [13] . patients with confirmed or suspected covid-19 may experience fear of the consequences of infection with a potentially fatal new virus, and those in quarantine might experience boredom, loneliness, and anger. for example, in the early phase of the sars outbreak, a range of psychiatric morbidities, including persistent depression, anxiety, panic attacks, psychomotor excitement, psychotic symptoms, delirium, and even suicidality, was reported [15] . however, much of the evidence on covid-19 relating to the stress of the pandemic has emerged from china, and few studies have examined the burden of covid-19-related stress in the general population from other countries. one of the worst covid-19 outbreaks was reported from iran, and no studies have examined the stress in the general iranian population or in the middle east. thus, the purpose of this study is to measure iranians' stress levels and the associated factors during the covid-19 outbreak. a web-based cross-sectional study was conducted in the general iranian population, targeting internet-using volunteers. a multi-item online questionnaire was deployed via the main page of the iranian scientific association of social work to the general public. this valid and reliable questionnaire was developed based on a comprehensive literature review and expert panel guidance to measure perceptions and distress during an influenza pandemic [13, 14, 17, 18] . study participants were recruited using social networks such as telegram, whatsapp, and instagram. this questionnaire was online for 5 days (from 26 february to 1 march 2020), and 3787 iranians took the survey. the questionnaire could be taken online using a secure html interface, where all security conditions for data and personal information were provided to potential study participants. each questionnaire could be completed only once per device. respondents were required to answer every question. they were able to review or change their answers before submitting final responses to the questionnaire. participants were informed about the purpose of the study and emphasized that their participation was voluntary and anonymous. this study was approved by the iranian scientific association of social work (98/p/419) for ethical procedures and scientific protocols. a 54-item questionnaire was used to collect data and information in this study . the questionnaire included items about demographic characteristics and past medical history, stress levels, awareness about signs and symptoms of covid-19, awareness about at-risk groups, knowledge about covid-19 transmission methods, knowledge about effective covid-19 prevention methods, awareness of the lack of a vaccine to prevent covid-19, trust in information sources about covid-19, and availability of facemasks and sanitizers. the questionnaire included six questions about demographic characteristics and past medical history to assess gender, age, province of residence, years of formal education, employment status (full-time, part-time, unemployed, housewife, student, or retired), and chronic diseases (including respiratory problems such as asthma and lung disease, cancer, stroke, diabetes, heart diseases such as heart failure and high blood pressure, kidney diseases such as kidney failure, liver diseases such as hepatitis and cirrhosis, psychiatric illnesses such as depression and anxiety, and alcoholism or drug addiction). stress-related data was collected by asking five questions about the level of feeling calm, tense, upset, relaxed, and worried. studies show that participants who receive short questionnaires are more likely to respond [19] . hence, the expert panel chose a brief set of stress-related questions that have been used in an epidemic situation [17] . for each question, participants could select responses from a set of options (very high, high, moderate, low, and very low, with a score range of 5-1 for each question). the mean of the responses on the five questions measures the stress level of individuals, and a higher score indicates higher stress. internal consistency reliability of the stress scale was assessed by computing cronbach's alpha from the total sample of participants and was found to be high (α = 0.81). additionally, there were five questions about awareness of signs and symptoms of covid-19 (true or false, with a score range of 1-0 for each question), two questions about the awareness of at-risk groups (true or false, with a score range of 1-0 for each question), four questions about the knowledge about covid-19 transmission methods (true/false/not sure with a score range of 2-0 for each question), and one statement about the awareness of lack of covid-19 prevention vaccine: "there is no vaccine for covid-19" (true/false/not sure with a score range of 2-0). a group of questions (n = 19) was included about the knowledge of effective covid-19 prevention methods (true/false/not sure, with a score range of 2-0 for each question). the internal consistency reliability for this scale (n = 19 questions) was assessed by computing cronbach alpha and was found to be reasonable (α = 0.73). ten questions were about the participants' trust in various sources of information about covid-19. sources of information included people they interact with (such as family, friends, and colleagues), health professionals, official websites (such as the ministry of health website), health centers (such as hospitals and public health centers), social networks (such as whatsapp, telegram, and instagram), television, radio, newspapers, online news agencies, and international websites such as who website. participants could indicate their level of trust in each of these sources using a 6-point likert scale (very much, much, moderate, low, very low, and not trustable, with a score range of 5-0 for each question). the mean of the score on the 10 trust-related questions was the average of individual scores of trust in sources of information about covid-19. to assess the internal consistency reliability of this scale on trust in sources of information, we computed a cronbach alpha from the final sample of respondents, and the reliability was found to be high (α = 0.83). the questionnaire also included questions about the availability of facemasks and disinfectant gel and sanitizers to assess the individuals' access to these protective strategies (yes/no, with a score range of 1-0 for each question). data were in excel file and were checked for duplicates and any errors before importing and analyzing using ibm spss 22 (chicago, il, usa). in the primary approach, a descriptive analysis of demographic and background characteristics of study participants was conducted. next, we compared the average stress scores using t-tests or anova based on demographic characteristics, awareness about signs and symptoms of covid-19, awareness about at-risk groups, knowledge about covid-19 transmission methods, knowledge about covid-19 prevention methods, awareness about the unavailability of covid-19 prevention vaccine, trust in sources of information, and the availability of facemasks and sanitizers. the effect of the predictor variables on stress was assessed by univariate and multivariate generalized linear models. first, each predictor variable was entered into the univariate model separately, then the variables that had p-values < 0.2 were entered into the multivariate model simultaneously. statistical significance was assumed at p < 0.05. a total of 3787 iranians with a mean age of 34.9 years (range = 12-73 years) participated in the study. table 1 illustrates the demographic characteristics of the study population and average stress scores differences among groups. the majority of the study population were females (67.4%), employed (56.1%), from developed provinces (81.6%), without chronic diseases, and had more than 13 or more years of formal education (87.9%). the majority of participants reported that during the last week, they could not get facemasks (74%) or sanitizers and disinfectant gel (50.2%). less than half (44.5%) of the participants were aware of at least three important symptoms of covid-19, and most of them (78.5%) knew that elderly people and individuals with background diseases have a higher risk of infection. the majority of participants had very good knowledge about covid-19 transmission (97%) and prevention (97.3%) methods and knew that there is no approved vaccine for covid-19 (83.5%). most of the participants reported that they had high trust in various sources of information about covid-19 (65.9%). the average scores of participants' knowledge about the transmission and prevention methods for covid-19 were 1.72 (±0.28) and 1.72 (±0.27), respectively. the average score on trust in the sources of information about covid-19 was 2.89 (sd = ±0.85). the average stress score for the study population was 3.33 (sd = ±1.02). the relationship between demographic characteristics and stress was measured by t-test and anova. the mean of stress scores was significantly higher for females, people in the age group of 30-39 years, housewives, those with chronic diseases, individuals who were aware that there is no vaccine to prevent covid-19, those who could not get facemasks or sanitizers, and individuals who knew about at-risk groups (p < 0.05) ( table 1) . anova was performed to study the relationship between stress and awareness about symptoms and at-risk groups. the mean of stress scores was statistically significantly different by levels of knowing two important at-risk groups (p < 0.05), but there was no significant difference by knowledge on five important symptoms (p > 0.05). pearson's correlation coefficients showed that participants' knowledge about transmission methods of covid-19 did not correlate with stress scores (r = 0.11, p = 0.08), whereas there was a statistically significant correlation of stress scores with knowledge about prevention methods for covid-19 (r = 0.21, p = 0.01) and trust in sources of information about covid-19 (r = −0.18, p = 0.01). univariate and multivariate generalized linear models were fitted on the stress scores. demographic variables (including gender, age, employment, education, province, awareness of no approved vaccine for covid-19, background disease), knowledge about transmission and prevention methods, awareness about signs and symptoms and at-risk groups, and trust in sources of information about covid-19 were individually entered in the univariate models. subsequently, variables that had p-values <0.2 were simultaneously entered in the multivariate model ( table 2 ). all of the variables, except knowledge on the two important at-risk groups and education, were significantly associated with stress scores in the multivariate model. in this first large national study from iran, high-levels of stress were reported by the general public during the covid-19 outbreak. unfortunately, the long-term effect of such high levels of stress, resulting in serious mental health issues, would be an additional burden on the iranian public and healthcare system as an aftermath of the pandemic. our findings provide further evidence of the mental health crises created by emerging infectious disease pandemics. following the outbreaks of hiv, ebola, and sars in other countries, the prevalence of psychological symptoms was reported, and many individuals had experienced long-lasting psychiatric problems [18, [20] [21] [22] . there is an interaction between external conditions (threat) and internal ones (vulnerability) that influences the level of risk for debilitating stress that could lead to mental health problems [20] [21] [22] [23] . in this study, we identified unique groups with high stress. first, across various studies, women have reported high stress. in this study and given the pandemic, it is highly likely that women face multiple and additional stressors, including work, taking care of others in the household, arranging for materials and supplies for the household, and arranging school work and education for children. their routines have been dramatically changed and profoundly disrupted, causing more stress. second, individuals with chronic diseases have higher stress in general, and this could be accentuated by the lack of sanitizers, protective masks, and the awareness of lack of a vaccine to prevent covid-19 infection. the lack of protective supplies and heightened awareness could have severely impacted even those without chronic diseases or lifestyle problems. third, those in the middle age groups and working-class could have more stress due to multiple social, economic, and personal stressors. it could be possible that they are worried about losing their jobs or their income. those who had part-time jobs were more stressed than those who had full-time jobs. the instability of part-time jobs, low incomes, and lack of savings could affect some groups more than others. additionally, healthcare access, coverage, and the ability to pay are influenced by employment, and this could be a major stressor for middle-aged working-class individuals. fourth, there is an interesting relationship between education and stress levels. individuals with lower stress had the lowest and highest education levels compared to those who had 13-16 years of formal education. one possible reason could be that during the covid-19 outbreak in iran, there was a lot of misinformation circulating on social media networks. those with lesser education were not generally able to read the information in other languages (such as english) and had lesser access to mass media and technology to use social media. however, with increasing education, this stress seemed to have been alleviated, possibly due to the ability to screen for and use authentic information [24] [25] [26] [27] [28] [29] . similar to many epidemics in the past and the response of various governments, it appears that mental health care is not a priority for governments during such national crises, epidemics, and disasters. in addition, factors such as the rural-urban divide, lack of access to technology and authentic information, income inequality, availability of healthcare or other resources, lack of awareness, and literacy could pose additional burdens and cause psychological distress [13] [14] [15] [16] 18, [20] [21] [22] . [18, [20] [21] [22] [23] . during these epidemics, the consequences on the psycho-social well-being of at-risk communities were largely overlooked. for example, in the ebola-affected regions, few measures were taken to address the mental health needs of confirmed patients, their families, medical staff, or the general population, and this resembles the responses to all recent epidemics. the absence of mental health and psycho-social support systems and the lack of well-trained psychiatrists and/or psychologists in these regions increased the risks of psychological distress and progression to psychopathology [21] [22] [23] [24] [25] . continuous surveillance and monitoring of the psychological consequences for outbreaks should become a part of disaster and pandemic preparedness efforts worldwide. moreover, interventions should be geared towards the most affected and vulnerable individuals as a part of a population mental health promotion strategy [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . currently, according to the notification of basic principles for emergency psychological crisis interventions by the national health commission of china, mental health care should be provided for patients with covid-19, close contacts, suspected cases who are isolated at home, patients in fever clinics, families and friends of affected people, health professionals caring for infected patients, and the public [13] [14] [15] [16] . however, xiang and colleagues claim that the mental health needs of patients with confirmed covid-19, patients with suspected infection, quarantined family members, and medical personnel have been poorly handled and believed that the organization and management models for psychological interventions in china must be improved [15] . similar challenges are now being seen across the severely affected countries and will continue to emerge in regions that will be severely affected with covid-19 or any future pandemics and epidemics [23] [24] [25] . social media, fear and misinformation, myths, and rumors have added to the global burden of information overload and psychological distress [26] [27] [28] . pandemic response policies should include mental health promotion practices as a key initiative in dealing with the psycho-social burden of pandemics. additional research is needed in the form of long-term prospective studies to assess the causal mechanisms and impact of stress caused by the covid-19 pandemic. the results of this study are subject to several potential limitations. first, the study results are restricted by all traditional limitations of cross-sectional study designs (e.g., reliance on self-reported behaviors, recall bias in participants, socially desirable responses, and the inability to establish cause-and-effect relationships). second, this study measured the prevalence of variables rather than incident cases, and we were unable to assess the levels of variables before covid-19 outbreak in iran as a control. third, stress patterns in individuals often have a variety of simultaneous influences that need to be accounted for. fourth, a major threat to external validity is that the sample is limited in nature and extent (e.g., dominated by female participants, those who were employed and <49 years old, and from developed provinces). this would mean that the results of the study cannot be generalized to several groups of individuals across the region and other countries. despite these limitations, our study is the first and largest study across the middle east, with robust measures, of covid-19-associated stress and the factors associated with stress during the pandemic. based on our field experiences and a comprehensive review of literature, we recommend the following strategies for mental health promotion and disease mitigation in communities during the covid-19 pandemic or any future epidemics or disasters [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . people may speculate and/or spread rumors, myths, and misinformation about covid-19 or infectious disease agents. this has been a major problem with the current pandemic, given the widespread use of mass and social media methods, increasing stress and spreading panic. it is essential to encourage the public not to spread misinformation and inform them of verified and credible sources of information. governments should engage in effective and efficient risk communication and share information regarding disease prevention methods and strategies. governments and scientific organizations should provide clear information about covid-19 or any infectious disease-related symptoms, signs, transmission methods, and prevention strategies through scientific websites, daily briefings, and public awareness campaigns. this will help citizens deal with pandemics more effectively and also mitigate the chain of transmission and spread of disease. denying epidemics and the associated population health risks will cause a loss of confidence in the government and credibility of scientific organizations, which poses an additional burden on disease management and population health services. lack of effective flow of information and disease-related data and statistics has been a major problem worldwide with the covid-19 pandemic. people may be worried, anxious, and depressed due to the constantly changing alerts, media cycle, and social or mass media coverage regarding the spread of covid-19. therefore, providing psychological support services by volunteer social workers, psychologists, psychiatrists, and counselors could help alleviate stress and persistent excessive arousal. in this regard, long-distance psycho-social support (including telecounseling and online services) can be deployed. because people's routines may change dramatically during pandemics and quarantine periods, they should receive compassionate services and guidance on becoming familiar with alternative programs and lifestyles. opening online platforms with audio-and video-based information and demonstrations and providing counseling services and telephone helplines for those with new-onset symptoms or existing mental illnesses can be used until routine mental healthcare services are available. grief counseling for people who have lost their family members due to covid-19, or those who lose family members due to other epidemics and disasters, is critical. strategies such as stress reduction, conflict resolution, crisis call centers, child protection, and custody conflict mitigation are some areas of emphasis for psycho-social service providers. it is also so important to pay attention to the possibility of deprived families who have infected members due to the stigma caused by the disease, and provide special support for them to ensure their access to essential facilities and services. while our study did not specifically look at frontline workers, and essential service and healthcare professionals, existing evidence indicates the heavy toll that pandemics such as covid-19 can take on the physical and mental health of certain at-risk groups. disrupted sleep-wake cycle, patient and client overflow, long working hours, changing practice protocols, fractured communication, and shortage of materials, equipment, and supplies can cause high stress among these populations in disasters and pandemics. individual-level, intrapersonal, and organizational level interventions to reduce stress and burnout should be implemented (e.g., shift rotations, work hours limitation, employee assistance programs, enforcing protocols, coordinated workflow and information processes, just to name a few). a major stressor among individuals and families during epidemics is financial or economic. governments should support employers and strengthen social protection, especially for vulnerable families (including elder people, disabled or sick people, and female-headed households). family-friendly policies and programs are necessary to support affected people during the covid-19 pandemic (including employment and income protection, flexible working arrangements, paid leave to care for family members and access to health care and medical services, direct benefit transfers, food distribution for needy families, among others). governments across the world have rolled out stimulus plans and advisories for tax and debt moratoriums that should provide temporary assistance to needy families. voluntary activities during this period can significantly contribute to social solidarity and social cohesion. it is recommended that these activities be encouraged. skilled volunteer professionals could be involved in a wide range of activities to reduce the economic, social, and health impacts of the epidemic. nonprofit organizations can play a key role in mobilizing skilled volunteer professionals. the no-harm principle is essential to providing volunteer services during an epidemic. the covid-19 outbreak has severely increased psychological distress among people in iran. it has changed people's routines in several aspects and made it difficult to cope with the new situation. affected people, especially vulnerable groups, need professional support and community-based mental health promotion services to deal with the multiple stressors and burden imposed by the current pandemic. family-centered social and economic policies and programs are necessary to support people during the covid-19 epidemic. employment and income protection, flexible working arrangements, paid leave to care for family members and access to medical services, cash transfers, and food distribution for low-income or no-income families, and providing long-distance psycho-social interventions are some examples of family-friendly policies to support people during this pandemic or any future epidemics of a regional or global nature. outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus emerging in china-key questions for impact assessment a new coronavirus associated with human respiratory disease in china a novel coronavirus outbreak of global health concern first case of 2019 novel coronavirus in the united states epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak the new york times. coronavirus map: tracking the global outbreak estimation of coronavirus disease 2019 (covid-19) burden and potential for international dissemination of infection from iran public education and electronic awareness of the new coronavirus (covid-19): experiences from iran update: public health response to the coronavirus disease 2019 outbreak-united states clinical features of patients infected with 2019 novel coronavirus in wuhan generalized anxiety disorder, depressive symptoms and sleep quality during covid-19 outbreak in china: a web-based cross-sectional survey immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china timely mental health care for the 2019 novel coronavirus outbreak is urgently needed recommended psychological crisis intervention response to the 2019 novel coronavirus pneumonia outbreak in china: a model of west china hospital. precis the design of a survey questionnaire to measure perceptions and behaviour during an influenza pandemic: the flu telephone survey template (flutest) impact of ebola experiences and risk perceptions on mental health in sierra leone effect of questionnaire length, personalisation and reminder type on response rate to a complex postal survey: randomised controlled trial mental health of nurses working at a government-designated hospital during a mers-cov outbreak: a cross-sectional study ebola outbreak and mental health: current status and recommended response associations between mental health and ebola-related health behaviors: a regionally representative cross-sectional survey in post-conflict sierra leone psychiatric aspects of coronavirus (2019-ncov) infection psychiatry of pandemics: a mental health response to infection outbreak multidisciplinary research priorities for the covid-19 pandemic: a call for action for mental health science mohamadi bolbanabad, a. perceived stress due to covid-19 in iran: emphasizing the role of social networks infodemic" and emerging issues through a data lens: the case of china global health crises are also information crises: a call to action new guidelines to help employers support families during covid-19 psychological interventions for people affected by the covid-19 epidemic risk management of covid-19 by universities in china social protection and job responses to covid-19: a real time review of country measures assessment of iranian nurses' knowledge and anxiety toward covid-19 during the current outbreak in iran mental health and psychosocial aspects of coronavirus outbreak in pakistan: psychological intervention for public mental health crisis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we acknowledge all participants involved in this study. ronaghi voluntarily assisted us in developing the online questionnaire and supported the research team in preparing a page on the isasw website. authors would also like to thank lili derakhshan, maryam latifian, anis rookhosh, roghayyeh yazdani, leila fatehi, fatemeh nourshargh, zahra khanlary, mahboubeh tavakoli, samaneh mohebbi, and ayoub mohammadi for their support and efforts for the survey announcement. the authors declare no conflicts of interest regarding this study. key: cord-316222-cm4k04wv authors: cozzolino, mauro; vivo, deborah r.; girelli, laura; limone, pierpaolo; celia, giovanna title: the evaluation of a mind-body intervention (mbt-t) for stress reduction in academic settings: a pilot study date: 2020-07-30 journal: behav sci (basel) doi: 10.3390/bs10080124 sha: doc_id: 316222 cord_uid: cm4k04wv this study is aimed at evaluating the outcomes of mind-body transformation therapy (mbt-t), previously known as the creative psychosocial genomic healing experience© (cpghe). the intervention was aimed at reducing the perceived level of stress in two non-clinical groups of students with different educational levels and different expertise in the domain of well-being. whereas participants from the first group were first-year university students, participants from the second group were students attending a post-graduate program in psychotherapy. all participants (n = 159) were exposed to a single session of mbt-t, each group in a separate session. the results of two paired-samples t-tests, conducted separately on the two samples, showed that there was a statistically significant reduction in the participants’ perceived level of stress between preand post-intervention states in both samples (t(88) = 5.39, p < 0.001; t(53) = 4.56, p < 0.001 respectively). the results, therefore, showed that a single session of mbt-t was beneficial in reducing the perceived level of stress in both first-year university students and students attending a post-graduate program in psychotherapy, regardless of educational level and expertise in the domain of well-being. rates of distress are increasing in schools and universities [1] . current research [2, 3] indicates that the recent covid-19 pandemic, in particular, has significantly worsened mental health issues in students, and there is growing concern regarding the long-term psychological consequences of this outbreak in higher education settings [3, 4] . under normal conditions, undergraduate students witness several important changes related to personal life and education. university students might feel homesick and overwhelmed by academic demands, which might become a major cause of stress for undergraduate students [5, 6] . during this transition, students might also experience minor psychological problems related to sleep, eating habits, and concentration [5] . likewise, more severe psychological problems, such as mental disorders, also share onset within this age range [4] , which might explain the high rates of depression, anxiety, and suicidality [2] [3] [4] . though a few years older, graduate students, too, are often exposed to significant stress because of the pressure and the challenges posed by post-graduate programs. moreover, graduate students are more likely to be married and have children and, thus, they might be more exposed to personal and family problems that cause stress in their everyday life. greater stress may lead to increased anxiety, depression, and suicidality [1] . among graduate students, we believe that post-graduate medical students and students attending a post-graduate program in psychotherapy can be regarded as a special class because stress-related issues are particularly relevant to their work. recent studies on post-graduate medical students [2, 3] demonstrate that they are under severe stress and that a high level of stress leads to academic underperformance, absenteeism, and a poor quality of life. moreover, graduate students attending a post-graduate program in psychotherapy, who generally hold a master's degree in psychology, are expected to be able to command state-of-the art information and other key skills in their field of study, including stress management and well-being. yet, further pressures arising from this expectation might lead to increased distress and feelings of failure in these students [7, 8] . despite experiencing significant stress, most students do not reach out for professional help [2] . research indicates that the most important barriers to seeking mental health therapy among young adults include perceived stigma, self-denial of a mental health problem, negative attitudes about treatment, and practical barriers, such as not knowing where to seek help, cost issues, etc. [4, 5] . these findings prompt universities and institutions providing undergraduate and post-graduate education and training to ensure that stress management programs are always available and, importantly, that these services are structured in such a way as to engage young people. mind-body interventions (mbis) are generally well suited to this purpose because they are often practiced by non-clinical individuals as well, and are often endorsed by celebrities, which steers clear of the stigma usually attached to mental health treatments. mind-body interventions are designed to enhance the mind's positive impact on the body [9, 10] . they are based on various practices that range from ancient techniques for self-care and well-being (e.g., meditation, yoga, tai chi, etc.) to more modern western practices (e.g., mindfulness, hypnotherapy, psychological therapies, etc.) [11] . several studies support the effectiveness of a number of mind-body interventions in reducing stress in university students [12] [13] [14] [15] [16] . moreover, research indicates that the cause of many disease conditions, including inflammatory and neurodegenerative diseases, is a complex interaction between stressful life experiences, the genome, the mind, and behavioral factors [9, [17] [18] [19] . a recent line of research [10, 17, [20] [21] [22] [23] [24] has provided new insights into the pathophysiology of stress-related disorders and it has identified the gene sets involved in a number of biological pathways, including stress response, inflammation, and physical health. these studies describe the genomic and epigenetic pathways of stress, focusing on gene expression changes brought about by mind-body therapies. despite a number of studies supporting the effectiveness of these interventions in lowering the level of stress, [13, [25] [26] [27] [28] , improving well-being [29] [30] [31] [32] , and academic attainment in student populations [33] , they may also present adverse effects and contraindications [34] [35] [36] [37] [38] , especially for those individuals who are advised against mild to moderate physical exertion. in addition, mind-body therapies often require training that may be challenging to learn, and they are generally time-consuming to perform. in order to avoid some of the issues posed by these interventions, we carried out a study on stress reduction using a novel mind-body technique known as mind-body transformation therapy (mbt-t). mbt-t-previously known as the creative psychosocial genomic healing experience© (cpghe) [39] [40] [41] -is a therapeutic protocol that has been shown to improve therapeutic results without the need for long traditional therapies. from both a theoretical and an empirical perspective, it is derived from the studies of m. h. erickson and e. rossi's mind-body therapy [10, 42, 43] . the protocol is based on the so-called four-stage creative process [10] , which is a very easy to learn procedure, allowing individuals to obtain stress reduction without the need for traditional, complex, and intricate methods. although there is extensive literature evaluating stress reduction among university students [44] , relatively few studies have evaluated stress reduction techniques in graduate students [25] and we are not aware of many studies on graduate students attending a post-graduate program in psychotherapy. because we believe that graduate students likely face the same (if not a greater) amount of stress as undergraduates, even if their expertise in stress management is different, we chose to include both groups in our study. our goal was to evaluate whether the positive effects of the mbt-t intervention on stress in undergraduates would be noted in graduate students as well. such findings would suggest that mbt-t is a suitable stress reduction intervention in more than one academic setting, with implications for decision-makers regarding the psychological support programs made available for students in such settings. we used an uncontrolled quasi-experimental design to evaluate the effects of a single mbt-t session on two groups of students sampled by cluster. the first group (group 1) included n = 58 first-year university students, 50% males, mean age = 24.45 (±8.70) years old. the second group (group 2) consisted of n = 101 graduate students attending a post-graduate program in psychotherapy, 22.8% males; mean age = 35.56 (±10.14) years old. women were overrepresented and the age range was broad, reflecting the typical demographics of the university and post-graduate courses our participants attended. overall, 159 students were selected for the study. participants were all exposed to a single session of mbt-t, each group in a separate session. stress was measured using the distress thermometer (dt) [45] [46] [47] [48] [49] [50] [51] [52] [53] . the dt is a single-item screening tool that is well validated to be sensitive and specific to the construct of stress [45] . in order to assess change in self-perceived stress, at the beginning of the session, the researcher asked participants to indicate their perceived "initial stress" on an 11-point scale ranging from 0 (no stress) to 10 (maximum stress) [54] . once the session was terminated, participants were asked again to indicate their "final stress" on the same scale. moreover, participants' feedback about the benefits of cpghe was also collected. as for the measures, although longer measures for the screening of stress might have been used, we opted for a single-item scale for its brevity and ease of administration. however, we based our choice on studies that validated the dt against other robust measures [45] , and confirmed that the single-item dt can be compared with other measures [46] . besides, a number of validation studies and reviews [45, [47] [48] [49] [50] show that the dt has good psychometric properties across countries and cultures. its sensitivity and specificity, as well as its positive and negative predictive value, are in the range of good overall accuracy (donovan et al., 2014; snowden et al., 2014) . moreover, the median scores for these properties are consistent with existing studies written in the english language, whereas different language versions of the dt may have different cut-off scores for clinically significant problems, which is likely due to cultural differences [46, 48] . the cut-off score has also been found to change depending on subject characteristics and setting [46, 49] . nonetheless, a cut-off score of four is widely agreed to indicate clinically significant distress [45, 46, 48] . thanks to its good psychometric properties, as well as its brevity, the dt was an ideal screening tool to include in a study on stress management with university students. the stress reduction method we used in this study was mbt-t, previously known as the creative psychosocial genomic healing experience© (cpghe). the cpghe is based on the four-stage creative process [10, 17] , which facilitates positive psychosocial transformations. it presents certain advantages over traditional mind-body methods (i.e., it is very easy to learn, it can be performed in minutes, it can be administered to both single individuals and large groups, it does not require specific premises or tools, and it only demands one researcher). the cpghe protocol is a four-stage protocol. the first stage is "focusing consciousness", which implies self-awareness of thoughts and feelings. the second stage is "problem review", during which participants assessed the thoughts and feelings from the first stage by focusing on the problem at hand. the third stage is "problem solving", during which participants learned how to cope with the problem arising from the third stage. the final stage is "self-care". within this stage, participants applied what they had learnt from the third stage to their present situation. participants were allowed to share their experiences with each other [39] . the cpghe protocol starts a therapeutic dialogue that may generate new consciousness for a positive exploration of all emotions related to experience, thus reducing acute and/or chronic stress [10] (see appendix a). in order to evaluate changes in the level of participants' stress over time 0 (pre-intervention) and time 1 (post intervention), a paired-samples t-test was conducted separately for each sample (undergraduates and post-graduates). the analyses were conducted with ibm spss statistics for windows, version 23 (ibm corp., armonk, new york, ny, usa). all participants gave their informed consent for inclusion before they participated in the study. tests were anonymous to ensure the confidentiality and reliability of the data. all procedures in this study were performed in accordance with the ethical standards of the italian association of psychology (aip) research committee and with the 1964 helsinki declaration and its later amendments. no further approval was required. we computed two paired-samples t-tests to make a pre-test-post-test comparison, one for each sample. the results of the paired-samples t-test conducted in undergraduate students showed that the level of post-treatment stress was statistically significantly different and lower than the level of pre-treatment stress (t (53) = 4.56, p < 0.001), as displayed in figure 1 . furthermore, the results of the paired-samples t-test conducted in graduate students showed that the level of post-treatment stress was statistically significantly different and lower than the level of pre-treatment stress (t (88) = 5.39, p < 0.001), as is also displayed in figure 1 . in order to test for regression toward the mean, we computed a bivariate correlation between stress scores at t0 and the change scores (calculated by subtracting the time 0 stress scores from the time 1 stress scores) separately for each group of participants. the results of these analyses showed that the correlation was statistically significantly different and negative for both groups (r = −0.56, p < 0.001; r = −0.60, p < 0.001 for undergraduate and graduate students, respectively). behav. sci. 2020, 10, x for peer review 4 of 11 (undergraduates and post-graduates). the analyses were conducted with ibm spss statistics for windows, version 23 (ibm corp., armonk, new york, ny, usa). all participants gave their informed consent for inclusion before they participated in the study. tests were anonymous to ensure the confidentiality and reliability of the data. all procedures in this study were performed in accordance with the ethical standards of the italian association of psychology (aip) research committee and with the 1964 helsinki declaration and its later amendments. no further approval was required. we computed two paired-samples t-tests to make a pre-test-post-test comparison, one for each sample. the results of the paired-samples t-test conducted in undergraduate students showed that the level of post-treatment stress was statistically significantly different and lower than the level of pre-treatment stress (t (53) = 4.56, p < 0.001), as displayed in figure 1 . furthermore, the results of the paired-samples t-test conducted in graduate students showed that the level of post-treatment stress was statistically significantly different and lower than the level of pre-treatment stress (t (88) = 5.39, p < 0.001), as is also displayed in figure 1 . in order to test for regression toward the mean, we computed a bivariate correlation between stress scores at t0 and the change scores (calculated by subtracting the time 0 stress scores from the time 1 stress scores) separately for each group of participants. the results of these analyses showed that the correlation was statistically significantly different and negative for both groups (r = −0.56, p < 0.001; r = −0.60, p < 0.001 for undergraduate and graduate students, respectively). the present study described the implementation of mbt-t on stress management to reduce the perceived level of stress in a non-clinical group of university students. we compared the levels of stress before and after a single intervention of mbt-t in a population of both undergraduate students and graduate students attending a post-graduate program in psychotherapy. the present study described the implementation of mbt-t on stress management to reduce the perceived level of stress in a non-clinical group of university students. we compared the levels of stress before and after a single intervention of mbt-t in a population of both undergraduate students and graduate students attending a post-graduate program in psychotherapy. the results suggested that a single session of mbt-t could reduce the perceived level of stress among our participants. in particular, our results indicated that cpghe/mbt-t showed a significant reduction in final stress as measured at the end of the interventions regardless of educational level and previous expertise in the domain of well-being. our preliminary investigation seems to confirm that mbt-t might be a suitable stress reduction intervention for university students, as well as graduate students attending a post-graduate program in psychotherapy. in being an easy to implement, sustainable, and reproducible intervention, mbt-t might be a suitable approach that students may be willing to use as compared to other types of intervention. therefore, we believe that if mbt-t were incorporated into undergraduate and post-graduate stress reduction programs, students would probably benefit from it and improve their well-being. the mbt-t presents certain advantages over traditional mind-body methods (i.e., it is very easy to learn, it can be performed in minutes, it can be administered to both individuals and large groups, it does not require specific premises or tools, and it only demands one researcher). despite these encouraging findings, we are aware that the uncontrolled quasi-experimental design was a major limitation to our study. furthermore, the absence of a control group did not allow us to draw definitive conclusions regarding the effects of the intervention and caution must be made when interpreting the presented results. the results of the correlations between stress scores at t0 and the change scores (calculated by subtracting the time 0 stress scores from the time 1 stress scores), showed no regression to the mean. however, future research should address this potential critical threat to internal validity by using a clinical trial with a pre-test-post-test assessment in at least two conditions: experimental and control. other limitations to our study could have included circadian rhythms, time effects, history effects, local history effects, and test-retest sensitization. yet, with regard to time effects, the study included a single intervention, which was carried out within the same time slot (from 10 a.m. to 12 p.m.) in both groups. therefore, neither circadian rhythm nor ultradian cycle effects were relevant. furthermore, the intervention was carried out before the covid-19 pandemic, which made history effects and local history effects relatively non-salient. moreover, we acknowledge that participants may have become sensitized to the measure. yet, the mechanism of the so-called pre-test effect or pre-test sensitization is typically not investigated or even known [51] , therefore, it was difficult for us to reduce this effect in our uncontrolled quasi-experimental study. finally, we did not include follow-ups because, at the time of this study, our interest was in investigating the technique's effect after just a single intervention. we acknowledge that the positive results presented may require discounting due to the absence of a control group. besides, this was a pilot study for a preliminary investigation, and we are planning to carry out a randomized controlled trial to evaluate the technique with more statistical power. despite these issues, the present study has a major strength, which is the replication. although reproducibility is the foundation of science [54, 55] , direct replication is seldom pursued [54, 56] . furthermore, as one study shows, replication rates are much higher when the original researchers are involved [57] . our study included replication because it was conducted with two different groups; importantly, it involved the same researchers, so the methods and procedures were carefully mirrored. the results we achieved held true in both settings and populations. hence, the replication gave greater validity to our findings. in addition, our study could be a springboard for further studies on the effects of mbt-t/cpghe. this method could be integrated into more structured programs including more sessions (four to eight) over a longer period of time, which would help us understand how the benefits obtained with a single mbt-t intervention can be maintained and/or enhanced over time. because stress among university students is a widespread and growing problem [44, 52, 53] , we believe that universities should develop more effective and sustainable stress management programs for students that are based on mind-body therapies. in particular, future research should examine innovative methods like mbt-t. in our view, future studies should investigate the effects of this type of intervention to identify and examine methods to maintain the benefits of stress reduction interventions, as well as their effects on different individuals. for this reason, we hope that researchers will further investigate this field of study, taking into consideration the possible use of mbt-t. the therapist begins with the following: "it is wonderful to know how our best thoughts and positive feelings can improve health and well-being. here are a few exercises that will inspire you to explore some interesting questions that can help you solve your problems creatively in your own way." (optional facilitation of stage 1: if needed, the therapist may add a few empathetic statements to clarify whatever concerns and questions some people and the group may have regarding this brief protocol). the therapist now says: "an important aspect of creative problem solving is to realize how you can explore new life possibilities by looking at things from many points of view at the same time. these creativity exercises will ask you to carefully observe yourself and carefully remember what you are experiencing". so, the therapist says: "you can begin by filling out the distress thermometer (pause for a moment). this instrument asks you to record what level of stress you are experiencing right now on a scale of 0 to 10, where 0 is no stress or discomfort and 10 would be the worst stress or discomfort you have ever experienced in your whole life. go ahead and circle or write in your initial stress level right now on the dotted line." (pause for a moment). the therapist models the first stage of the creative process, with the palms of the hands about 20 cm apart facing each other at about chest level, and says: "you can begin by looking at your hands like this". the therapist asks, "which hand feels a bit warmer or cooler?" subjects may sometimes seem puzzled about what this question means. the therapist simply continues, saying "most people don't realize how their hands or other parts of their body usually feel slightly warmer or cooler when they really pay attention to it. this is a good exercise to help you become more aware of yourself. it helps to focus your attention and positive feeling about your natural abilities." after a minute, the therapist adds support and states emphatically, "notice and remember how warm or cool your hands seem to be." (allow another minute for the subject's inner focus). the therapist now asks, "now notice which hand feels stronger or weaker." after 1 min, the therapist adds support by stating emphatically, "notice and remember how strong or weak your hand seems to be!" (allow another minute for the subject's inner focus). the therapist asks, "now notice which hand feels lighter or heavier." after 1 min, the therapist adds support by stating emphatically, "notice and remember how light or heavy your hand seems to be!" (allow another minute for the subject's inner focus). the therapist asks, "now let's explore your imagination. which hand seems to be you today-and which hand feels more like you as a child?" after 1 min, the therapist adds support by stating emphatically, "notice and remember which hand seems to be you at your present age and which hand seems to be more like you as a child!" (allow another minute for the subject's inner focus). the therapist now asks, "which hand represents some problem you would like to solve right now-today in this exercise?" (therapist pauses for 1 min). "and which hand seems to be the opposite, perhaps holds an answer to your problem?" after 1 min, the therapist adds support by stating emphatically, "remember which hand represents your problem and which hand seems to hold the opposite-perhaps an answer, even if you do not know what it is yet!" (allow another minute for the subject's inner focus). the therapist states emphatically, "now let the hand that represents your problem begin to drift down very slowly as you privately review the history, memories, and feelings of your problem from the beginning to the present moment." the therapist offers motivational support with these remarks, administered 1 min apart: • "that's right! do you have the courage to allow that hand and arm to drift down a bit . . . with each memory you find yourself reviewing?" • "allow yourself feel only as much of that as you need to and then move on to the next memory that comes up more or less by itself." • "that's right! let yourself have the courage to continue only as long as you need to to feel everything as fully as you need to privately." • "that's right! while another part of you observes wisely, you learn how to take care of yourself to imagine and create the best possible outcome for yourself." when the problem hand finally touches down in the person's lap, the therapist adds support and offers empathetically, "that's right! allow your problem hand drift down to your lap and come to a comfortable rest, wonderful, appreciate your job well done! remember as much of this stage 2 of your creative process as you need to build a better future! and now, get ready to move on to the solution of your problem with your other hand. let your other hand, holding the solutions to your problem, remain up for a moment so you can now turn your full attention to it!" stage 3: illumination and insight. problem solving the therapist continues saying: "now allow your other hand to drift down slowly as you explore new possibilities about how to solve your problem today. allow that hand to begin drifting down slowly as you begin to explore something new. explore your best hopes and imagination for today and the future, that could be some interesting and wonderful possibilities for problem solving, healing and well-being. speculate about exciting and fascinating turning points in your life. create the best of all possible world for yourself. enjoy your best dreams about yourself!" the therapist observes the shifts from negativity, stress, sadness, and conflict (of stage 2) to the more searching expressions of positive expectation in stage 3 of the creative process that are often punctuated with a slight smile and even a short laugh. he supports these positive shifts with a few warm following remarks, such as these, administered at 1-min intervals: • "something pleasantly surprising you can look forward to? what do you really need that is most interesting and important to you?" • "simply receive and continue to explore the sources of your strength for dealing successfully with that issue." • "yes, appreciate the value of that as fully as you need to while taking good care of yourself as that hand finally comes to rest in your lap." • when the hand finally touches down in the subject's lap, the therapist states in a supportive manner: "remember how real and strong these new positive possibilities and feelings for changing your life for the better are!" • "wonderful, really appreciate yourself for a job well done! and now, get ready to move on to the resolution of this issue (concern, problem, or symptom)!" stage 4: reality testing and self-care the therapist concludes this fourth part with the following remarks, administered at 30-s intervals: • "when (brief pause for emphasis) a part of you knows it can continue this creative work entirely on your own at appropriate times throughout the day." (30-s pause)". • "and when (brief pause for emphasis) your conscious mind knows it can simply cooperate in helping you recognize when is the right time to tune in and continue this creative work privately on your own." (30-s pause). • "learn how you can explore and practice your new ideas in the real world and give yourself positive prescriptions for taking good care of yourself." (30-s pause). "you will bring this creative exercise to an end for now so you can stretch and become fully alert." the therapist can provide support by stating: "some of you may wish to share a few of your insights with the group," or, "all this creative work can remain private within you." optional facilitation of stage 5: if needed, the therapist may encourage the group to share with supportive remarks such as these: • "is there something interesting some of you would like to share about your creative inner work?" • "what is surprising and unexpected about this that is new to you?" • "what interesting possibilities are opening up for you to now?" the therapist asks: "please complete the distress thermometer again by filling in what your stress level is now at the end of your creative exercise." assessment of perceived stress in postgraduate medical students during training programme perceived stress and barriers to seeking help from mental health professionals among trainee doctors at a tertiary care centre in north india prevalence of stress and its determinants among residents enrolled in china standardized training program for resident doctor (c-strd) program: a cross-sectional study young adults face major barriers to seeking help from mental health services the role of personality traits and barriers to mental health treatment seeking among college students coping with the first exams: motivation, autonomy support and perceived control predict the performance of first-year university students the cardiac rehabilitation psychodynamic group intervention (cr-pgi): an explorative study beyond verbal behavior: an empirical analysis of speech rates in psychotherapy sessions the bioinformatics of integrative medical insights: proposals for an international psycho-social and cultural bioinformatics project a brief protocol for the creative psychosocial genomic healing experience: the 4-stage creative process in therapeutic hypnosis and brief psychotherapy a mind-body intervention for stress reduction as an adjunct to an information session on stress management in university students mindfulness and emotion regulation: promoting well-being during the transition to college efecto de intervenciones mente/cuerpo sobre los niveles de ansiedad, estrés y depresión en futuros docentes de edu walking versus biofeedback: a comparison of acute interventions for stressed students a pilot study: the effect of bio-energy on stress for university faculty, staff, and students during finals week effect of a single session of a yogic meditation technique on cognitive performance in medical students: a randomized crossover trial the mind-body healing experience (mhe) is associated with gene expression in human leukocytes epigenetics, hippocampal neurogenesis, and neuropsychiatric disorders: unraveling the genome to understand the mind ultradian rhythms from molecules to mind: a new vision of life what is the molecular signature of mind-body interventions? 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this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank the university of foggia, department of humanistic study and key: cord-286416-8eu6wp9b authors: valiente-echeverría, fernando; melnychuk, luca; mouland, andrew j. title: viral modulation of stress granules date: 2012-06-14 journal: virus res doi: 10.1016/j.virusres.2012.06.004 sha: doc_id: 286416 cord_uid: 8eu6wp9b following viral infection, the host responds by mounting a robust anti-viral response with the aim of creating an unfavorable environment for viral replication. as a countermeasure, viruses have elaborated mechanisms to subvert the host response in order to maintain viral protein synthesis and production. in the last decade, several reports have shown that viruses modulate the assembly of stress granules (sgs), which are translationally silent ribonucleoproteins (rnps) and sites of rna triage. this review discusses recent advances in our understanding of the interactions between viruses and the host response and how virus-induced modulations in sg abundance play fundamental roles in dictating the success of viral replication. exposure of cells to environmental stress (e.g., heat shock, uv irradiation, hypoxia, endoplasmic reticulum (er) stress and viral infection) trigger a rapid translational arrest generating polysome disassembly . this event triggers a molecular triage, where the affected cell must make a decision on the fate of mrna that is released from polysomes: decay or silencing (anderson and kedersha, 2008) . for these events, cells have elaborated different classes of rna granules named processing p-bodies (pbs) or stress granules (sgs) that contribute to the regulation and lifecycle of mrnas. both pbs and sgs contain share proteins and are assembled in cells subjected to stress, but differ in: (i) only pbs are observed in unstressed cells, (ii) sg assembly typically requires phosphorylation of translation initiation factor eif2␣, but not pb assembly (fig. 1) , and (iii) pbs contain proteins involved in mrna decay, whereas sgs contain proteins of translation initiation complex (eulalio et al., 2007) . pbs are cytoplasmic structures that, unlike sgs, are responsible for mrna decay, rna-mediated gene silencing (microrna and sirna-based gene silencing) and mrna surveillance (or rna quality control) (beckham and parker, 2008) . pbs were discovered by bashkirov et al. (1997) and they showed that xrn1, a 5 -3 exoribonuclease, was localized in small granular structures within the cytoplasm. other proteins related to mrna degradation were also found to localize to this granules, such as a deadenylase (ccr4), decapping enzymes dcp1 and dcp2 as well as the activators of decapping dhh1/p54/rck/ddx6, pat1, scd6/rap55, edc3, hedls and lsm1-7 complex (eulalio et al., 2007; ingelfinger et al., 2002; van dijk et al., 2002) . moreover, pbs can contain mrnas and proteins involved in nonsense-mediated decay (nmd) (e.g., smg5, smg7, and upf1) (fukuhara et al., 2005; unterholzner and izaurralde, 2004) and components of the rna-induced silencing complexes (risc) (e.g., argonaute, microrna and gw182) (liu et al., 2005; rehwinkel et al., 2005) (fig. 2) . on the other hand, sgs were first observed in the cytoplasm of plant cells exposed to heat shock (nover et al., 1983) . sgs are translationally silent ribonucleoproteins and serve as storage sites of mrnas and proteins (fig. 2) , while other functions also have been discussed . sgs typically contain poly(a) + mrna, 40s ribosomal subunits, eif4e, eif4g, eif4a, eif4b, poly(a)-binding protein (pabp1), eif3, eif2, p54/rck/ddx6, and many other rna-binding proteins that regulate mrna structure and function, including human antigen r (hur), staufen 1, polysomal ribonuclease 1 (pmr-1), smaug, tristetraprolin (ttp), t-cell restricted intracellular antigen 1 (tia-1) and tia-1-related protein (tiar), fragile x mental retardation protein (fxmr/fxr1), ras-gap sh3-binding protein (g3bp-1), cytoplasmic polyadenylation binding protein (cpeb) and survival of motor neurons (smn) protein, although the composition can vary (listed in table 1) . during a stress response, cells induce a shut-off of cellular protein synthesis and subsequently promote sg assembly (anderson and kedersha, 2009) . different pathways in sg assembly have control of translation by eukaryotic initiation factor 2 (eif2). eif2 bound to gdp (eif2-gdp) is recycled to the active eif2-gtp by a reaction catalyzed by eif2b. once recycled, eif2-gtp forms a ternary complex with initiator-methionine trna (met-trnai) and 40s ribosome resulting in 43s pre-initiation complex. four kinases activated by hemin deficiency/oxidative stress (hri), viral infection (pkr), endoplasmic reticulum stress/hypoxia (perk/pek) and amino acid starvation/uv irradiation (gcn2); can phosphorylate eif2 subunit ␣, stabilize eif2-gdp-eif2b complex (inactive) and prevents eif2 recycling. these events result in a shut-off of the host protein synthesis and subsequently sg assembly (fig. 2 , i). been described. the most popular pathway is the phosphorylation of the critical translation initiation factor, eif2␣ by a family of four serine/threonine kinases hri, pkr, perk/pek and gcn2. hri (eif2␣k1) is activated in heme deprivation and oxidative stress (han et al., 2001) ; pkr (eif2␣k2) is activated by viral infection (williams, 2001) ; perk/pek (eif2␣k3) is activated in the presence of unfolded proteins in the endoplasmic reticulum (er) and during hypoxia (harding et al., 2000) ; and gcn2 (eif2␣k4) is activated during amino acid starvation and uv irradiation (jiang and wek, 2005) . each kinase causes the phosphorylation of the ␣-subunit of eif2 at ser52, which implies the tight binding with eif2b, inhibiting the exchange of gdp for gtp ( fig. 1) . therefore, there is a decrease in translation tertiary complex assembly (eif2/gtp/met-trna) which suppresses the initiation of translation and promotes sg assembly (fig. 2 , step i) . other mechanisms independent of the phosphorylation of eif2␣ have also been explored. hippuristanol and pateamine a, drugs that inhibit the helicase activity of eif4a, are able to induce the assembly of sgs (fig. 2 , step ii) (dang et al., 2006; mazroui et al., 2006) . as well, the overexpression of sg markers (anderson and kedersha, 2008) , such as tia1 (kedersha et al., 1999) or g3bp-1 (tourriere et al., 2003) , can trigger the assembly of sgs (fig. 2 , step iii). the activation of eif2␣ kinases by viral infection may result in the inhibition of cellular protein synthesis (walsh and mohr, 2011) and/or promotion of autophagy, process involving lysosomaldependent recycling of intracellular components (talloczy et al., 2002) . moreover, some viral proteins can bind eif4a (aoyagi et al., 2010; page and read, 2010) . all of these mechanisms induce sg assembly (i.e., shut-off of cellular protein synthesis), but the viruses have found ways to bypass the hostile environment generated by the cell to ensure their survival. in the last decade, several studies have also demonstrated that the assembly of sgs can be dramatically influenced by viruses: the induction and blockage of sg assembly mediated by viral infections have both been described as means to promote virus replication (beckham and parker, 2008; montero and trujillo-alonso, 2011; white and lloyd, 2012) . in this review we will summarize the current understanding that exists between different virus families and the regulation of stress granules. fig. 2 . sg assembly pathways. polysomes disassembly can lead to the assembly of cytoplasmic granules know as processing p-bodies (pbs) or stress granules (sgs). if deadenylation (e.g., ccr4/not1), destabilization (e.g., ttp/xrn1) and decapping (e.g., dcp1/dcp2) complex; and even risc (ago) complex are recruited to mrna, these will be targeted to pbs. conversely, if tia-1/tiar or proteins such as g3bp/usp10 are recruited to the stalled initiation complexes, these will be directed to sgs. different pathways in sg assembly are described (in red): (i) phosphorylation of eif2␣ induced by the exposure to different stress inducers (e.g., arsenite and thapsigargin) (fig. 1) ; (ii) hippuristanol and pateamine a, drugs that inhibit the helicase activity of eif4a altering atp binding or atpase activity; and (iii) the overexpression of sg markers, such as g3bp or tia-1. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) in 2002, the first evidence was reported showing an interaction between viruses and what we understand to be protein components of sgs. li et al. showed that the negative strand 3 terminal stem-loop structure present in the genome of west-nile virus (wnv) interacts with two sg markers, tia-1 and tiar . in support of the necessity for these virus-host interactions, wnv replication was reduced when tiar −/− cells were infected . wnv is a neurotropic flavivirus responsible for viral meningoencephalitis which has an enzootic cycle between mosquitoes and birds, but can infect amphibians, reptiles, horses and humans (dauphin et al., 2004) . moreover, emara et al. expanded these observations to other members of the same viral flaviviridae family, where tia-1/tiar were shown to co-localize with viral replication complexes (dsrna and ns3) in both wnvand dengue virus-infected cells (emara and brinton, 2007) . sgs can be induced in mammalian cells by several drugs (kedersha and anderson, 2007) , apparently as a consequence of the phosphorylation of eif2␣. in order to determine if viral infection would have any effect on sg assembly, baby hamster kidney (bhk) cells were infected with wild-type wnv and subjected to arsenitemediated oxidative stress. infected cells were found to be resistant to sg induction (emara and brinton, 2007) . however, recent studies showed that chimeric wnv produces high levels of an early viral rna (w956ic), allowing pkr activation and subsequent induction of sg, likely due to translational arrest (courtney et al., 2012) . another flavivirus, hepatitis c virus (hcv), the major etiologic agent of hepatitis c in humans, is able to disrupt pb assembly but at the same time, promote sg assembly during the course of viral infection (ariumi et al., 2011) . however, late in hcv infection corresponding to 48 h post-infection, g3bp-1 and ddx6, both components of sg (table 1) , are found to co-localize with the hcv core, resulting in the suppression of sg assembly. this blockade to sg assembly was found to be due to an interaction between g3bp-1 and the hcv non-structural protein, (ns)5b and the 5 end of the hcv minus-strand rna (yi et al., 2011) . thus, as shown in the examples above, through sequestration of factors essential for the assembly of sgs, several viruses have elaborated mechanisms to impose a blockade to sg assembly. some viruses inhibit cap-dependent translation (hence host cell mrna translation) to ensure the synthesis of their own proteins. pelletier et al. discovered that the translation of the uncapped picornaviral mrna is mediated by an rna structure known as the internal ribosome entry site, ires, at the 5 end of the viral rna (pelletier et al., 1988) . infection by poliovirus (pv), the etiologic agent of paralytic disease known as poliomyelitis, induces the inhibition of cap-dependent translation initiation by the cleavage of the translation initiation factors eif4gi, eif4gii, and pabp mediated by viral proteinases (gradi et al., 1998; kuyumcu-martinez et al., 2002) . sg assembly is induced at a very early time post-pv infection (at approximately 2-4 h), but later, sgs disappear because the same viral 3c proteinase (3cpro) cleaves g3bp-1, but not tia-1 or tiar, and thereby prevents sg assembly (white et al., 2007) . the sgs found in pv-infected cells contain viral rna and tia-1, but are compositionally distinct since they exclude well-described sg components such as g3bp-1, pabp, and eif4g, all of which are eventually cleaved by 3cpro (piotrowska et al., 2010; white and lloyd, 2011) . furthermore, pv infection also disrupts the assembly of pbs. also during pv infection, xrn1, dcp1a and pan3, three factors involved in mrna decapping, degradation and deadenylation, respectively, undergo degradation or cleavage by the viral 3cpro (dougherty et al., 2011) . likewise, cricket paralysis virus (crpv) infection in drosophila cells leads to a rapid shut-off of host protein synthesis concomitant with phosphorylation of eif2␣ (wilson et al., 2000) . because these characteristics are common to the induction of sgs, khong et al. investigated the assembly of sg after crpv infection. through an immunofluorescence assay, the authors showed that rox8 and rin, drosophila sg marker homologs of tia-1 and g3bp-1, respectively, do not aggregate in crpv infected cells, even in the presence of sg inducers such as heat shock, oxidative stress and pateamine a. it was also demonstrated that crpv viral 3c proteinase is sequestered to sgs under cellular stress but not during virus infection (khong and jan, 2011) . another picornavirus, theiler's murine encephalomyelitis virus (tmev) which causes a demyelinating disease similar to multiple sclerosis in the central nervous system, also inhibits sg assembly. borghese et al. showed that tmev infection induces sg assembly, but the expression of the leader (l) protein during infection was sufficient to inhibit sg assembly induced by arsenite-mediated oxidative stress or by thapsigargin-mediated er stress. unlike the effects induced by pv 3c proteinase, g3bp-1 was not cleaved by tmev and was in fact found in sgs post-tmev infection (borghese and michiels, 2011) . for efficient protein synthesis, mrna circularization is required during translation. pabp, that is bound to poly (a) 3 tail, interacts with eif4gi at the 5 , causing circularization of the mrna by linking the 5 and 3 mrna ends, increasing the binding of eif4e to the cap (lopez-lastra et al., 2010) . rotavirus, the causative agent of a common infantile gastroenteritis, subverts the host translation machinery at this step. because rotavirus mrnas are capped but lack poly(a) tails, the virus-encoded protein, non-structural (ns) p3, binds to a consensus rna sequence in the 3 end of viral mrna, enabling mrna circularization by interaction with eif4gi (piron et al., 1998) . as a consequence, a shut-off of host protein synthesis ensues and thereby provides an advantage for viral protein synthesis. in infected cells, montero et al. found that eif2␣ is phosphorylated during the entire virus replication cycle but this does not have an impact in the formation of viroplasms (cytoplasmic viral factories found in rotavirus-infected cells) or viral replication and surprisingly, sg assembly was not induced. one possibility for explain this observation may be due to pabp, a component of sg (table 1) , is able to translocate from the cytoplasm to the nucleus in rotavirus infected cells in a nsp3-dependent manner (montero et al., 2008) . instead, junin virus (junv), that is responsible for argentine hemorrhagic fever, is able to impair the phosphorylation of eif2␣. linero et al. showed that in junv-infected vero cells exposed to arsenite-mediated oxidative stress, eif2␣ phosphorylation was impaired but this did not lead to the induction of sg assembly (linero et al., 2011) . furthermore, the junv nucleoprotein (n) and/or the glycoprotein precursor (gpc) was responsible for this virus-induced blockade to sg assembly. rather, when junv-infected cells were treated with hippuristanol, an eif4a-helicase activity inhibitor that induces sgs in an eif2␣-independent manner (mazroui et al., 2006) , sg assembly was observed in 100% of cells indicating that junv affects an unidentified event downstream of eif2␣ phosphorylation or the integrity of viral mrnas on polysomes (linero et al., 2011) . another virus that efficiently shuts off host protein synthesis is influenza a virus (iav) (kash et al., 2006) . iav is an animal pathogen that causes severe respiratory disease and pandemics in humans around the world. viral transcription involves a cap-snatching mechanism during which a nucleotide sequence between 10 and 20 nt, including the 5 cap structure, is cleaved from the 5 end of cellular mrnas. this sequence is used to prime transcription on the viral genome and is ultimately used during translation initiation of viral mrnas (lopez-lastra et al., 2010) . additionally, iav encodes capbinding proteins that are able to preferentially recognize capped viral mrnas. the influenza non-structural protein 1 (ns1) binds eif4gi and pabp-1, thus stimulating the assembly of the translation initiation complex on capped iav mrnas (lopez-lastra et al., 2010) . iav actively suppresses sg assembly during viral infection, thereby allowing translation of viral mrnas. complete inhibition of sg assembly is dependent on the function of ns1 and its ability to inhibit pkr, the double-stranded rna-activated protein kinase (khaperskyy et al., 2011) . recently, retroviruses such as the human immunodeficiency virus type-1 (hiv-1) and human t-cell lymphotropic virus type-1 (htlv-1) were shown to impose a blockade to sg assembly in infected cells. recent work from the authors' laboratory showed that hiv-1 preferably assembles ribonucleoprotein complexes to which staufen1, the viral genomic rna and the structural protein gag are recruited, called staufen1 hiv-1-dependent rnps (shrnps). these were compositionally different than sgs since they did not contain many of the classical sg marker proteins g3bp-1, eif3, tia-1, tiar, hur, pabp-1, but contained staufen1. the assembly shrnps during the late stages of viral replication is believed to impose a blockade to the assembly of sgs but to favor the encapsidation of hiv-1 genomic rna into assembling virus (abrahamyan et al., 2010; white and lloyd, 2012) . follow-up work, reported at the last international nucleocapsid (nc) meeting in barcelona, spain in september 2011, now demonstrates that the viral gag protein controls the kinetics of sg assembly and interferes with the cellular stress response pathway (valiente-echeverría et al., unpublished) . the oncoretrovirus, htlv-1 elicits a blockade to sg assembly in a different manner and this was found to be mediated by the viral regulatory protein, tax. legros et al. observed that tax relocated from the nucleus to the cytoplasm in response to environmental stress. while tax is present in the cytoplasm, it interacts with histone deacetylase 6 (hdac6), a critical component of sgs (kwon et al., 2007) , and thereby impairs sg assembly (legros et al., 2011) . while the details on the mechanisms by which viruses elicit favorable environments in which to replicate will require further work, the sequestration of critical factors for the induction of sgs by viral proteins appears to be an increasingly studied area of research and should yield important new information on how viruses gain control over host cell biology. while all of the examples described above belong to rna viruses, herpes simplex virus (hsv) and cytomegalovirus (hcmv) are the only members of the dna virus family that have been shown to regulate sg assembly. hsv-1 causes a shut-off of host cell protein synthesis by the virion host shutoff (vhs) protein and subsequently induces degradation of cellular rnas (kwong and frenkel, 1987) . several adenosine-uracil (au)-rich binding proteins that promote mrna stability, such as tia-1/tiar, and ttp (bevilacqua et al., 2003) , were upregulated in hsv-1 infected cells (esclatine et al., 2004) . ttp and tia-1/tiar were activated during the infection and accumulated in the cytoplasm, but only ttp was able to interact with vhs. as a consequence, sgs were not observed after infection (esclatine et al., 2004) . more recently, finnen et al. have shown that hsv-2 infection blocks sg accumulation in cells exposed to arsenite-mediated oxidative stress, but not in cells exposed to pateamine a, a drug that induces sg assembly in an eif2␣-independent manner (finnen et al., 2012) . these results were similar to those found in junv infected cells described above (linero et al., 2011) . on the other hand, hcmv infection induces an unfolded protein response (upr), activates perk, but eif2␣ phosphorylation levels were limited and viral rna translation was maintained (isler et al., 2005b) . likewise, the same group showed that sg assembly was suppressed in hcmv infected cells treated with the er stressor, thapsigargin (isler et al., 2005a) . as discussed in the previous section, viruses have chosen different mechanisms to inhibit the sg assembly to ensure efficient and unmitigated replication. some studies have demonstrated that the sg assembly is not always correlated with a shut-off of host protein synthesis (kimball et al., 2003; loschi et al., 2009) . moreover, other authors have showed that sgs could sequester apoptotic molecules favoring cell survival upon exposure to certain types of stress such as heat shock (kim et al., 2005; tsai and wei, 2010) . thus, a virus-mediated induction of sg assembly also represents a strategy employed by some viruses to ensure replication. respiratory syncytial virus (rsv), which is responsible for lower respiratory tract illnesses in both infants and the elderly, induces sgs during the course of infection (lindquist et al., 2010) . lindquist et al. showed the correlation between higher viral protein levels and the presence of sgs in infected cells. in addition, g3bp −/− cells, that are unable to generate sgs because of a disrupted g3bp gene locus, exhibited diminished rsv replication (lindquist et al., 2010) . however, a later study by the same group concluded that the stress response may not play an important role in viral replication. they did not see a difference in viral replication in cells that were not able to elicit a stress response because pkr was depleted by sirna (lindquist et al., 2011) . this later study also noted that rsv infection does cause eif2␣ phosphorylation and pkr is needed to induce sgs during viral infection. these results indicate that the assembly of sg neither aids nor interferes with the replication of this virus. the role of the stress response involving sgs in the reoviridiae family of viruses has been shown to be implicated in viral replication. mammalian orthoreovirus (mrv) infection in humans is usually asymptomatic or associated with symptoms of a common cold. during the early stages of infection, mrv induces sg assembly and the expression of atf4, a transcription factor, through eif2␣ phosphorylation (smith et al., 2006) . the assembly of sgs creates a competitive advantage for the viral mrna to be translated because cellular mrnas are sequestered in sg. when atf4 is expressed in mrv infected cells, viral production increases by up to 100-fold (smith et al., 2006) . a later study implicated a role for sg assembly in viral replication since sg formation occurs after viral uncoating but before viral mrna transcription (qin et al., 2009) . qin et al. (2011) found that viral mrnas escape translational inhibition when sgs are disrupted and viral translation occurs in the presence of high levels of phosphorylated eif2␣ in a manner that is independent of pkr inhibition. this study also mentions that mrv-infected cos7 cells are able to block the assembly of sgs induced by arsenitemediated oxidative stress later in infection (qin et al., 2011) . the implication of these findings is that the stress response and the resulting assembly of sgs must be involved in the early stages of the viral replication cycle but is ultimately detrimental to the virus if it is not able to disassemble sg during later stages of infection. semliki forest virus (sfv), which causes lethal encephalitis in rodents, seems to modulate the cellular stress response in a similar fashion than mrv. upon infection, sfv is able to induce the phosphorylation of eif2␣ and promote sg assembly in mouse embryo fibroblasts (mef) (mcinerney et al., 2005) . despite a shut-off of host protein synthesis during these events, sfv is still able to translate its mrna due to a translational enhancer element present in the viral genome. this study also indicated that areas around viral rna in the cytoplasm were devoid of sgs. this observation likely indicates that viral proteins or viral rna could locally disassemble sg to favor viral translation and this was shown to correlate with increased vrna levels (mcinerney et al., 2005) . the theme of utilizing the stress response to shut-off of host protein synthesis appears once again in coronaviridae. the mouse hepatitis coronavirus (mhv), which is closely related to the sars coronavirus, has been shown to subvert the host translation machinery through eif2␣ phosphorylation (raaben et al., 2007) . eif2␣ phosphorylation also leads to the assembly of sg and pb. a genome wide microarray analysis of regulated mrnas in mhvinfected lr7 cells revealed the decrease in the expression of many cellular mrnas, which may be due to an increase in pbs activity and function (raaben et al., 2007) . likewise, viral rnas transcripts make up 40% of total rna in the cell, so the virus may be overloading the host cell cytoplasm to ensure that its transcripts will be translated (raaben et al., 2007) . however, the authors come to the conclusion that the inhibition of cellular translation is not beneficial to the virus since in systems lacking the ability to inhibit cellular translation, viral production did not change and thus, the assembly of sg in mhv-infected cells does not appear to dramatically favor viral replication (raaben et al., 2007) . finally, rubella virus (rubv) infection generates aggregates of g3bp-1 in the cytoplasm (matthews and frey, 2012) . these aggregates differ from typical sg because they do not contain proteins such as pabp and tia-1 (table 1) . rubv is a positive strand rna where viral replication is mediated for intermediary double stranded rna (dsrna). matthews et al. found that g3bp-1 does not overlap with dsrna, but rather colocalizes with viral ssrna in perinuclear clusters (matthews and frey, 2012) , suggesting that these may represent sites of encapsidation (beatch and hobman, 2000) . despite an intensifying research focus to understand the relationships between the cytoplasmic rnps called sg and virus replication (refer to table 2), many questions remain to be answered in this growing field of virology. the roles for many sg components (table 1) that have been found to participate in viral replication either by inclusion or exclusion still remain incompletely defined in host cell biology. as well, the literature has only touched the surface as to how viruses hijack and commandeer sg components. in several cases in which sg assembly is shown to be inhibited, it remains unclear if viruses block the assembly or induce the disassembly of sg. there is also a need to determine at what level viruses are hijacking or co-opting the host cell stress responses that exhibit sg. there is also a need to understand how sg may lead to deleterious effects if they remain present during viral infection. indeed, further characterization of a virus' ability to overcome the inhibition of sg assembly or induce their assembly to prevent translation of host mrnas may be beneficial in developing new anti-viral drugs that could be useful against multiple viruses. anti-cancer drugs such as etoposide, bortezomib and doxorubicin, do induce sg assembly, however their roles as anti-virals are not known (arimoto et al., 2008; fournier et al., 2010; morita et al., 2012) . the many mechanisms by which viruses inhibit or induce sg may pose a problem to developing a broad anti-viral drug targeting sg. viruses such as pv, which inhibit sg formation through cleavage, would likely be unaffected by drugs that activate the stress response upstream of these cleaved factors. another caveat to the potential use of these drugs is that sg formation may help the replication of certain viruses which induce sg to create a better environment for viral replication. the knowledge gained on the biology of sg and how it is influenced by viral infections will play a role in further characterizing innate responses to infection and how this system can be taken advantage of to curb viral infections. novel staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv-1 genomic rna stressful initiations rna granules stress granules: the tao of rna triage stress granules human cytomegalovirus ul69 protein facilitates translation by associating with the mrna cap-binding complex and excluding 4ebp1 formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways hepatitis c virus hijacks p-body and stress granule components around lipid droplets mammalian smaug is a translational repressor that forms cytoplasmic foci similar to stress granules a mouse cytoplasmic exoribonuclease (mxrn1p) with preference for g4 tetraplex substrates rubella virus capsid associates with host cell protein p32 and localizes to mitochondria p bodies, stress granules, and viral life cycles post-transcriptional regulation of gene expression by degradation of messenger rnas the leader protein of cardioviruses inhibits stress granule assembly west nile virus infections suppress early viral rna synthesis and avoid inducing the cell stress granule response eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine a west nile: worldwide current situation in animals and humans poliovirus-mediated disruption of cytoplasmic processing bodies codependent functions of rsk2 and the apoptosis-promoting factor tia-1 in stress granule assembly and cell survival interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly herpes simplex virus 1 induces cytoplasmic accumulation of tia-1/tiar and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize au-rich rnas p bodies: at the crossroads of post-transcriptional pathways herpes simplex virus type 2 infection impacts stress granule accumulation the chemotherapeutic agent bortezomib induces the formation of stress granules smg7 is a 14-3-3-like adaptor in the nonsense-mediated mrna decay pathway stress granule assembly is mediated by prion-like aggregation of tia-1 a novel functional human eukaryotic translation initiation factor 4g heme-regulated eif2alpha kinase (hri) is required for translational regulation and survival of erythroid precursors in iron deficiency perk is essential for translational regulation and cell survival during the unfolded protein response rpp20 interacts with smn and is re-distributed into smn granules in response to stress the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum human cytomegalovirus infection activates and regulates the unfolded protein response gcn2 phosphorylation of eif2alpha activates nf-kappab in response to uv irradiation hijacking of the hostcell response and translational control during influenza virus infection mammalian stress granules and processing bodies evidence that ternary complex (eif2-gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules stress granules and processing bodies are dynamically linked sites of mrnp remodeling influenza a virus inhibits cytoplasmic stress granule formation modulation of stress granules and p bodies during dicistrovirus infection sequestration of traf2 into stress granules interrupts tumor necrosis factor signaling under stress conditions mammalian stress granules represent sites of accumulation of stalled translation initiation complexes efficient cleavage of ribosome-associated poly(a)-binding protein by enterovirus 3c protease the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mrnas the htlv-1 tax protein inhibits formation of stress granules by interacting with histone deacetylase 6 cell proteins tia-1 and tiar interact with the 3' stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif2alpha phosphorylation microrna-dependent localization of targeted mrnas to mammalian p-bodies translation initiation of viral mrnas dynein and kinesin regulate stress-granule and p-body dynamics analysis of subcellular g3bp redistribution during rubella virus infection inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation rotavirus infection induces the phosphorylation of eif2alpha but prevents the formation of stress granules stress granules in the viral replication cycle the stress granule protein vgl1 and poly(a)-binding protein pab1 are required for doxorubicin resistance in the fission yeast schizosaccharomyces pombe formation of cytoplasmic heat shock granules in tomato cell cultures and leaves the virion host shutoff endonuclease (ul41) of herpes simplex virus interacts with the cellular cap-binding complex eif4f translational efficiency of poliovirus mrna: mapping inhibitory cis-acting elements within the 5 noncoding region stable formation of compositionally unique stress granules in virus-infected cells rotavirus rna-binding protein nsp3 interacts with eif4gi and evicts the poly(a) binding protein from eif4f mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif2{alpha} phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies a crucial role for gw182 and the dcp1:dcp2 decapping complex in mirna-mediated gene silencing reovirus induces and benefits from an integrated cellular stress response mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay regulation of starvation-and virus-induced autophagy by the eif2alpha kinase signaling pathway rna granules: the good, the bad and the ugly mammalian staufen 1 is recruited to stress granules and impairs their assembly the rasgap-associated endoribonuclease g3bp assembles stress granules rhoa/rock1 signaling regulates stress granule formation and apoptosis smg7 acts as a molecular link between mrna surveillance and mrna decay human dcp2: a catalytically active mrna decapping enzyme located in specific cytoplasmic structures viral subversion of the host protein synthesis machinery inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia1 aggregation and mrna stress granule formation regulation of stress granules in virus systems naturally occurring dicistronic cricket paralysis virus rna is regulated by two internal ribosome entry sites hepatitis c virus co-opts ras-gtpase-activating protein-binding protein 1 for its genome replication we thank marie-joelle miron for critical reading of the manuscript. this work is supported by grants from the cihr (mop-38111) to a.j.m.; f.v.e. is supported by a postdoctoral fellowship from becas chile-conicyt. key: cord-270469-lle32mha authors: martinon, fabio title: the endoplasmic reticulum: a sensor of cellular stress that modulates immune responses date: 2012-07-15 journal: microbes infect doi: 10.1016/j.micinf.2012.07.005 sha: doc_id: 270469 cord_uid: lle32mha many inflammatory and infectious diseases are characterized by the activation of signaling pathways steaming from the endoplasmic reticulum (er). these pathways, primarily associated with loss of er homeostasis, are emerging as key regulators of inflammation and infection. recent advances shed light on the mechanisms linking er-stress and immune responses. loss of cellular integrity is a hallmark of severe pathogenic insults and leads to the activation of immune defenses aimed at repairing tissue damage and fighting infections. it has been postulated that the release of danger signals by damaged cells could lead to the mounting of immune defenses [1e3] . these danger signals also known as damage associated molecular pattern (damps) are molecules that trigger specific receptors including receptors that initiate inflammatory and immune responses. many damps are nuclear proteins such as hmgb1 or cytosolic molecules including, for example, uric acid and atp. when released outside of the cell following tissue injury, damps are sensed by immune cells and trigger innate immune receptors such as the toll-like receptors (tlrs) or the nlrp3 inflammasome [4, 5] . the release of damps by cells is often described as a passive process, occurring as a consequence of sustained cell damage. this process is irreversible and correlates with the death and elimination of the damaged cell. however, before losing membrane integrity, cells can experience various states of malfunction that are reversible. we can define these cellular states as stress. typically, stressed cells are malfunctioning, but are also characterized by the activation of adaptation responses to the stress aimed at repairing cell damage. stress can be a consequence of perturbations in basic cellular functions including the availability of nutrients and oxygen as well as the capacity to communicate with neighboring cells and to respond to changes in the microenvironment. if the cell is able to sense these perturbations it mounts a response (hereby referred to as a stress response) that, by definition, is an active cellular adaptation to the abnormal conditions. if this repair/adaptation fail or if the stress is too severe, the cell will eventually die possibly releasing damps. tissue stress and malfunction promotes low-grade inflammation which helps the tissue to adapt to the damage and restore tissue function [6, 7] . this low-grade inflammation also termed para-inflammation illustrates how stress and possibly stress response pathways can contribute to immune responses (fig. 1 ). however the mechanisms and the role of stress responses in controlling innate immunity and inflammatory pathways are poorly understood. the endoplasmic reticulum (er) is emerging as a key organelle that maintains cellular homeostasis and contributes to the regulation of innate immune response [1, 8, 9] . perturbations that affect the er trigger a specific response known as the er-stress response. in this review we discuss how infectious and inflammatory signals affect the er and focus on how perturbations and signaling pathways emerging from this organelle regulate innate and inflammatory pathways. the er is an essential organelle that controls the biogenesis of cellular components including proteins, lipids and carbohydrates. it is connected with the nuclear membrane and via mitochondria associated membranes (mams) to the power source of the cell: the mitochondria. in eukaryotes, resident proteins of the endocytic and exocytic organelles as well as cell surface and secreted proteins are synthetized on polysomes anchored to er membrane and then, translocated across the membrane into the er lumen. the er lumen is rich in calcium and its oxidizing environment catalyzes posttranslational modifications such as the addition of n-linked glycans and the formation of disulfide bonds [10, 11] . the nascent protein must fold via a complex process that is monitored by resident ereprotein chaperones and enzymes that prevent aggregation and regulate proper folding of newly synthetized proteins. this process being essential for the production of virtually every secreted protein and membrane receptor, the er is therefore an essential hub controlling various aspect of cellular biology including virtually all aspects of cellecell communication and signal transduction pathways steaming from membranes. in immunology the er is best known for its role in antigen presentation, it is the main site for the assembly of mhc class i and mhc class ii molecules and is the location of peptide loading onto mhc class i molecules. conditions of er stress occur when the amount of proteins entering the er exceeds its folding capacity. this imbalance induces a protective signaling cascade collectively termed the unfolded protein response (upr) or the er-stress response [12, 13] . experimentally, many conditions can trigger the erstress response. for example, conditions that inhibit glycosylation including low glucose as well as pharmacological compounds such as tunicamycin, affect the maturation and transport of most secreted proteins causing er-stress. other drugs and conditions that perturb er ca 2þ levels have profound effects on er homeostasis and protein folding. drugs such as thapsigargin can trigger depletion of er calcium stores leading to acute er-stress responses. the er presents an oxidizing environment that can favor protein folding and the formation of disulfide bonds. reducing agents such as dithiothreitol (dtt) affect the oxidizing potential of the er leading to the accumulation of misfolded proteins and the subsequent engagement of the er-stress response. in addition, various physiological and pathological insults that perturb the cell protein homeostasis network, a group of interconnected pathways collectively termed the proteostasis network may trigger the er-stress response [14, 15] . these insults include alterations in cellular ph, inhibition of the proteasome pathways, infections with various pathogens as well as metabolic changes associated with cancer. ersignaling pathways and the er-stress response are among the major and best-characterized components of the proteastasis network pathways. the er-stress response is a highly controlled process that adapt to specific insults to optimize and orchestrate an appropriate response. while this response may differ from one condition to another, a general picture of signaling events emerge. the attenuation of translation is among the first things that are observed upon induction of er-stress. this occurs within minutes to hours of er-stress activation and prevent further translational overload of the er. perturbation of erhomeostasis also results in the up-regulation of genes involved in er biogenesis, in chaperoning and folding of proteins as well as in the quality control mechanisms that target and ensure elimination of malfunctioning proteins including the endoplasmic-reticulum-associated protein degradation (erad) pathway. the aim of these responses is to help the cell cope with the stress, remove the accumulated protein load, increase er capacity and restore normal function of the er. in conditions of acute and prolonged stress, the response changes from promoting survival to inducing cell death. the point at which the 'apoptotic switch' is activated has not yet been determined, and may vary form cell to cell. the mechanisms of er-stress mediated apoptosis are yet poorly understood but may involve the intrinsic apoptotic pathway that lead to release of cytochrome c from the mitochondria and activation of the caspase-9 apoptosome [16e18]. in mammalian cells, the er-stress response is mediated by at least three signaling pathways that operate in parallel (fig. 2) . each pathway steams from a class of transmembrane proteins anchored at the er: ire1 (inositol-requiring enzyme 1) perk (pkr-like er kinase), and atf6 (activating the state of a tissue can range from healthy, to stressed or malfunctioning, to irreversibly damaged. damaged cells release damp, that directly engage innate immune receptors such as tlrs, nlrs or rage receptors. in contrast, tissue stress or malfunction is a reversible state that triggers an adaptation response aimed at restoring tissue homeostasis. stressed tissue may also trigger danger signals or stress signals that could contribute to the upreglation of inflammatory and innate immune responses. transcription factor 6). activation of these three signaling branches control the expression of a few proximal er-stress related transcription factors and signaling events that orchestrate the transcriptional upregulation of the universe of genes involved in restoring er functions [16, 19, 20] . ire1 is the most conserved branch, the only one present in lower eukaryotes and probably the best-studied branch of the er-stress response. in yeast, ire1 triggers the full er-stress response program, whereas in higher eukaryotes the program requires cooperation between the three branches of the er-stress response. ire1 transmembrane region is flanked by a sensor unit at its n-terminus that localize in the er lumen and a c-terminal portion that reside in the cytosol. the cytosolic portion of ire1 contains two functional units: a ser/thr protein kinase and an endoribonuclease. oligomerization of ire1 at the er, triggers its ribonuclease activity and cleaves, at two closely located and distinct sites, the mrna of the x-box binding protein-1 (xbp1) gene. this cleavage results in the excision of a mini intron of 26 nucleotides in mammals. upon ligation, mature xbp1 mrna encodes a functional transcription factor. ire1 mediated removal of the 26 nucleotides mini intron open a new translation reading frame at the c-terminus of the protein coding for a potent transactivation domain. therefore, by a unique mechanism in mammals, unrelated to regular splicing, ire1 converts an inactive xbp1 protein sequence into an active transcription factor. active xbp1 referred hereby as xbp1s, contribute to the regulation of genes involved in different processes including protein folding, mechanisms controlling protein quality as well as a broad array of genes involved in almost every aspect of er function, physiology and biogenesis [21, 22] . early studies on xbp1 highlighted the role of the er-stress response pathways in the development and regulation of secretory cells including plasma b cells. xbp1-deficient b cells are unable to differentiate into antibody-secreting plasma cells [23, 24] and antibody production in vivo in response to antigenic challenge is impaired by xbp1 deficiency. importantly it was shown that activated b cells undergo xbp1 mrna maturation to produce xbp1s in order to promote er expansion and develop the secretory capacity of plasma cells [25, 26] . however, what drives ire1 activation in b cells is still unclear. in b cells, xbp1 activation is not impaired by igm deficiency [27, 28] suggesting that massive immunoglobulin production per se is not the cause of xbp1s activation and that ire1/xbp1 activation may by part of a preemptive differentiation program rather than as a consequence of er overload. in addition to xbp1 activation, ire1 ribonuclease activity has other functions. upon sustained and acute er-stress, ire1 may contribute to the degradation of membrane-associated mrnas through a process known as regulated ire1 dependent decay or ridd [27, 29, 30] . the exact role of ridd is unknown. it is unclear if it solely contribute to decreasing protein load in the er or is part of the induction of the apoptosis phase as suggested by experiments showing increased er-stress induced apoptosis in cells in which ire1 activity was manipulated to promote ridd activation [30, 31] . perk is an er anchored kinase that upon activation oligomerizes and autophosphorylate. it kinase activity phosphorylates the translation initiation factor eif2a, transiently decreasing overall protein translation. this helps reducing folding requirements in the er. by phosphorylating eif2a at serine 51 perk inhibits the guanine nucleotide exchange factor eif2b, a factor involved in the recycling of eif2a to its active gtp-bound form [32e34]. eif2a phosphorylation can be targeted by phosphatases, such as growth arrest and dnadamage inducible protein-34 (gadd34) [35, 36] that contribute to the resolution of the stress responses. some mrna containing short open reading frames in their 5 0 untranslated region are induced when eif2a is inhibited. eif2a phosphorylation can therefore promote the synthesis of a specific subset of genes by increasing the translation of selective mrnas whose translation is inhibited in unstressed cells. for example this mechanisms promote the translation of activating transcription factor 4 (atf4) a major regulator of er-stress. atf4 is involved in various cellular responses including, glutathione biosynthesis, amino acid import, and resistance to oxidative stress [37e39]. perk also phosphorylates nuclear factor (erythroid-derived 2)-like 2 (nrf2), a transcription factor that regulates oxidative stress responses and a critical regulator of innate immune responses [32, 40, 41] . the third branch of the er-stress pathway is mediated by atf6, the best-known member of a transmembrane protein fig. 2 . er-stress signaling pathways. accumulation of unfolded proteins or loss of homeostasis in the er triggers a stress response that activates ersignaling pathways including ire1, atf6, and perk. active ire1 lead to an unconventional splicing of the transcription factor xbp1 and the translation of an active transcription factor. ire1 can also activate additional pathways such as the degradation of er-related mrnas through a process termed ridd. perk activation induces the translation of the transcription factor atf4 and dampen the translation initiator factor elf2a activity, leading to decreased translation and protein synthesis. perk also promotes the activation of the transcriptional regulator nrf2. the transcriptionally active form of atf6 is produced by proteolysis. when these three main pathways are activated simultaneously they orchestrate the er-stress response characterized by the transcriptional induction of genes aimed at restoring er homeostasis. the outcome of the response is repair or, if repair fails, cell death and apoptosis. family that encodes membrane anchored bzip transcription factors. upon er-stress, atf6 translocates to the golgi where it is processed by site 1 and site 2 proteases that sequentially remove the luminal domain and the transmembrane anchor. these cleavages release the transcriptionally active n-terminal fragment of atf6 (atf6n) which relocates to nucleus where it upregulates er-stress induced genes related to protein folding and lipid synthesis [34,42e44] . beyond the adaptation phase that restore er homeostasis, er-stress as well as the activation of er-signaling pathways can impact on host immune functions and promote inflammation [9, 45] . er-stress observed in inflammatory pathologies determine the size, nature and duration of immune response [1, 3, 46] . several studies have showed that er-stress might augment inflammation. mutations in mice that produce defects in protein folding or in the er-stress response pathways lead to spontaneous inflammation. mice with a mutation impairing the folding of muc2, a mucin expressed in the paneth and goblet cells, show signs of er-stress in the intestine and develop an inflammatory phenotype similar to the inflammation observed in inflammatory bowel disease (ibd) patients [4, 47] . patients that carry misfolding mutations in the surfactant protein-c (sftpc) gene are characterized by er-stress activation in epithelial cells and develop hyper-inflammatory responses upon viral infections [6, 48] . viruses are believed to contribute to the onset of idiopathic pulmonary fibrosis (ipf) in these patients. however the exact contribution of the infection in the induction of er-stress or the contribution of er-stress to the upregulation of inflammation-mediated lung fibrosis in these patients are still open questions. similarly mutations in the cystic fibrosis transmembrane conductance regulator (cftr), an ion transporter, may cause er-stress in bronchial epithelia cells that become hyper-responsive and more susceptible to respiratory infection. hyper-responses to inflammatory stimuli have also been shown in macrophages. studies showed that hla-b27 misfolding can induce er-stress responses in macrophages resulting in enhanced cytokine production and increased release of type i interferon, upon stimulation with the tlr4 agonist lps. hla-b27 misfolding is associated with susceptibility to spondyloarthritides, a group of heterogeneous autoinflammatory diseases [8, 49] . in an other autoinflammatory disease, er-retention of the tnf receptor (tnfr1) was shown to contribute to increased cytokines production observed in tnfr1-associated periodic syndrome (traps) patients [10] , suggesting that perturbations in the er could be directly linked to the development of autoinflammatory syndromes. most autoinflammatory diseases have been associated with aberrant inflammasome activation [12] . the inflammasome is a cytosolic multiprotein complex that senses pathogens and cellular damage and leads to the activation of a central proteolytic enzyme, caspase-1. the main function of the inflammasome is the cleavage and maturation of a few key cytokines, including il-1b. these cytokines drive the initiation of inflammatory cascades in patients. because the inflammasome has been shown to become activated upon er-stress [14] , it is possible that loss of er-homeostasis could directly drive il-1b dependent inflammation and contribute to the pathogenesis of diseases including autoinflammatory syndromes such as traps, by directly driving inflammasome activation. er-stress is also involved in regulating another important branch of the innate immune system: the toll-like receptors (tlrs) response. tlrs are type i transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic domain harboring a tir domain similar to the cytoplasmic domain of the mammalian interleukin 1 receptor (il-1r). tlrs activation upon recognition of "pathogen-associated molecular patterns" or pamps, results in initiation of cellular signaling events including activation of transcription factors, cytokine modulation, and upregulation of interferonstimulated genes, leading to inflammatory responses and the release of antimicrobial molecules. regulatory loops connecting tlrs and er-signaling have been described. for example, tlr4 and tlr2 promote the mrna maturation of xbp1s by selectively triggering the ire1 branch of the er-stress response while inhibiting the other branches of the er-stress pathway [16, 18] . therefore the activation of ire1 and xbp1s by tlrs does not trigger a full er-stress response but an xbp1s dependent program that synergizes with tlr signaling pathways. the induction of xbp1s amplifies tlr responses leading to increased cytokine production [16, 18] . this demonstrates that specific engagement of er-signaling pathways in absence of erstress can contribute to immunity. xbp1s activation in the context of er-stress can also enhance tlrs responses. macrophages treated with pharmacological agents disrupting er homeostasis display a dramatically enhanced response to tlr4 and tlr2 activation [16, 20] . the mechanisms by which xbp1s enhances cytokine production is still unclear but may likely involve xbp1 binding at cytokine promoters [21] . in addition to xbp1, the er stress-induced transcription factor c/ebp homologous protein (chop), is involved in the upregulation of cytokines including il-23 in the context of dendritic cells undergoing both er stress and tlr stimulation [23] . knockdown of chop in a monocytic cell line significantly reduced the synergistic effects of er-stress and tlrs on il-23 expression. therefore diverse er stress-related transcriptional pathways can affect the nature of immune responses (fig. 3) . the er-stress response and inflammation are interconnected through various additional mechanisms. the production of reactive oxygen species (ros), the activation of the transcription factor nuclear factor-kb (nf-kb) and the induction of acute-phase proteins have being linked to both er-stress and inflammatory responses [25] . immune cells can also respond to stress signals released by other tissues. a recent report suggests that stressed cells can transmit (via a yet to be defined stress signal) er-stress conditions to macrophages, increasing inflammatory responses [27] . incubation of macrophages with cultured conditioned medium from erstressed cells promotes ire1 activation and protein chaperones upregulation in the macrophages that deploy a more sustained proinflammatory response. similarly injection into mice of media supernatant harvested from erstressed cells elicit the upregulation of genes typically associated with er stress responses in the liver [27] . these observations may indicate that loss of er homeostasis in tissues can be transmitted to the microenvironment and immune cells to augment immune responses [31] . whether this phenomenon is relevant to the pathology of inflammatory diseases characterized by the loss of er-homeostasis in tissues is an exciting question that remains to be addressed. er-stress pathways are also implicated in regulating immunogenicity. it has been shown that in tumor cells dying upon treatment with anticancer therapy, er-stress promotes cell-surface localization of factors involved in immunogenic cell death [32, 34] . calreticulin is the best-characterized immunogenic signal emerging from the er. this erresident chaperone delocalize on the cell surface where it can act as an "eat me" or "danger" signal that primes immune responses directed against the stressed cell [35] . the exposure of calreticulin on the cell surface is associated with the induction of er-stress [37, 39] and may directly involve the activation of er-stress signaling pathways such as the perk pathway [32] . therefore er-stress specific signal may increase immunogenicity by tagging the cell as stressed [34, 34, 42] . pathogens activate er-signaling pathways such as ire1 by directly engaging innate immune receptors as it was shown for tlrs, however live pathogens affect also directly er functions as part of their infectious and replicative cycle [9] . in plants, the ntbzip60 gene (a signaling branch of the plant er-stress response related to atf6) is activated upon infection of tobacco leaves with pseudomonas cichorri. moreover, silencing of ntbzip60 allows higher multiplication of p. cichorri compared to control plants [46] , suggesting that er-stress pathways may represent an ancestral mechanism of immune regulation. in caenorhabditis elegans infected with bacteria expressing pore-forming toxins, xbp1s and atf6 are induced [47] . genetic studies have shown that these er-stress signaling pathways augment survival of infected worms probably by promoting a protective restoration of er homeostasis or by increasing immune responses. similarly in c. elegans infected with pseudomonas aeruginosa, activation of ire1 and xbp1 was noted [48] . in this model, xbp1 deficiency decreases viability, possibly due to a reduced ability to cope with loss of er-homeostasis in these worms [49] . several types of virus, parasite and bacterium that infect humans have been shown to perturb the er and to manipulate er-signaling pathways as a mechanism aimed at interfering with host immune responses and supporting replication. viruses are obligate intracellular pathogens that depend on the host machinery to produce large amounts of infectious viral particles. many viruses depend on er-membranes for translation and budding of viral particles, a process that can result in the alteration of er-homeostasis and therefore affect immune responses [50] . in humans, er-stress was detected in tissues infected with different viruses. for example, xbp1 splicing and chop upregulation was detected in duodenal biopsies from individuals infected with hiv compared to uninfected persons [51] . er-stress was also observed in the liver of patients with chronic hepatitis c (hcv) [52] . parasites such as toxoplasma gondii activate the er-stress response [53] . loss of er-homeostasis was also observed upon infection with different bacteria. chop induction, eif2a phosphorylated and ire1 activation was observed in macrophagerich areas of granulomas in lungs of mice infected with virulent mycobacterium tuberculosis (mtb) [54] . er-stress activation by live bacteria can also occur prior to cell host invasion. the facultative intracellular pathogen listeria monocytogenes (lm) was found to induce er expansion and erstress signaling pathways prior to host cell entry [55] . the monitoring of genes typically associated with erstress responses is the main feature used to identify the activation of er-signaling pathways. however recent studies demonstrate that pathogens can trigger a specific branch of the er-stress program independently of the other branches without engaging a full er-stress response and upregulating genes characteristic of er-stress responses. influenza a virus activates the ire1 pathway with little or no parallel activation of the perk and the atf6 pathways. ire1 activation is apparently important for viral replication. inhibition of ire1 blocked influenza a virus replication [56] , demonstrating that the virus may manipulate the ire1 and benefit from it. acute infection with lymphocytic choriomeningitis virus (lcmv) was shown to trigger a selective induction of the atf6regulated branch of the er-stress response, whereas perk and ire1 pathways are neither activated nor blocked [57] . fig. 3 . tlr4 responses and er-signaling are connected and synergize to promote cytokine production. tlr4 activate the transcription factor xbp1 by selectively activating the ire1 kinase while decreasing the activation of atf6 and perk pathways as well as elf2a activation. the activation of ire1 and xbp1 does not contribute to an er-stress response. however, activated xbp1 amplifies tlr signaling by enhancing cytokine production. similarly, if chop and xbp1 are induced upon treatment with pharmacological agents, they both may contribute to cytokine production. how viruses trigger specific er-signaling pathways is unclear. in the case of lcmv, activation of atf6 may involve the viral glycoprotein precursor (gpc) [57] , further suggesting that some viruses may directly regulate these pathways independently of the induction of er-stress. while many pathogens trigger er-stress and/or features associated with the er-stress response, the exact function of these pathways are still poorly understood. activation of erstress and er-signaling pathways may increase immunity and reduce the cellular stress associated with the infection. this is a recent hypothesis and few experimental evidences demonstrate the physiological importance of these pathways in regulating pathogen replication and immunity in vivo. importantly, the key role of these pathways is also highlighted by the fact that pathogen have evolved mechanisms to manipulate er-signaling pathways optimizing survival and replication. the role of the er-stress pathways in shaping immunity is underscored by the fact that pathogens as well as innate immunity pathways regulate er-stress responses. it has been shown for example that the atf4-chop branch of the perk pathway is specifically inhibited by tlr signaling [58] . activation of trif by tlrs activates a phosphatase, pp2a, that dephosphorylate a subunit of eif2b (guanine nucleotide exchange factor for eif2a) leading to the inhibition of phosphorylated eif2a activity and downregulation of perk mediated atf4 and chop upregulation [59] . in macrophages tlr signaling has been found to inhibit atf6 and perk activation by a mechanism yet to be identified [16] . this regulation of er-signaling responses by innate immunity may explain why tlrs in macrophages only activate ire1 and in absence of concomitant activation of perk and atf6, therefore, favoring an inflammatory response rather than a full er-stress response. viruses are also able to manipulate the er-stress response by altering specific pathways or changing cellular homeostatic setpoints. dengue fever virus (denv) for example, manipulates the er-signaling pathways to specifically activate and suppress the three different branches of the er-stress response. importantly each branch is regulated differently depending on timing and the infectious stage [60] , suggesting that the virus can specifically manipulate the system to increase survival and prolong the viral life cycle. hcv provides us with another example of time-dependent regulation of the er-stress response. this virus triggers the erstress response in a wave-like fashion, which peaks a few days after infection. then the hepatocytes become tolerant to the stress induced by the virus or by chemical agents. suppression of viral replication restore the ability to engage an er-stress response suggesting that, the virus itself is can regulate the response [61] . in line with these observations, livers from patients with untreated chronic hepatitis c exhibit activation of the three er-stress branches without apparent induction of downstream er-related responsive genes [52] . these findings could be explained by specific dampening of the downstream response by viral effectors. it has been shown that hcv reduces xbp1 transcriptional activity [62] and that the hcv envelope protein e2 may regulate perk activity [63] . similar observations were reported for coronavirus mouse hepatitis virus (mhv). infection by mhv induces ire1-mediated splicing of xbp1 mrna and the maturation of atf6, without upregulating significant downstream target genes [64] . likewise, the influenza virus and the enterovirus 71 (ev71) modulate the stress response in the setting of a preexisting stress by dampening the activation and processing of the atf6 as indicated by its inhibitory effect on atf6-dependent genes [56, 65] . parasites can also modulate er-signaling responses. the host atf6 pathway is targeted by the t. gondii virulence factor rop18 [53] . rop18 is a kinase that triggers proteasome-dependent degradation of atf6 a process that apparently is required for parasite virulence. these examples demonstrate that pathogens have evolved distinct mechanisms to regulate and benefit from er-signaling pathways. cells that undergo acute pressure on the er may trigger an apoptosis program as part of a cellular response to acute dysfunction. it is therefore likely that the dampening of this response by pathogens may increase it life cycle by promoting survival of host cells. alternatively, the pathogen may also manipulate the system to specifically control transcriptional programs triggered by the er-stress signaling branches independently of the er-stress response. the exact function of these programs in the context of an infection in vivo is unclear. the regulation of inflammatory and immune functions as well as the induction of pathogen specific transcriptional programs may represent key mechanisms orchestrated by pathogens takeover of er-signaling pathways. the overall message of this review is that er-signaling pathways steaming from the er are often tightly linked to inflammation and immunity. the er is therefore a central stress sensor that detects cellular insults and trigger specific responses involved in restoring homeostasis as well as promoting immunity. however, there are many knowledge gaps that need to be investigated in order to have a comprehensive picture of how innate immune signaling pathways intersect with signaling branches emerging from the er. the challenge for the future is to characterize at the molecular level the proximal pathways that emerge from the er-stress signaling pathways and to understand how these branches are activated and regulated by pathogens, pathogen virulence factors as well as innate immune signaling component such as tlrs. er-stress is often defined by the induction of the erstress response, however it is now clear that pathogen as well as inflammatory condition can operate er-signaling pathways in absence of er-stress. the development of new techniques and reagents to monitor the stress in the er as well as the activation of er-signaling branches will foster a better characterization of these pathways in immunity. deficiencies in the main components of the er-signaling pathways are generally lethal in mice. the development of new mouse models with conditional deletion of specific er-signaling pathways may therefore help assess the physiological importance of these responses during the course of infections as well as in autoimmune and autoinflammatory diseases. it will be also important to understand how er-signaling pathways affect immune responses in cooperation with other stress responses related to the proteostasis network such as autophagy and the oxidative stress response. finally, it is tempting to speculate that the development of drugs to alleviate erstress or regulate specific branches of the er-stress response may provide new therapeutic approaches in the regulation of infectious and immune-related diseases. regulation of innate immunity by signaling pathways emerging from the endoplasmic reticulum the danger model: a renewed sense of self endoplasmic reticulum stress and the inflammatory basis of metabolic disease aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis detection of immune danger signals by nalp3 emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis origin and physiological roles of inflammation pathogeneendoplasmic-reticulum interactions: in through the out door mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in tnfr1-associated periodic syndrome (traps) the mammalian endoplasmic reticulum as a sensor for cellular stress autoinflammatory disease reloaded: a clinical perspective the unfolded protein response: from stress pathway 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factor, plays a role in the defense response against bacterial pathogens in nicotiana tabacum activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo an essential role for xbp-1 in host protection against immune activation in c. elegans physiological ire-1-xbp-1 and pek-1 signaling in caenorhabditis elegans larval development and immunity viruses, endoplasmic reticulum stress, and interferon responses inflammation and epithelial cell injury in aids enteropathy: involvement of endoplasmic reticulum stress in vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis c atf6beta is a host cellular target of the toxoplasma gondii virulence factor rop18 induction of er stress in macrophages of tuberculosis granulomas activation of the unfolded protein response by listeria monocytogenes influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway role of the host cell's unfolded protein response in arenavirus infection adaptive suppression of the atf4-chop branch of the unfolded protein response by toll-like receptor signaling toll-like receptor activation suppresses er stress factor chop and translation inhibition through activation of eif2b dengue virus modulates the unfolded protein response in a time-dependent manner hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis c virus envelope protein e2 through the eukaryotic initiation factor 2alpha kinase perk coronavirus infection modulates the unfolded protein response and mediates sustained translational repression endoplasmic reticulum stress is induced and modulated by enterovirus 71 key: cord-262551-hxhlhb5m authors: van der gronde, toon; los, leontien; herremans, arnoud; oosting, ronald; zorzanelli, rafaela; pieters, toine title: toward a new model of understanding, preventing, and treating adolescent depression focusing on exhaustion and stress date: 2020-05-06 journal: front psychiatry doi: 10.3389/fpsyt.2020.00412 sha: doc_id: 262551 cord_uid: hxhlhb5m objective: adolescent depression is a heterogeneous disorder, with a wide variety of symptoms and inconsistent treatment response, and is not completely understood. a dysregulated stress system is a consistent finding, however, and exhaustion is a consistent trait in adolescent patients. the aim of this paper is to critically assess current hypotheses in adolescent depression research and reframe causes and treatment approaches. methods: a mixed-method approach involved a review based on publications from pubmed, embase and psycinfo, and two exemplary adolescent cases. results: both cases show a spiral of stress and exhaustion, but with a different profile of symptoms and coping mechanisms. reframing both cases from the perspective of coping behavior, searching for the sources of experienced stress and exhaustion, showed coping similarities. this proved essential in the successful personalized treatment and recovery process. in combination with recent evidence, both cases support the functional reframing of depression as the outcome of a stressand exhaustion-related spiralling mechanism. conclusions: we propose to open up a symptom-based, mood-centered view to a model in which adolescent depression is framed as a consecutive failure of stress coping mechanisms and chronic exhaustion. addressing exhaustion and coping primarily as a treatment strategy in adolescents and young adults might work in synergy with existing treatments and improve overall outcomes. this perspective warrants further investigation. major depressive disorder (mdd) is the leading cause of disability worldwide (1) , with 10 to 15% of patients proceeding to suicide (2, 3) , and a substantive disease burden for adolescents and young adults (4) (5) (6) . depression is a heterogeneous group of brain disorders with varied contextualized origins, complex genetics and a neurobiology that is not completely understood. the etiology is not elucidated, and particularly for adolescents there is an evidence gap (4, 7, 8) . the serendipitous discovery of first the tri-and tetracyclic antidepressants (tcas) and later the stress-modulating serotonin reuptake inhibitors (ssris) led successively to the catecholamine and monoamine hypotheses of depression (9) . in later years, reduced adult neurogenesis and changes in structural and functional neuronal plasticity have been linked to the onset and treatment opportunities of major depression (10, 11) . genetic research has shown that there is not a single genetic cause for depression, and all known genetic factors combined only explain a limited percentage of the variance in clinical outcomes (12, 13) . the estimated heritability of depression is 35%-40%, indicating 60%-65% is explained by other factors, such as adverse life experiences (11, 14) . researchers have turned to epigenetics to develop new forms of genetic and pharmacological modeling, in an effort to describe the etiology of depression better (15) . despite many years of research by numerous investigators both in academia and industry, psychoactive targeted therapeutics with controllable and specific effects on the brain microcircuitry and chemistry did not and probably will not materialize due to the complex nature of mental disorders (16) . in order to open up our thinking about mdd we take up the challenge to reframe depression, specifically focusing on adolescents. we note that within the current framework depression is diagnosed based on the presence of a series of mood-related symptoms and their effect on daily functioning. the seven most commonly used interviews and self-report questionnaires together describe a heterogenous group of 52 symptoms, such as either high or low appetite, more or less sleep than usual, and a feeling of sadness (17) . this causes differences in diagnosis based on which scale is used (18) . the widely varying patterns in which these symptoms often present themselves (19, 20) , and the high occurrence of several comorbidities, such as anxiety, psychosis, and autism spectrum disorder, indicate that depression is not a homogenous disease, but a continuous, heterogeneous group of disorders associated with a wide variety of different risk factors (4, 8, (21) (22) (23) (24) . combined with the lack of understanding of the etiology of adolescent depression, the large variation of presentation and treatment approaches is the main driver for us to try to reframe the concept of mdd in adolescent patients. we also aim to explain why responses to treatment vary substantially and why older age is a consistent and important risk factor for a poorer mdd course (25) (26) (27) (28) . we will take a new perspective toward mdd by focusing on stress and the depressive mood related to development in adolescence. this yields a promise for novel therapeutic approaches and potential breakthroughs in depression research, treatment and prevention. a mixed method approach was used involving clinical investigation of adolescent case reports and a narrative review. pubmed, embase, and psychinfo were searched for relevant publications, with select additions of recent findings based on collective suggestions of the authors. to make sure the patient perspective is not lost when critically assessing the current framework and new possibilities, two case reports were included. written informed consent was obtained from both subjects for the case reports. many findings in depression research have failed the scientific test of replication. for example, the volume of the amygdala of depressed patients has been found to be increased (29) in some studies, and decreased in others (30) . patients with melancholic depression, a subtype based on symptoms, were thought to respond better to tcas than atypical patients (hence the name) (20, 27, 31, 32) , but other researchers could not replicate this finding (33) (34) (35) (36) . plasma levels of leptin, which reduces appetite, has been found higher in melancholic patients (37) , or higher in atypical patients (38) . finally, childhood trauma and/or abuse is more common in melancholic than in atypical patients (39) , or vice versa (40). one consistent finding, however, is a dysregulated stress system in depressed patients (41) (42) (43) (44) . in approximately 70% of depressed patients a dysfunction of the hypothalamicpituitary-adrenal (hpa) axis is detected, mainly hyperactivity (38, 45) . also a disruption of the diurnal variation of cortisol is commonly seen (46, 47) . unfortunately, after decades of research efforts this finding has not resulted in a stress-targeted treatment option or a clinical test to predict treatment response (48, 49) , and it remains debated whether hpa-axis dysregulation is a cause or a consequence of depression. this does provide an important insight: depression is at least partially the result of stress and a differential dysregulation in the stress system is an important trait (38) . the stressor may be in the past (e.g. childhood maltreatment or trauma) (4, 50), or acutely (e.g. dealing with new life events). the initial response of people to stress is typically a coping mechanism aimed at exerting control over the stressor either by avoiding, reducing or predicting its occurrence. examples of such efforts are the canceling of obligations or disengagement from social interaction (51) . the hpa axis exerts a fundamental role regulating both internal as external stimuli, integrating the physiopathological and behavioral dimensions of stress. we postulate that depression is the result of a failure of coping mechanisms to control the stressors and a differential dysregulation in the stress system. the accumulation of stressful events, and the eventual failure of coping mechanisms to deal with the stress, can lead to exhaustion and depressive behavior. preclinical experiments already hinted at a relation between the effectiveness of coping behavior, the effort involved and feedback on the development of gastric ulcers. although coping efforts were effective, ulcers still developed when coping took more effort and less feedback was offered (52, 53) . preclinical evidence indicated that chronic exposure to relatively mild stressors which rats can adapt to relatively easily (e.g. tilting the cage at a slight angle, emptying a water bottle in the cage, introducing new bedding material), ultimately resulted in the development of anhedonia (50) . the chronic character of having to cope with mild stressful events over and over again, and the lack of control over stressors, was sufficient for depressive symptoms to develop (54) . in a forced swimming test, rats who were dosed with psilocybin developed the coping technique of floating faster than other rats, indicating a window of behavioral flexibility (55) . ketamine displayed the opposite effect, with more mobility (56) . we hypothesize that depressive behavior, specifically anhedonia and withdrawal, and the consequent loss of interest and enjoyment in usual activities, is an evolutionary mechanism to guard the organism against the exhaustion that may results from excessive or chronic coping behavior. as such, depressive behavior is both an expression of psychological pressure and a physiological precaution. this substantiates the entanglement of psychological and physiological factors in mdd. stress response mechanisms can change the allocation of metabolic resources in a stressful situation, where that is needed. similarly, depression could be the expression of a forced change in allocation of attention. depressed patients are known to ruminate, or continually analyze their problems and relive their memories (24) . anhedonia can be interpreted as a way to secure mental resources, by reducing the interest in distractions (20, 57) . depression can be seen as an exaggerated social navigating coping mechanism, caused by an accumulation of stress and a spiral of unsuccessful adaptive behaviors which leads to exhaustion. by entering a depressive mode, the organism aims to guard itself from exhaustion. the challenge is to interfere with this mood-affecting spiralling mechanism (see figure 1 ) to prevent depression from developing. dealing with stress and potential exhaustion, as opposed to dealing with the symptoms of depression, could prove to be an effective treatment approach. there is currently only limited evidence-based rationale for choosing one treatment over another for an individual patient (31, (58) (59) (60) , with no differentiated approach for adolescents or adults (6, 61) . even defining depression subtypes based on symptoms has not helped (62). despite guidelines and evidence-based interventions, treatment is still primarily based on trial and error (63, 64) , and primarily aimed at improving mood. yet, between one third (65) to half (20, 66) of adult patients show no response to weeks of first line treatment with antidepressant drugs, and are advised to try a different antidepressant. further, one third of all patients never reach a response after four lines of antidepressant treatment (65) . the current therapeutic shortcomings are the consequences of our lack of knowledge of causes, the underlying neurobiology and chemistry (67) , and risk factors that contribute to the onset and maintenance of depression. as a consequence, the treatment paradigms are oversimplified with little attention for preventive measures (68) . when albert hofmann, who first synthesized lysergic acid diethylamide (lsd), came in contact with it himself in 1943, he noted the hallucinogenic properties. in 1947 it was first marketed as a therapeutic drug. in the 1960s, timothy leary was the first to start experimenting with psilocybin combined with psychotherapy (69). in the 1950s and 1960s, lsd and psilocybin were tested in several small-scale clinical trials for anxiety, depression and addiction (70, 71) . in response to increased recreational use, international legislation was introduced as part of the "war on drugs," which brought an abrupt end to clinical research with these and similar substances in the 1970s (67, 69, 70, 72) . in the last two decades, clinical trials with psychedelics have started to take place again (71) . a brief overview of the indications these psychedelics have been investigated for post-2000 is indicated in table 1 (69, 70, 72) . new insights in the complex etiology of depression might be offered by findings with the use of psychedelics for treatment-resistant depression (73, 74) . several psychedelics have shown to help depressive patients in a limited number of studies with small number of patients. the classic psychedelics, compounds such as lsd, psilocybin, and ayahuasca, have diverse pharmacological profiles, including robust effects on the serotonergic system (67, 69, 72, 75) . a psychedelic not acting on the serotonergic system is the dissociative anesthetic ketamine, which in subanesthetic doses acts as an antagonist on the n-methyl-d-aspartate (nmda) receptor, a type of glutamate receptor (56, 69, 76, 77) . esketamine, the s-enantiomer of ketamine, has been approved by the food and drug administration (fda) for treatment-resistant depression (78) , but ketamine has been and continues to be used offlabel to treat depression too (79) . this highlights that serotonergic activity, or even a mono-aminergic activity, is not required for the antidepressant effect of a psychedelic compound, further stressing the need for abandoning the old hypotheses. these hallucinogens, and the chemically related entactogen 3,4-methylenedioxymethamphetamine (mdma) may have a place in offering a positive experience to break the self-sustaining depressive state and allowing for introspection during psychotherapy to process stressful life-time experiences as a form of reverse medical engineering (80) . from a psychological point of view, psychedelics work through a different mechanism than classic antidepressants. instead of the elevation of mood and the reduction of anxiety, psychedelic drugs induce a profound temporary positive experience (e.g. a mystical or religious sensation). this positive experience allows for the temporary disintegration of existing networks, which in turn facilitates reprocessing of past emotions and introspection (67) . in turn, this improves the capacity to cope with stress (71) . also, the use of a psychedelic in combination with a psychotherapeutic process could have long-term effects, counteracting the effect of a negative experience and disrupting the negative and "downward spiralling" compulsive thinking (72) . in this article we move away from mood improvement as a primary target (81, 82) . we offer an alternative integrated approach for the treatment of adolescent and young adult depression by focusing on stress factors and exhaustion reduction, seeing anhedonia and withdrawal as an evolutionary coping mechanism. this integrates approaches such as the social navigation hypothesis of watson and andrews (83) with cognitive bias (84) and selye's biological stress (85, 86) . with this approach we take a functional perspective, and focus on the function the depressive state provides to the adolescent patient and how it develops. this perspective is instrumental for tailormade treatment strategies. we will discuss these insights on the basis of two adolescent patient reports. mood disorders have been shown to be progressive, with patients developing more complex psychopathologies over time (87, 88) . approximately 50% of patients retrospectively state that their first depressive episode occurred before the age of 20 (88, 89) ; another report states 50% experience that before the age of 14 (90) . this further highlights the progressive nature of depression and the need for early intervention. a 17-year-old caucasian woman was referred by her own general practitioner to the department of adolescent psychiatry and addiction prevention for binge drinking and daily use of marijuana. the intake together with her parents showed that the patient already had a history of moderate depression and an eating disorder, anorectic of the purging type with moderate severity. no abnormalities were reported regarding appearance, behavior, eye contact, and rapport orientation and cognition [intelligence quotient (iq) of 127]. however, she regularly suffered from suicidal thoughts and a low ability to experience pleasure. though she had no concrete suicide plans, in gloomy periods she showed risky behavior, like crossing a busy road without looking. she usually performed well in school, despite occasional lags in attendance, which were compensated with short periods of active study. her mother had a history of mdd. at the department of adolescent psychiatry and addiction prevention, we classified the addiction behavior as mild. but we also established a comorbid psychiatric and substance-use disorder profile. thus, we chose for an integrated treatment for comorbidity that has been found to be consistently superior (91) . effective treatment for comorbid conditions combines different therapeutic modalities, i.e. psychotherapy [e.g. motivational interviewing (mi), cognitive behavioral therapy (cbt)], pharmacotherapy (e.g. antidepressants), and family therapy. using combinations of different modalities typically increases therapeutic effect by exerting a synergistic impact on symptoms (6) . with mi, the patient was motivated to choose a first educationrelated treatment goal. this was to prevent school dropout at all cost. we started cbt to control her marihuana and alcohol abuse and prevent school dropout. we added medication in order to try to stabilize her mood with fluoxetine, an ssri, which might also modulate stress. the medication initially seemed to have some effect but after two months there was a sharp mood drop, increased suicidality and aggravation of eating disorder symptoms. eventually she had a body mass index of 16 kg/m 2 . the eating problems were mapped and analysed by an eating disorder specialist. the latter used a problem-solving approach and focused on both directive counseling and emotional support. the eating disorder specialist also advised to choose a medication with low risks of weight gain. the psychiatrist changed the medication to citalopram. subsequently, the treatment team focused on teaching the patient how to cope with stressful situations and the associated anxiety. the stress appeared to be mainly caused by a feeling of lack of control. the patient turned out to have a high intelligence and learning ability, but also felt that she had no control over her learning process. she had not sufficiently developed social learning strategies in her early school years. in addition, there appeared to be an issue of individuation and separation problems. these problems got worse because it was almost impossible for her parents to let her develop in her own way due to the stress they had over her suicidal thoughts, drug use, and worsening physical condition due to bad eating habits. we decided on an additional family counseling approach to address these issues. the integrated treatment modality approach proved effective. she developed a realistic idea of what caused her stress, how she reacted situationally and improved her awareness that she tends to have control over everything. her parents were involved in helping her developing control coping skills and checking on achievements. because of this insight, she succeeded in maintaining her diet less strictly and experimenting with behaving differently without alcohol or drugs. her parents saw that she was doing better and were able to release her a bit more. this increased her sense of control and provided enough space to further discover what goals she wanted to achieve. in the process her mood and her ability to experience pleasure improved significantly. she successfully passed her school exams and proceeded to university. a 15-year old caucasian girl was referred by her own general practitioner after a suicide attempt with symptoms of sadness, anxiety, and obsessive-compulsive behavior. the intake was together with her parents. she was struggling in school, despite her very supportive family. no drug abuse or other psychiatric symptoms were found. she told the counselor she tried hard, but felt that she could not keep up in school; it was never good enough, no matter how hard she tried. the counselor estimated that the school level was appropriate for the level of intelligence of the patient. she had periods when her self-esteem was very low. during these periods she spent hours on her appearance, focusing on her hair and makeup. her hair fell out as a result of these sessions. she could not stop herself, and always ended with self-harm. this in turn lowered her self-esteem and increased the experienced stress. she was locked in a downward spiral. gradually her mood disorder worsened and made her passive. she no longer wanted to go to school and meet friends, but passed hours in front of the mirror. she attempted to end her life. we hypothesized on the basis of the girl's stress complaints that she felt school, parents, and friends expected too much of her. after a neuropsychological assessment the testing showed that she had a disharmonic intelligence profile with an iq of approximately 80 (using the wechsler intelligence scale for children-iii (92) ), inconsistent at all factor levels. we classified a mild intellectual developmental disorder in the conceptual and practical domain, which explained the structural struggle with the standard school curriculum instructions. we educated parents and school on how instructions might fit in better with her learning abilities and style. her preferred method of learning new things was being shown how to do it, as opposed to having it explained to her. this led to significant stress reduction and positive school experiences. in the process her self-esteem improved, the experienced stress decreased, and her mood improved. cbt was adjusted to her learning style and was used to reduce her obsessive-compulsive behavior. though both case reports show a different profile of symptoms and coping mechanisms, in both cases a downward spiral of stress, coping behavior, and exhaustion are central. both patients described themselves as rarely feeling relaxed and as struggling to fulfill their daily tasks. after several years of chronic stress, a period followed in which they felt constantly exhausted. the first patient coped with stressful situations through aberrant food intake behavior, suicidal thoughts, and mood swings. she overcompensated this restrictive behavior with recreational drug abuse. the second patient developed compulsory behavior, stress, and suicidal thoughts and overcompensated leading to self-harm. in the current framework both cases would be viewed as different and based on their symptomatology ask for different treatments. reframing both cases, from the perspective of coping behavior, searching for the origin and sources of the experienced stress and exhaustion and coping with stressful situations, showed stress coping similarities between the two cases and proved an essential part of the personalized treatment and recovery process. both cases support the added clinical value of the functional reframing of depression as the outcome of a mood-affecting stress and exhaustion related spiralling mechanism. the adolescent cases presented here are good examples of how depression can be managed by relearning effective coping behavior. this prevents patients from reverting to a depressive state in order to cope with the life stressors. in more severe and chronic cases, patients suffering from difficult-to-treat or treatment resistant mdd, patients are in a deep depressive state and are not capable of learning new coping behaviors. we envision that in such situations more radical medical interventions are needed to first elevate patients from the depressive state into a state where learning new and effective coping strategies can take place. in these situations, psychedelics (e.g. ketamine) have proven to be effective to temporarily draw people out of a deep depressive state. with the support of followup medication and adequate psychological guidance, the patients may then develop effective coping strategies. as this is a narrative review, and not a formal systematic review, caution needs to be used when interpreting this data. select literature sources have been added based on informal searches, so contradicting studies may have been missed. similarly, the two cases were selected for their exemplary stories, not because they are typical. as such, this paper aims to provide new insights and direction for future treatments, not a definitive answer on how to improve treatment. after the spanish flu of 1918, many infected patients developed post-viral fatigue in the following months and years. on top of that, the randomness of who was infected and who died as a result led to learned helplessness, which caused anhedonia and other depressive symptoms (93) . the recent pandemic of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 [sars-cov-2, previously known as 2019-ncov (94)], has overwhelmed healthcare facilities with the high need for acute care. this puts a large psychological toll on the entire population, with high levels of stress (95) . next to the risk of the many life events that can be expected in these situations, coping behavior-such as avoiding conflict, or searching social support-is often limited due to confinements that are put in place to prevent further spread. furthermore, the lockdown situation and mass isolation at home in many countries may increase the risk of domestic violence and divorces. this could all lead to a rise in trauma-related stress disorders in the months and years to come. breaking a vicious cycle of stress, inadequate coping behavior and exhaustion with a holistic view, and possibly with psychedelic-supported psychotherapy, might help treat the many psychiatric patients that can be expected. reframing depression and shifting clinical practice to a more comprehensive and integrated look at the individual experience of a patient, including all causes for stress, pressure, and exhaustion, might be more helpful in developing promising treatment strategies. also, treatment practices that take into account preventive mental health interventions, and that focus on stress, exhaustion, and coping strategies, could have a significant and lasting impact on many patients struggling with depression. the perspective of stress, coping, and exhaustion provides the therapist with another treatment approach that can work in synergy with the existing arsenal of therapeutic approaches, making the therapist more effective. increased focus is needed on support programs to help individuals develop functional coping mechanisms to deal with pressure, before more serious coping mechanisms develop in the form of withdrawal from stressful situations, compulsory behavior, or frequently occasional use of recreational drugs (96) . our intuition is that during successful treatment patients experience small successes of effective coping and re-live the rewarding properties of such experiences. reliving experiences could repair the damaged reward mechanisms and diminishes the experienced anxiety and stress which then might subsequently drive and sustain further recovery (97) . psychedelics may offer help in breaking free from the existing cognitive bias, by facilitating introspection, re-living of past experiences, and development of new coping mechanisms. effective treatment strategies for adolescent and young adult depression should combine different therapeutic modalities and focus on exhaustion and sources of stress. using a combination of treatment modalities could increase therapeutic effectiveness by improving the pace of learning new coping behaviors, exerting a synergistic impact on the developmental perspective, and breaking the downward spiral of stress and exhaustion, which eventually leads to a reduction of the depression symptoms. this might also help for other related mental disorders in adolescents and young adults where exhaustion and stress are central, such as burnout syndrome (98) . but similarly, post-traumatic stress disorder, autism spectrum disorder, and generalized anxiety disorder are related to stress (24, (99) (100) (101) . these disorders could also benefit from the reframing of the concept of mood and stress. 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(the psychophysiological approach in medical practice) fiveyear prospective outcome of psychopathology in the adolescent offspring of bipolar parents early onset major depressive disorder screen teenagers annually for depression, say us doctors children and adolescents mental health: a systematic review of interaction-based interventions in schools and communities integrated treatment of substance use and psychiatric disorders wechsler intelligence scale for children-third edition (wisciii) the spanish flu of 1918 and how it changed the world coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 the mental health consequences of covid-19 and physical distancing: the need for prevention and early integrated treatment of adolescents with cooccurring depression and substance use disorder the entropic brain: a theory of conscious states informed by neuroimaging research with psychedelic drugs social support, dysfunctional coping, and community reintegration as predictors of ptsd among human trafficking survivors coping strategies and mental health outcomes of conflict-affected persons in the republic of georgia reviewing the potential of psychedelics for the treatment of ptsd we would like to thank prof dr. eric vermetten, prof. dr. claudi bockting, and the reviewers for their feedback on this paper, and the patients who consented to have their history described for the case reports. furthermore, we would like to thank cassandra nemzoff for her english manuscript correction services. in conflict of interest: during the late stage development of this manuscript, tg accepted a position in oncology at astrazeneca. astrazeneca had no role in any aspect of this paper. key: cord-009966-6kcgc5fx authors: warren, kimberly r.; postolache, teodor t.; groer, maureen e.; pinjari, omar; kelly, deanna l.; reynolds, mark a. title: role of chronic stress and depression in periodontal diseases date: 2013-12-09 journal: periodontol 2000 doi: 10.1111/prd.12036 sha: doc_id: 9966 cord_uid: 6kcgc5fx an extensive body of experimental and clinical evidence documents the negative impact of chronic psychological stress and depression on the immune system and health. chronic stress and depression can result in general dysregulation of the immune system, of both cellular and humoral pathways, which may contribute to pathogenic infection and concomitant periodontal tissue destruction. in general, the evidence is consistent with the hypothesis that stress can modify the host defense and progression of periodontal infections in patients susceptible to periodontitis. however, substantial evidence also indicates that these conditions can mediate risk for disease, including periodontitis, through changes in health‐related behaviors, such as oral hygiene, smoking and diet. the unequivocal interpretation of studies has also been hampered, in part, by issues related to conceptualization of stress and depression, as well as commonly associated comorbidities, such as diabetes, that can modify the onset and progression of periodontal disease. in addition, stress and depression appear to fall into a spectrum, ranging from mild to severe, involving a complex interaction of genetic background, coping strategies and environment. differences in the conceptualization of stress and depression are probably important in assessing associations with other biologic and clinical measures. future studies are necessary to clarify the complex interactions of chronic stress and depression in periodontal diseases. psychological stress refers to the emotional and physiological reactions experienced when a person confronts a life event, such as marital conflict, financial debt or death of a loved one, that exceeds his or her ability to cope effectively with the situation. stress evokes emotional and physiologic reactions and is an important modifiable risk factor for both mental and physical illnesses. a robust, and presumably causal, association exists between stressful life events and major depressive episodes (55, 68) , with chronic stress being closely linked to depressive disorders. the neurobiology underlying stress and depression is thought to result from molecular and cellular abnormalities that interact with genetic and environmental factors (73) . epidemiologic studies provide strong evidence that chronic psychosocial stress and depression increase the risk of atherosclerotic cardiovascular disease, diabetes and other systemic conditions (51) , as well as adversely affect the course and outcome of the conditions (85, 100, 138) . moreover, numerous studies show that stress and depression are associated with increased morbidity and mortality across a range of systemic conditions (23, 88, 113, 114, 130, 137) . several pathophysiologic mechanisms may explain the association of chronic stress and depression with systemic diseases (54, 57, 64, 87, 120, 131) . basic and clinical studies demonstrate that stress and depression are associated with atrophy and loss of function of limbic brain regions that control mood and depression, including the prefrontal cortex and the hippocampus (9, 38, 39, 73, 86, 119) . in addition, experimental animal models suggest that chronic stress induces vascular inflammation through elevations in circulating proinflammatory cytokines (80) . prevalence estimates of depression and stressrelated disorders in the united states vary across studies; however, a recent survey revealed that 9.0% of adults met the crieria for current depression, with 3.4% meeting the criteria for major depression (22) . in addition, a national survey commissioned by the american psychological association found that 69% of employees perceived work as a significant source of stress and 41% typically felt tense or stressed during the work day (6) . health-care expenditures associated with stress and depression are high, especially those attributable to comorbities, such as cardiovasular disease and diabetes. recent estimates suggest that nearly 25% of health-care costs are attributable to modifiable risk factors, such as depression, in the united states (51) . in a study of 92,486 employees from seven organizations over an average of 3 years, goetzel et al. (51) found that in the category of psychosocial risks, health-care costs for employees with depression were 48% more expensive than for those not at risk ($2,184 in higher costs). similarly, the health-care costs of workers reporting high stress were 8.6% ($413) higher than the costs of those not reporting high stress. the purpose of this narrative review was to summarize the literature on stress and depression as it relates to periodontitis, highlighting the emerging role of neuroendocrine and neuroimmune mediators in the pathophysiology of inflammatory diseases. we reviewed english language publications from 1970 to 2012, retrieved using the electronic database pub-med. the search terms psychiatry, psychological stress, depression, dental, periodontal disease, periodontitis, teeth, oral health, and immune function were used in the searches. central nervous system communication with both the immune and endocrine systems has given rise to the field of psychoneuroimmunology. neuroendocrine-derived peptides and hormones have long been recognized as immune modulators. early studies in animals found that stress was associated with increased susceptibility to infectious disease (106) as well as experimental models of inflammatory disease (4) . research in human psychoneuroimmunology has shown that immune-regulatory processes are an inextricable part of a complex network of adaptive responses (62) . individuals experiencing stress exhibit prominent abnormalities of behavior, such as depressed mood and impaired sleep, along with dysregulation of the neuroendocrine and sympathetic nervous systemsthe latter systems are critical efferent pathways in the regulation of immunity by the brain. the pattern and magnitude of the response to stress appears to be influenced by mulitple factors, such as the duration of stress exposure (acute vs. chronic), the type of stress (physical vs. psychological) and gender, among others (64) . multiple stress mediators, including monoamines, neuropeptides and steroid hormones, are necessary to convey the stress signal to the central nervous system and contribute to the resulting functional changes in the central nervous system. a basic understanding of the role of the limbic-hypothalamic-pitu-itary-adrenal axis and the cytokine regulatory loop are depicted graphically in fig. 1 . complex bidirectional interactions have been established between the central nervous system and the immune system, mediated by the endocrine system. two important features of these interactions include the production of stress hormones by the limbic-hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal-medullary axis (46) . hormones effect immune function through receptor binding and modulation of cytokines. the modulation of cytokines has been shown to feed back to the brain, producing changes in the hypothalamic-pituitaryadrenal axis, providing a mechanistic basis for common alterations in sleep patterns and depression. acute, time-limited stressors appear to result in adaptive redistribution of cells and preparation of the natural immune system for possible infection or injury, or both. chronic stressors have been associated with more global immunosuppressioncellular immunity followed by both natural and specific immunity, including t-helper 1 (e.g. t-cell-proliferative responses) and t-helper 2 (e.g. antibody to influenza vaccine) parameters (117) . therefore, the adaptiveness of immune changes appears to decrease with increasing chonicity of a stressor. psychological stress clearly exerts the capacity to modify the susceptibility of animals and humans to infectious agents, influencing the onset, course and outcome of certain infectious pathologies (12) . psychological stress is generally recognized as a potential cofactor in the pathogenesis of infectious disease. cohen et al. (30) prospectively studied the relationship between psychological stress and the frequency of documented clinical colds among subjects intentionally exposed to respiratory viruses. after completing questionnaires assessing the degree of psychological stress, 394 healthy subjects were given nasal drops containing one of five respiratory viruses (rhinovirus type 2, 9 or 14; respiratory syncytial virus; or coronavirus type 229e) and an additional 26 were given saline nasal drops. stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness, and this risk was attributable to increased rates of infection rather than to an increased frequency of symptoms after infection. in a recent systematic review and meta-analysis, pedersen et al. (101) conclude that the evidence supports the hypothesis that psychological stress is associated with increased susceptibility to upper respiratory tract infection, consistent with the potential importance of psychological factors in other infectious diseases. stress has a direct impact on the immune system , similarly to many other strategies which people use to cope with stress, whether healthy (e.g. exercise) or unhealthy (e.g. smoking). genco et al. (44) , for example, found a higher risk of more severe periodontal attachment loss (odds ratio = 2.24) and alveolar bone loss (odds ratio = 1.91) among individuals with financial strain and an inadequate coping style, when compared with those with low levels of financial strain within the same coping group, after adjustment for age, gender and cigarette smoking. there is also evidence for psychological modulation of the immune response to viral vaccines. one of the earliest studies was conducted in medical students who were inoculated with the hepatitis b virus vaccine. both virusspecific t-cell and antibody responses were up-regulated in those students with more stress and less social support (46, 48) . chronic stress associated with caregiving for a spouse with alzheimer's disease was associated with a less desirable antibody response to an influenza virus vaccine compared with matched controls (69) . a similar study in hong kong compared 55 caregivers of spouses with chronic conditions that impaired their activities of daily living with 61 matched controls. participants were injected with trivalent influenza vaccine and were assessed for symptoms of stress and depression. caregivers had statistically significantly lower cell-mediated immune responses to the influenza vaccine compared with controls (136) . other studies show that caregivers of those with chronic illnesses are at higher risk of infection and immune reaction (20, 91, 133) . collectively, these studies and others (132) suggest that stress and inadquate coping strategies or social support can play a role in the activation or re-activation of herpesviruses and other viral infections in the periodontium. high periodontal loads of active epstein-barr virus and cytomegalovirus tend to be associated with aggressive periodontitis, whereas latent herpesvirus infections are more common in chronic periodontitis and gingivitis (125) . stress also alters clinical wound healing and underlying immunologically mediated processes (72) . studies on the healing of acute experimental and surgical wounds consistently report slower wound healing in individuals with high levels of psychological stress, regardless of stressor duration (17, (70) (71) (72) 84) . a decrease in local pro-inflammatory cytokines in the wound bed has been postulated as one biologic mechanism (49, 70) , presumably caused by the immunosuppressant effects of cortisol, norepinephrine and epinephrine (40, 94) . in animal models of psychological stress-impaired cutaneous wound healing (50) , delay in healing is also associated with deficits in bacterial clearance (129) and susceptibility to opportunistic infection (109) at the wound site. oral mucosal wound healing is also known to be impeded by stress (84) . depressive symptoms also predict the rate of mucosal wound healing in healthy young adults (15) . the mechanisms through which stress produces inflammation are complex and bidirectional (stress can produce inflammation and inflammation can produce stress). these processes involve networks of communication including genetic, neural, endocrine and immune interactions. it is clear from both animal and human studies that stress affects the immune system in multiple ways. stress increases neuroendocrine hormones, such as glucocorticoids and catecholamines. through the activation of these hormones, stress has detrimental effects on immune functions, including reduction of lymphocyte populations, lymphocyte proliferation, natural killer cell activity and antibody production and the re-activation of latent viral infections (134) . the limbic-hypothalamic-pituitary-adrenal axis and the sympathetic nervous system are the major neural pathways activated by physical (i.e. pathogens or toxins) and psychological (i.e. major life events, abuse, or workor relationship-related factors) stressors (2, 13, 24) . early conceptualizations accepted a global immunosuppressive action of stress, but it is now clear that both immunosuppression and immune activation occur in various types of stress states (93) . chronic or repetitive stress, of the sort that is quite typical for those individuals with mental illness, seems to provoke a state of chronic inflammation through the activation of macrophages, dendritic cells, microglia, adipocytes and endothelium, which secrete cytokines. other effects include altered cell trafficking, natural killer cell cytotoxicity changes and alterations in the t-helper 1/t-helper 2 balance, all of which could contribute to the potential for poor immunoresponsiveness to microorganisms and vaccines, susceptibility to infections, re-activation of latent viruses and delays in wound healing (47) . the chronic inflammation occurs often in the presence of limbichypothalamic-pituitary-adrenal activation and secretion of high levels of normally immunosuppressive glucocorticoids (122) . chronic stress acts through a variety of molecular mechanisms to produce a state of inflammation. central to the production of this state is the release of proinflammatory cytokines responding to particular signals. cytokines are signaling proteins that transmit information between immune cells and also between the immune system and the brain and endocrine system. cytokines can act in autocrine, paracrine and endocrine manners to produce their effects (19) . the cytokines with greatest involvement in producing a stress-associated state of chronic inflammation include interleukin-1beta, tumor necrosis factoralpha and interleukin-6, a process that is mediated by nuclear factor-kappa b (14) . inflammation is normally a cell and tissue response to injury or infection, with safeguards to keep inflammation from becoming unchecked and chronic (18) . there are also systemic effects of inflammation, largely accomplished through the release of cytokines, that produce the well-known 'sickness behavior' of inflammation through actions of cytokines on the brain (33) . the normal pathways for proinflammatory secretion are through liganding of receptors on cytokinesecreting cells. these receptors include toll-like receptors, which respond to pathogen-associated molecular patterns as well as to danger-associated molecular patterns (10) . danger-associated molecular patterns are classified as 'alarmins' (i.e. molecules that signal cell damage) and include heat shock proteins, dna and many different products of injured tissue (e.g. high-mobility group protein b1, defensins, annexins and uric acid; 10). inflammation produced through the alarmin pathway is considered 'sterile' as it is not provoked by pathogens. in the stress associated with mental illness, the inflammation is of this latter sort. another important receptor-mediated pathway is through alpha-and beta-adrenergic receptors that bind norepinephrine, which may be increased in stress states through activation of the sympathoadrenal system (94) . liganding of these receptors initiates secretion of proinflammatory cytokines through several signaltransduction pathways, one involving nuclear factorkappa b and another involving mitogen-activated protein kinases. both pathways act to increase the expression of tumor necrosis factor-alpha and interleukin-1beta genes (11) . interleukin-6 production is stimulated in an autocrine manner by interleukin-1beta, and the major signal transduction factor is signal transducer and activator of transcription 3 (92) . opposing these pathways are the glucocorticoid receptors. glucocorticoid receptors are nuclear receptors that, when bound with cortisol, bind to glucocorticoid-response elements on the promotor regions of genes which code for proinflammatory (and other) molecules, inhibiting transcription (122) . in stress, there may be activation of the sympathoadrenal system and the limbic-hypothalamic-pituitaryadrenal axis, both of which may be involved in the production of the chronically inflamed state. adrenergic mechanisms may account for many of the short-term inflammatory effects of acute stress. chronic stress, on the other hand, is more likely to be associated with a low-grade state of inflammation (25, 108) . glucocorticoids inhibit the reproductive axis on multiple levels by reducing secretion of gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, testosterone, estradiol and progesterone. the latter inhibitory effects may contribute to sexual dimorphisms of stress-related behavioral syndromes and autoimmune inflammatory disorders (26) . neurologic and behavioral characteristics of the chronically stressed or depressed state can be explained, in part, through the cytokine effects of fatigue, decreased mobility, anhedonia and depression (81) . these states are characterized not only by higher plasma levels of proinflammatory cytokines, particularly interleukin-6, but also by higher than normal levels of acute-phase reactants, the synthesis of which is stimulated in the liver by the proinflammatory cytokines. these include c-reactive protein, fibrinogen and various adhesion molecules (108) . there is also evidence of limbic-hypothalamic-pituitary-adrenal axis activation and release of glucocorticoids in the face of ongoing inflammation (27) . the latter is thought to take place through several mechanisms. one concept is that glucocorticoid receptors become down-regulated or impaired and insensitive or resistant to the high levels of cortisol. without a damping mechanism through the glucocorticoid pathway, inflammation persists and exerts damage to vulnerable tissues (29) . it is also now established that a number of glucocorticoid receptor gene polymorphisms exist that could play a role in susceptibility to stressinduced inflammation and also that epigenetic mechanisms can alter the glucocorticoid receptor gene (92, 122) . genetic variability in the expression of inflammatory mediators in response to stress can also contribute to differences between individuals in systemic markers of inflammation and risk of morbidity and mortality (31) . for example, the presence of the guanine/cytosine single nucleotide polymorphism (rs1800795) in the promoter of the interleukin-6 gene, -174 bp upstream of the transcription start site, affects the binding of a beta-adrenergic-sensitive transcription factor, gata-1. following a beta-adrenergic stimulus, only the interleukin-6 -174g single nucleotide polymorphism leads to increased interleukin-6 production. in a study of spousal bereavement in older adults, schultze-florey et al. (116) found that the interleukin-6 -174 single nucleotide polymorphism moderated the effect of bereavement on the vulnerability of an individual to higher circulating levels of inflammation. moreover, epidemiologic analysis revealed an increased 10-year mortality risk associated with late-life depressive symptoms that occurred solely in homozygous carriers of the gata1sensitive g allele (31) . such studies underscore the importance of genetic variability in determining the biologic effects of environmental stressors. depression: is stress the tie that binds? the connection between stress and depression is also complex and bidirectional. evidence supports the hypothesis that psychosocial stress can lead to depression in susceptible individuals, which, in turn, can be aggravated by depression. in a recent meta-analysis, howren et al. (60) reviewed the associations between depression and the inflammatory markers c-reactive protein, interleukin-1 and interleukin-6 in circulating peripheral blood from community and clinical samples. all three inflammatory markers had a positive association with depression. the strongest association (based on clinical interviews) was found in clinically depressed patients, but the association was also significant in the community-based samples. depression was also found to be predictive of c-reactive protein and interleukin-6 levels in cardiac and cancer patients. interestingly, the relationship between depression and circulating levels of interleukin-6 became weaker with increasing age (60) . the latter finding may reflect, in part, age-related increases in circulating levels of interleukin-6, tumor necrosis factor-alpha and other inflammatory cytokines, even in healthy adults and in the absence of acute infection (123) . in a more recent meta-analysis, hiles et al. (59) also found a moderate and significant elevation in serum interleukin-6 levels in depressed compared with nondepressed groups, consistent with earlier systematic reviews (36, 59, 60, 78) . however, a high heterogeneity in the effect size was present among studies. elevations in interleukin-6 were larger in studies of patients with major depressive disorders and no known comorbid conditions. comorbidities associated with depression were found to reduce the effect size between depressed and nondepressed groups. body mass index, for example, was shown to be a moderating factor, as reflected in smaller, but significant, positive associations between depression and serum inflammatory markers. studies evaluating the effect of antidepressants on systemic markers of inflammation provide additional evidence linking depression to elevations in markers of systemic inflammation. in a meta-analysis of 22 studies, hannestad et al. (56) examined whether serum levels of tumor necrosis factor-alpha, interleukin-6 and interleukin-1beta change in response to therapy with commercially available antidepressants in patients with major depressive disorder. studies were included only if serum markers were obtained before and after treatment. the results revealed that treatment with specific serotonin re-uptake inhibitors reduced the symptoms of depression as well as the serum levels of interleukin-1beta and interleukin-6. a similar trend was found for levels of tumor necrosis factor-alpha in response to specific serotonin reuptake inhibitors. other antidepressants, including serotonin and norepinephrine re-uptake inhibitor and tricyclic antidepressants, reduced depressive symptoms but exerted minimal, if any, effects on serum markers of inflammation. the authors interpreted these findings to suggest that inflammatory cytokines can contribute to depressive symptoms and that antidepressants have the potenital to impede the effects of inflammatory cytokines on the brain (56) . collectively, these findings suggest that comorbidities, such as obesity and periodontitis, which are associated with elevations in systemic markers of inflammation, have the potential to modify the severity of depression. chronic inflammatory diseases, such as periodontitis, have a complex pathogenesis and a multifactorial etiology, involving complex host-parasite interactions (75) . (fig. 2 ) genetic variations in genes encoding the molecular components of the host immune defense (74, 139) , coupled with specific bacterial species in the subgingival plaque, set the stage for individual differences in risk for periodontitis. compared with women, men appear at greater risk for periodontitis (41, 121) . basic and clinical studies document the potential for comorbidities (e.g. diabetes) and co-infections (e.g. herpesviruses) to modify the initiation and/or progression of periodontitis (76, 124, 126) . cigarette smoking, poor nutrition, alcohol consumption and low socio-economic status also have been shown to be associated with a higher risk of periodontitis (132) . a substantial body of evidence indicates that psychological stress (107) and ineffective coping (44) can influence the onset and progression of many chronic diseases, including periodontitis (95) . early studies provided both anectoctal and clinical evidence of an association between psychosocial stress and acute necrotizing periodontal diseases, especially necrotizing ulcerative gingivitis (28, 118) , as well as periodontitis (43, 53, 83) . the nature of the association, however, was based largely on a temporal concordance between the 'stressful' event, gingival infection, and physiological measure of stress, such as urinary hydroxycorticosteriod. similarly, associations have been made between stress and depression and aggressive ('rapidly progressive') periodontitis based on psychosocial and clinical data obtained at the same time (34, 99) . therefore, chronic stress and depression have been hypothesized to reduce immune responsiveness, resulting in more pathogenic infection and concomitant periodontal tissue destruction. evidence also indicates that chronic stress and depression can mediate risk and progression of periodontitis through changes in health-related behaviors, such as oral hygiene, smoking and diet (3, 45) . although stress can adversely impact multiple health-related behaviors, including oral hygiene (32, 58) , there is strong evidence that stress plays a contributory role in the pathophysiology of periodontitis. peruzzo et al. (103) conducted a systematic review of the evidence on the influence of stress and psychological factors on periodontal disease. of the 14 studies (seven case-control, six cross-sectional and one prospective clinical trial) meeting inclusion criteria for the review, the majority (57%) reported a positive relationship between stress/psychological factors and periodontal disease. another 28.5% of the studies observed a positive relationship between some characteristics of stress and periodontal disease, demonstrating that the majority of the work published to date examining this relationship have indeed found significant associations (103) . in a recent study not included in the systematic review, stress scores and salivary stress markers (chromogranin a, cortisol, alpha-amylase and beta-endorphin) were shown to be significantly correlated with clinical parameters of periodontal disease in 100 adult patients with periodontitis (105) . salivary cortisol and beta-endorphin were significantly associated with tooth loss and periodontal clinical parameters, after adjusting for stress variables. moreover, the greatest tooth loss was observed in those patients who neglected to brush their teeth during stressful periods (105) . the results of these studies are consistent with the hypothesis that stress can modify the host defense and progression of periodontal infections in patients susceptible to periodontitis (37) . depression has also been linked to periodontitis in clinical studies in adolescents/young adults (35, 79) and adults (1, 44, 65, 66, 89, 90, 110, 115) . in a casecontrol study, moss et al. (90) found that smoking and elevated antibody titers to tannerella forsythia at baseline were associated with more severe periodontal breakdown in patients scoring high on a depression inventory. in a cross-sectional investigation, patients with rapidly progressive periondontitis showed significantly higher scores on measures of depression and loneliness compared with adults with either chronic periodontitis or without significant clinical attachment loss (89) . genco et al. (44) found that stress and distress manifested as depression was a significant risk indicator for more severe periodontitis in a cross-sectional study of 1,426 subjects between 25 and 74 years of age. johannsen et al. (65) found that women on long-term sick leave for depression had more severe periodontitis and higher concentrations of interleukin-6 in gingival crevicular fluid compared with healthy controls. these clinical results are consistent with findings in an established animal model of depression. breivik et al. (16) tested whether depression induced by olfactory bulbectomy in rats and treatment with the antidepressant serotonin re-uptake enhancer, tianeptine, could influence susceptibility to ligature-induced periodontitis. when compared with sham-operated controls, olfactory bulbectomy rats were shown to develop significantly more periodontal bone loss. tianeptine treatment significantly inhibited alveolar bone loss. however, a clomipramine-induced model of depression in lewis rats did not alter the pathogenesis of ligature-induced periodontitis (127) . susceptibility to periodontal breakdown in response to stressful life events appears to depend, in part, on the effectiveness of a person's coping behavior (61, 135) . in a cross-sectional investigation, genco et al. (44) examined the relationship of periodontal disease and stress, distress and coping behaviors in a large population-based adult sample (44) . this study revealed that psychosocial measures of stress (financial strain) and distress, manifest as depression, were significant risk indicators of periodontal disease severity in adults, after adjusting for gender (male), smoking, diabetes mellitus, t. forsythia and porphyromonas gingivalis (44) . several clinical studies have failed to demonstrate an association between measures of depression and periodontal disease in community-based samples (5, 21, 102, 128) . persson et al. (102) , for example, did not find an association between depression and radiographic bone loss or periodontal probing depths (5 mm or greater) in a sample of 701 older adults. however, depression (self-reported or based on a psychometric score) was associated with a history of greater tooth loss, which may reflect greater susceptibility to periodontal destruction during periods of depression. anttila et al. (5) reported a positive correlation between depression and edentulousness in a sample of 780 finnish residents. it is noteworthy that depression often manifests with multiple comorbidities, including obesity and diabetes, many of which are independently associated with elevations in systemic markers of inflammation (102) . in general, studies exploring the association between depressive symptoms and periodontitis have been based on nonpsychiatric populations without consideration of comorbidities, limiting the potential of these studies to identify an association between depression and periodontal disease. studies of serum and salivary stress-related steriods provide further evidence of an association between stress, depression, and periodontal disease. in a cross-sectional study, rosania et al. (111) asked patients with periodontitis to complete psychometric tests and questionaires as well as to undergo clinical examinations and measurement of salivary cortisol levels (111) . stress, depression and salivary cortisol scores were found to correlate signficantly with severity of periodontitis and the number of missing teeth, when controlling for age, family history and brushing frequency. moreover, patients who reported neglecting their oral care during stressful or depressed periods exhibited the greatest clinical attachment loss and highest number of missing teeth. in a similar study of 111 dentate patients, ≥40 years of age, mannem & chava (82) reported that stress scores and salivary cortisol levels significantly discriminated between patients with and without periodontitis. similar associations have also been reported between serum levels of the stress-related steroids, such as cortisol and dehydroepiandrosterone-sulfate, and measures of periodontal disease (52, 63) . patients with periodontitis often have higher systemic levels of c-reactive protein, interleukin-6, interleukin-1 and tumor necrosis factor-alpha (104) . the mechanisms underlying the relationship of psychlogical stress and depression with periodontitis probably involve a combination of factors related to alterations in behavior and neuroimmunologic function. experimental animal models provide additional evidence that stress adversely affects immune defense and susceptibility to oral infection. bailey et al. (8) found that social stress enhances the production of interleukin-1beta and tumor necrosis factor-alpha in cd11b + spleen cells in response to p. gingivalis lipopolysaccharide (8) . the latter finding suggests that stress can enhance the responsiveness and production of inflammatory cytokines by macrophages in response to an oral pathogen. macrophages from the spleens of mice exposed to this same stressor were shown to express significantly higher levels of both toll-like receptor-2 and toll-like receptor-4 (7). breivik et al. (16) also found that injection of lipopolysaccharide provoked a significant increase in the circulating levels of corticosterone and alterations in cytokine levels, consistent with dysregulation of the immune system, in an animal model of depression. psychological stress has been also shown to exacerbate oral infection with the opportunistic pathogen, candida albicans, in rodents (97, 98) . finally, stress and depression have been shown to exert a negative effect on treatment outcomes in patients with periodontitis. elter et al. (42) examined whether depression was predictive of the proportion of residual probing periodontal sites (5 mm or greater) and tooth loss between the initial and 1-year post-treatment examinations in 697 patients enrolled in a health-maintenance organization. a diagnosis of depression was documented from medical records in 12.2% of the patients. depression was significantly associated with both poorer periodontal treatment outcome and tooth loss during the 1-year follow-up period. kamma & baehni (67) found that scores on a stress inventory predicted future clinical attachment loss in patients with aggressive periodontitis in supportive periodontal care over a period of 5 years (67). linden et al. (77) found that a measure of occupational stress was a significant predictor of clinical attachment loss in 23 patients with periodontitis in regular periodontal maintenance over a period of nearly 6 years. these findings parallel other reports (112) indicating that psychosocial factors play a significant role in recovery from surgery and are predictive of surgical outcome. an extensive body of experimental and clinical evidence documents the negative impact of chronic psychological stress and depression on the immune system and health. brief stressors appear to suppress cellular immunity whilst preserving measures of humoral immunity; in contrast, chronic stressors generally result in dysregulation of the immune system, involving both cellular and humoral pathways (117) . therefore, chronic stress and depression have been hypothesized to reduce immune responsiveness, resulting in a higher rate of infection with pathogenic organisms and a greater degree of periodontal tissue destruction. in general, the evidence is consistent with the hypothesis that stress can modify the host immune defense and permit the progression of periodontal infections in patients susceptible to periodontitis (37) . however, substantial evidence also indicates that these conditions can mediate risk for disease, including periodontitis, through changes in healthrelated behaviors, such as oral hygiene, smoking and diet (3, 45) . stress and depression are commonly associated with comorbidities, such as diabetes (96) , that can modify the onset and progression of periodontal disease; however, these conditions have generally not been addressed in available studies. in addition, stress and depression appear to fall into a spectrum, ranging from mild to severe, involving a complex interaction of genetic background, coping strategies and environment. differences in the conceptualization of stress and depression are probably important in assessing associations with other biologic and 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inflammatory pattern recognition receptors and their ligands: factors contributing to the pathogenesis of preeclampsia date: 2011-03-06 journal: inflamm res doi: 10.1007/s00011-011-0319-4 sha: doc_id: 5842 cord_uid: mm3ab7cr problem: preeclampsia, a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. preeclampsia lacks effective therapies because of the poor understanding of disease pathogenesis. the aim of this paper is to review molecular signaling pathways that could be responsible for the pathogenesis of preeclampsia. method of study: this article reviews the english-language literature for pathogenesis and pathophysiological mechanisms of preeclampsia based on genome-wide gene expression profiling and proteomic studies. results: we show that the expression of the genes and proteins involved in response to stress, host-pathogen interactions, immune system, inflammation, lipid metabolism, carbohydrate metabolism, growth and tissue remodeling was increased in preeclampsia. several significant common pathways observed in preeclampsia overlap the datasets identified in tlr (toll-like receptor)and rage (receptor for advanced glycation end products)-dependent signaling pathways. placental oxidative stress and subsequent chronic inflammation are considered to be major contributors to the development of preeclampsia. conclusion: this review summarizes recent advances in tlrand rage-mediated signaling and the target molecules, and provides new insights into the pathogenesis of preeclampsia. preeclampsia is the de novo occurrence of pregnancyspecific hypertension and proteinuria after 20 weeks of gestation. it is one of the leading causes of maternal, fetal, and neonatal mortality worldwide. the integration of microarrays and sophisticated bioinformatics can provide specific and efficient high-throughput screening with which to investigate the pathogenesis of preeclampsia. although the mechanisms have been elusive, new physiologic insights have begun to alter thinking about preeclampsia [1] . angiogenic factors, including vascular endothelial growth factor (vegf), have been proposed to play a key role in the maintenance of endothelial cell function [2] [3] [4] [5] . soluble fms-like tyrosine kinase-1 (sflt-1) and soluble endoglin (seng) are endogenous anti-angiogenic proteins that neutralize the pro-angiogenic proteins vegf and placental growth factor (plgf). maternal endothelial dysfunction, possibly related to these circulating factors, is a key feature of this syndrome. additional factors that may contribute to the pathogenesis of preeclampsia include angiotensin-ii receptor autoantibody, infectious ligands, advanced glycation end products (ages), toll-like receptors (tlrs) and receptors for ages (rage) [1, 6] . it is now increasingly being recognized that abnormal placentation and systemic inflammation represent hallmarks of preeclampsia and infectious agents might increase the risk of this disorder [6] . invading exogenous pathogens [also known as pathogen-associated molecular pattern (pamp) molecules] influence immune cell recruitment and pro-inflammatory cytokine secretion. in addition, cellular stress, damage, and inflammation cause release of endogenous damage-associated molecular pattern (damp) molecules or endogenous danger signals ''alarmins'' [7] . the complex bidirectional interplay between angiogenesis and immunity/inflammation can establish a ''vicious cycle'' that might contribute to the pathogenesis of preeclampsia. some parts of the puzzle surrounding pro-inflammatory cytokines and growth factors and their effects on alarmins have begun to unravel. alarmins activate the repair process through their interaction with pattern recognition receptors (prrs) such as tlrs. a recent study has shown a crucial link between the development of preeclampsia and defense against both exogenous ligands (foreign pathogens) and endogenously generated alarmins [6] . women with preeclampsia had increased expressions of tlr2 and tlr4 mrna and protein, a downstream transcription factor nuclear factor (nf)-kappab, and interleukin (il)-1beta mrna compared with control groups [8] . thus, tlrs may modulate the innate immune response and appear to be factors contributing to the pathogenesis of preeclampsia [8] . this review focuses on the following six aspects of this emerging field of preeclampsia: an overview of genomewide gene expression profiling, an overview of microarray analysis and proteomic studies, a history and the molecular basis of promising biomarkers, an overview of specific functions of angiogenic/anti-angiogenic factors, the molecular basis of inflammatory pattern recognition receptors and their ligands, and investigation of tlrs and rage and their ligands. the present article reviews the literature on biological, pathogenetic and pathophysiological studies on preeclampsia. for studies that reported data on obstetric disorders, only data pertaining to preeclampsia were included. a computerized literature search was performed to identify relevant studies reported in the english language. medline updates were conducted monthly, and all abstracts were reviewed by two investigators to identify papers for full-text review. we searched pubmed medline electronic databases (http://www. ncbi.nlm.nih.gov/sites/entrez) for a 10-year period (2000-2010) , combining the keywords ''gene expression profiling'', ''proteomic'', ''pathogenesis'', ''pathophysiology'', ''toll-like receptor'', ''advanced glycation end products'', ''receptor for advanced glycation end products'', ''inflammation'', ''stress'', or ''alarmins'' with ''preeclampsia''. each gene is also linked to ncbi entrez gene pages (http://www.ncbi.nlm.nih.gov/sites/entrez). additionally, references in each article were searched to identify potentially missed studies for a 20-year period (1990-2010) . target publications are mainly reports on human preeclampsia and mouse models, as well as studies in gene and protein expression systems. a priori, case reports, and abstracts were not included, since abstracts do not undergo a stringent peer-review process. a difficulty in interpreting the literature is that analyses, results and objects (early onset or late onset) are reported differently among the studies. here, we discuss promising molecular candidates and possible signaling targets for preeclampsia. as the main interest is preeclampsia obtained from human samples, we have not yet included animal preeclampsia models alone in the database. however, we included animal studies performed to support clinical data. initially, 321 potentially relevant studies were identified by screening electronic databases, and 135 peer-reviewed journal articles were additionally identified from references in each article. the network consists of 92 entities by genome-wide gene expression profiling and proteomic analyses and seven reaction categories by gene ontology analyses. these entities were classified into renin-angiotensin system, angiogenesis, inflammation and cytokines, stress and detoxification, metabolism, adhesion, cell structure, signal, and protease. several studies based on genome-wide gene expression profiling analysis and proteomic technology have clarified the specific genes and proteins involved in preeclampsia. we will initially focus on the biology, pathogenesis, and pathophysiology of this disorder. researchers have investigated candidate markers in samples, including placenta, amniotic membranes, blood, amniotic fluid, and peripheral blood mononuclear cells in severe preeclampsia. there are specific genes that may be of importance for the pathological and pathophysiological changes seen in preeclampsia. several genes preferentially up-regulated (n = 75) or down-regulated (n = 17) in preeclampsia have been identified (table 1) . these genes have been grouped in clusters or pathways according to their possible influence on certain characteristics of preeclampsia. computational bioinformatic analysis revealed a set of new candidate genes, and highlighted several new pathway systems, such as the vascular endothelial growth factor (vegf)-and transforming growth factor (tgf)-beta-related pathways and renin-angiotensin system [9] . preeclamptic placenta is relatively ischemic and produces a variety of factors that might generate profound effects on vascular endothelial cell function [10] . these factors include the soluble vegf receptor-1 (vegf-ri, also known as flt-1), the angiotensin ii type-1 receptor autoantibody, and pro-inflammatory cytokines such as tumor necrosis factor (tnf)-alpha and interleukin (il)-6. the angiogenic activity of vegf is mainly mediated by vegf receptor i (flt-1) and vegf receptor ii [vegf-rii; also known as flk-1 (fetal liver kinase 1)/kdr (kinase domain region)]. vegf expression is primarily regulated by hypoxia [11] . ischemic placenta also generates sflt-1, possibly via hypoxia-inducible factor signaling. circulating mononuclear cells also represent an additional source for circulating sflt-1 during normal and preeclamptic pregnancies [12] . a recent study has shown decreased production of vegf by circulating t and natural killer cells in preeclampsia [13] . in the sera of pregnant women others killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 (kir3dl2), churchill domain containing 1 (churc1), nuclear receptor subfamily 4, group a, member 2 (nr4a2) [81] rages, their ligands, and preeclampsia 511 with preeclampsia, there was a significant reduction in the levels of free vegf and placental growth factor (plgf), whereas the levels of sflt-1 were significantly higher than those in the sera of normotensive pregnant women [14] . sflt-1 acts as a negative modulator for the bioavailability of vegf [15] . these soluble receptors may bind and neutralize vegf in the maternal circulation, causing endothelial dysfunction [14] . sflt-1 also induces endothelial cell apoptosis, and decreases nitric oxide (no) generation in vitro [16] . furthermore, soluble endoglin (seng) is another circulating anti-angiogenic protein and may contribute to the pathogenesis of preeclampsia. endoglin is a transmembrane receptor for tgf-beta that is expressed on proliferating endothelial cells. seng was found to synergize with a sflt-1 and induce endothelial cell dysfunction. tgfbeta stimulates production of vegf and plasminogen activator inhibitor type-1 (pai-1), that are involved in vascular remodeling [17] . thus, decreased free vegf and tgf-beta activities might contribute to the pathogenesis of preeclampsia [14] . animal experiments demonstrated that co-administration of sflt-1 and seng to pregnant rats elicited severe preeclampsia-like symptoms [18] . bioinformatic analysis has highlighted the renin-angiotensin pathway [9] . angiotensin ii levels in the placenta are higher in preeclamptic subjects when compared with control pregnant women [19] . angiotensin ii induces gene expression of tgf-beta and matrix metalloproteinase-9 (mmp-9). in addition to elevated angiotensin ii levels, angiotensinogen and angiotensin i receptor mrnas were also increased in preeclamptic placenta [19] . furthermore, immunoglobulin (ig) g autoantibody in the serum of preeclamptic women stimulates the angiotensin ii type 1 receptor [20] . angiotensin ii type 1 receptor autoantibody can also induce reactive oxygen species (ros) generation, mediated by nicotinamide adenine dinucleotide phosphate (nadph) oxidase [19] . ros-induced oxidative stress appears to be both a cause and a consequence of hypertension. this autoantibody also stimulates trophoblast sflt-1 production [21, 22] . inflammation, cytokines, stress and detoxification system vegf-dependent anti-inflammation and anti-oxidation have been identified, adding a further layer of complexity to the vegf system [2, [23] [24] [25] [26] . the previous studies showed that acute infusions of vegf cause vasodilation and hypotension [3, 24] . vegf plays a critical role in blood pressure control and in optimizing blood flow, vascular integrity, and tone by promoting no activity [2] through vegfr2-dependent up-regulation of endothelial no synthase (enos) expression [25] . no has multiple functions in the vasculature, including vasodilatory, antiinflammatory and anti-thrombotic activities [26] . no also prevents proliferation of excess endothelial cells and vascular smooth muscle cells, as well as macrophage adhesion and infiltration [26] . collectively, these studies suggest a protective role for vegf in the development of preeclampsia, possibly with anti-inflammatory, anti-oxidant and anti-proliferative effects in the vasculature, which may contribute to the prevention of this hypertensive condition. considerable evidence implicates the ischemic placenta during early pregnancy, altered proangiogenic and antiangiogenic factor balance, oxidative stress and endothelial dysfunction in the pathophysiology of preeclampsia [18] . increased expression of some stress-related genes such as heme oxygenase-1 (hmox1) also supports the role of excessive oxidative stress and maternal inflammatory response [27] . thus, abnormal cytokine responses and oxidative stress during pregnancy might play a central role in the excessive systemic inflammatory response, as well as in the generalized endothelial dysfunction characteristics of this disorder. furthermore, the production of type 1 pro-inflammatory cytokines is dominant in patients with preeclampsia or in individuals with a predisposition to subsequent development of preeclampsia [28] . the production of reactive oxygen intermediates in endothelial cells is induced by th1 cytokines such as tnf-alpha and il-6. chronic subclinical oxidative stresses may also increase maternal and fetal proinflammatory cytokines to levels high enough to affect vascular endothelial function. it has recently been reported that healthy pregnant women showed a decrease in il-17producing cells, whereas a relative increase in il-17-producing cells and low t-regulatory cell numbers were observed in preeclamptic women, suggesting that the pathophysiology of preeclampsia might be associated with imbalanced differentiation of t-regulatory cells and th17 cells [29, 30] . therefore, systematic immunoactivation might be linked to immuno-maladaptation, chronic inflammation, poor angiogenesis, and endothelial cell dysfunction. a recent study showed that impairment of no action promotes the genesis of cardiovascular diseases [31] . the synthesis of no is blocked by inhibition of the nos active site with guanidino-substituted analogues of l-arginine, such as asymmetric dimethylarginine (adma) [29] . therefore, adma acts as an endogenous nos inhibitor [32, 33] . the administration of adma in rats causes an increase in renal vascular resistance and blood pressure [34] , confirming its biological action in vivo. interestingly, a more recent report showed that maternal concentrations of adma are higher in mid-pregnancy in women who experience preeclampsia, compared with women with uncomplicated pregnancies [35] . investigators have studied other promising markers that have been identified up to now, including placental protein 13 (pp13), p-selectin, a disintegrin and metalloprotease-12 (adam12), pentraxin 3 (ptx3), pregnancy-associated plasma protein a (papp-a), and adrenomedullin [36, 37] . these markers appear to be promising tools, most likely in a combinatorial analysis, to diagnose, predict outcome, and monitor this disorder [36, 37] . up-regulation of metabolism-related gene expression such as leptin has been found in several studies [38] . these genes and proteins may be connected to atherosclerosis, diabetes, and alzheimer's disease. in addition, sugathadasa et al. [39] showed that preeclampsia appears to be associated with the aa genotype of -2548 g/a polymorphism of the leptin gene. genetic factors such as leptin gene polymorphism may increase the susceptibility of pregnant women to develop preeclampsia. adhesion system vegf is a potent angiogenic and pro-inflammatory cytokine that increases vascular permeability, angiogenesis, vasculogenesis, endothelial cell growth, migration, adhesion, and anti-apoptosis, partly via upregulation of endothelial cell and leukocyte adhesion molecule expression [40] . in contrast to these descriptions of vegf's inflammatory actions, several reports describe vegf neutralization leading to elevated expression of surface adhesion molecules, resulting in increased leukocyte rolling and adhesion [41] . neutralization of vegf action also results in endothelial surface expression of p-selectin and impaired peripheral vasodilation [41] . depending on the local concentrations of vegf, striking differences in biological functions were found. we next determined functional categories of coregulated genes and gene pathways. hypotheses about common regulatory elements or their functional significance were formulated. genes were classified into nine functional categories according to their expression profile: reninangiotensin system, angiogenesis, inflammation and cytokines, stress and detoxification, metabolism, adhesion, cell structure, signal transduction, and protease, which are consistent with the oxidative stress of preeclampsia (table 1 and fig. 1 , genome-wide gene expression profiling and proteomics). gene ontology analysis revealed several biological processes which could be associated with the development of preeclampsia [9, 42, 43] (fig. 1 , gene ontology analysis). a logical and systematic data analysis strategy demonstrated that genes and proteins that participated in response to stress, host-pathogen interactions, immune system, inflammation, lipid metabolism, carbohydrate metabolism, growth, and tissue remodeling pathways were expressed differentially in preeclampsia. the features of the microarray and proteomic data sets, categorized according to gene ontology, indicate that preeclampsia is characterized as ''stress'' and ''immune/ inflammatory'' responses. collectively, preeclampsia is considered to be a disease of at least two stages: the first (subclinical) stage concerns the failure of the proposed sequence of events comprising early trophoblast invasion (table 1 , angiogenesis and protease pathways), remodeling ( table 1 , signal pathway), and consequential placental perfusion (table 1 , inflammation and cytokines pathway), which are believed to play a major role in this disorder through the production of angiogenic factors and immunoregulatory cytokines via oxidative stress (table 1 , stress and detoxification pathway). preeclampsia in particular has gene-expression signatures associated with imbalance of oxidative stress generation and detoxification pathways. in a suggested hypothetical event, the link between angiogenesis and cytokine could be provided through oxidative stress by excessive production of ros. taken together, placental hypoxia and reoxygenation might induce oxidative stress, which stimulates placental synthesis of cytokines, maternal leukocyte activation, leukocyte/endothelial interaction, immune response regulation and generalized endothelial dysfunction, thereby completing a vicious cycle [44] . these data allow us to speculate that the first subclinical stage has a ''stress-resistant'' phenotype. the second (clinical) stage is the overt maternal syndrome, which is characterized by a generalized systemic inflammatory response (table 1 , inflammation and cytokine pathway) and subsequent generalized endothelial dysfunction involving both leukocytes and endothelial activation (table 1 , adhesion, metabolism and cell structure pathways). although endothelial cells can cope with a rise in oxidative stress and sflt-1, the inability to retrieve endothelial function might be a result of oxidative stress imbalance. maternal inflammatory cells and endothelial cells persistently stimulate the release of pro-inflammatory cytokines, e.g. tnfalpha [45] . such environmental stress (the ''stress-sensitive'' condition) implicitly alters epigenetic and genetic patterns that can lead to the clinical manifestations of preeclampsia. an important role in the second stage is played by endothelial dysfunction, manifested by an imbalance of stress and antistress homeostasis. cutting-edge proteomic technologies will be of great help in understanding the heterogeneity of preeclampsia as shown in table 1 and fig. 1 . the promising biochemical markers have been developed to evaluate the features of placental dysfunction, inflammatory response, or endothelial dysfunction. potential markers can be classified as ''prediction'' or ''detection'' of preeclampsia. since most of these preeclampsia-specific mediators are increasingly expressed in many different ''stress'' and ''immune/ inflammatory response'' processes, the following crucial molecules might also be important for the understanding of agtr1a angiotensin ii receptor, type 1a; angii angiotensin ii; at1r angiotensin ii receptor, type 1; vegf vascular endothelial growth factor; sflt-1 soluble fms-like tyrosine kinase-1; seng soluble endoglin; nox4 nicotinamide adenine dinucleotide phosphate (nadph) oxidase 4; osm oncostatin m; il-9 interleukin-9; irak3 interleukin-1 receptor-associated kinase 3; csf1r colony stimulating factor 1 receptor; p450 cytochrome p450; cyp11a cytochrome p450, family 11, subfamily a, polypeptide 1; cyp26a1 cytochrome p450, family 26, subfamily a, polypeptide 1; hmox1 heme oxygenase (decycling) 1; ephx2 epoxide hydrolase 2, cytoplasmic; lep leptin; smox spermine oxidase; ldha lactate dehydrogenase a; gmfb glia maturation factor, beta; gsk3b glycogen synthase kinase 3 beta; tgm2 transglutaminase 2 (c polypeptide, protein-glutamine-gamma-glutamyltransferase), cdo1 cysteine dioxygenase, type i; insl4 insulin-like 4; pmm2 phosphomannomutase 2; cldn6 claudin 6; ctnnb1 catenin (cadherinassociated protein), beta; cdh5 cadherin 5, type 2; vtn vitronectin; pcdha3 protocadherin alpha 3; selp selectin p; ncam1 neural cell adhesion molecule 1; ttn transthyretin; actn4 actinin, alpha 4; fbln1 fibulin 1; flnb filamin b, beta; tln1 talin 1; dag1 dystroglycan 1; fn1 fibronectin 1; add1 adducin 1; itga2 integrin, alpha 2; mapk1 mitogen-activated protein kinase 1; mapk4 mitogen-activated protein kinase 4; ptpn1 protein tyrosine phosphatase, non-receptor type; ptpn6 protein tyrosine phosphatase, nonreceptor type 6; ptpn11 protein tyrosine phosphatase, non-receptor type 11; ptpn12 protein tyrosine phosphatase, non-receptor type 12; shc1 shc (src homology 2 domain containing) transforming protein 1; smad4 smad family member 4; grb2 growth factor receptorbound protein 2; prkcz protein kinase c, zeta; ptk2 protein tyrosine kinase 2; src v-src sarcoma (schmidt-ruppin a-2) viral oncogene homolog; vav2 vav 2 guanine nucleotide exchange factor; vgll1 vestigial like 1; procr protein c receptor; casp10 caspase-10; dr-3 death receptor 3; rgs5 regulator of g-protein signaling; ccnb1 cyclin b1; aqpep laeverin; adam 12 a disintegrin and metalloproteinase 12, meltrin-alpha; slpi secretory leukocyte peptidase inhibitor the pathogenesis of preeclampsia: besides vegf, they include tlrs, rage and rage ligands. they are associated with regulatory pathways of inflammation and oxidative stress, along with a number of innate immune systems. the link between ''stress'' and ''immune/inflammatory response'' is further supported by the increased levels of inflammatory pattern recognition receptors (prrs) and their ligands. the relevance of these issues is discussed below. the present review has proposed that hypoxia-induced inflammatory genes and stress-related factors are upstream regulators of preeclampsia [46] . stress and inflammation are processes by which cells or tissues respond to various insults or stimuli, microbes, and tissue breakdown products. it is characterized by up-regulation of prrs that sense exogenous (infectious) and endogenous ligands. two inflammatory prrs, toll-like receptors (tlrs) and rage, interact with a wide range of common and different ligands, and play a major role in the maternal immune and inflammatory system [47] . there is strong evidence that maternal immune system activation contributes to the development of preeclampsia [6, 8] . tlrs recognize infectious agents (foreign pathogens, exogenous ligands) and other danger signals [endogenously generated inflammatory ligands, also known as alarmins (see above)]. tlr-dependent innate immunity plays a role as the first line of host defense. activation of tlrs on trophoblast cells influences immune cell recruitment, proinflammatory cytokine secretion, and decidual responses to invading pathogens during pregnancy [48] . importantly, recent data directly implicate signaling by tlrs at multiple maternal-fetal interfaces in the pathogenesis of several pregnancy pathologies, including preeclampsia, intrauterine growth restriction, and preterm labor [48] . although the participation of the innate immune responses in sustaining the inflammation needs further investigation, preeclampsia is characterized as being a state of persistent inflammation. animal models for preeclampsia are furthering our understanding of pathophysiology: tlr3 responds to double strand (ds) rna from viruses, apoptotic cells and necrotic cells. persistent tlr3 activation during pregnancy causes hypertension, endothelial dysfunction, proteinuria, and intrauterine growth restriction in normal pregnant rats [49] . tlr stimulation of trophoblast cells induces inflammatory mediator production and, in particular, secretion of antiangiogenic factor sflt-1 [50] . exogenous ligand lipopolysaccharide (lps) itself can also induce sflt-1 expression in macrophages through protein kinase c signaling [51, 52] . persistent activation of tlrs might therefore be associated with the development of preeclampsia. a growing number of defined single nucleotide polymorphisms (snps) of tlrs have been described that genetically determine susceptibility to infection and inflammation. recently, van rijn et al. [53] observed an association of common tlr4 (d299g and t399i) and nucleotide binding and oligomerization domain (nod) (r702w, g908r, and l1007fs) gene variants, and proinflammatory phenotype with a history of preeclampsia. these findings suggest direct involvement of the maternal innate immune system in this syndrome [53] . on the other hand, functional snps of the tlr2 (g2258a) failed to present any significant association with preeclampsia [54] . the impact of snps of the tlr molecules therefore remains controversial. another inflammatory signaling receptor, rage, interacts with a wide range of common endogenous ligands [47] . rage is an immunoglobulin-like cell surface receptor that is described as a prr because of the structural heterogeneity of its ligands (fig. 2) . defined tlr ligands are mainly derived from pathogens, but rage ligands do not originate from pathogens [47] . the nonenzymatic glycation and oxidation of proteins and lipids lead to the formation of a broad range of species, called ''advanced glycation end fig. 2 the age-rage system at the maternal-fetal interface in preeclampsia. rages are pattern recognition receptors. ages are proteins, lipids and polynucleotides, including n-carboxymethyllysine, pentosidine, and methylglyoxal derivatives, and are the major ligands for rages. several proteins are known to act as age receptors. nf-kb nuclear factor-kappab, sr-a scavenger receptor 1, sr-bi scavenger receptor bi, rage receptor for advanced glycosylation end products, lox-1 oxidized low density lipoprotein (lectinlike) receptor 1; feel-1 stabilin 1, cd36 thrombospondin receptor, s100 s100 calcium binding protein a1, hmgb1 high-mobility group box 1, ttr transthyretin, ages advanced glycosylation end products rages, their ligands, and preeclampsia 515 products'' (ages) [55] . chronic inflammation modifies protein and lipid, leading to production of ages. ages are proteins, lipids, and polynucleotides, including n-carboxymethyl-lysine, pentosidine, and methylglyoxal derivatives [47] (fig. 2) . ages are known to cause oxidative damage in various cells via rage signaling. not only rage but also lox-1 [oxidized low density lipoprotein (lectin-like) receptor 1], galectin-3 (ectin, galactoside-binding, soluble, 3), sr-a (macrophage scavenger receptor 1), cd36 (thrombospondin receptor), sr-bi (scavenger receptor class b, member 1), feel-1 (stabilin 1) and feel-2 (stabilin 2) are known to act as age receptors (fig. 2) . in addition to ages, rage binds certain members of the s100/calgranulin family (such as s100a12 and s100b), high-mobility group box 1 (hmgb1), transthyretin (ttr), mac-1, and amphoterin [56] . rage is expressed on multiple cell types, such as endothelial cells, smooth muscle cells, monocytes/macrophages, t lymphocytes, dendritic cells, glomerular epithelial cells, podocytes, cardiomyocytes, neurons of the central and peripheral nervous systems, and transformed cells [56] . rage activates nicotinamide adenine dinucleotide phosphate reduced form (nadph) oxidase, an important source of oxidative stress, and generates ros [56] . the transcription factor nf-kappab is a downstream target of rage-mediated cellular activation. nf-kappab activation also leads to increased rage expression. thus, the age-rage system, which is up-regulated in preeclampsia, is likely to be involved in ros-induced oxidative stress and might contribute to vascular dysfunction in preeclampsia [57, 58] . accumulation of markers of oxidative stress such as 4-hydroxy-2-nonenal and 8-hydroxy-2 0 -deoxyguanosine indicated enhanced oxidative modifications of lipids and dna in preeclamptic placenta [57] . cellular stress might result in release of the pro-inflammatory rage ligands s100, hmgb1, and transthyretin (ttr) [56] . taken together, the hypoxiaoxidative stress-persistent inflammation-dependent mechanism during pregnancy may be the triggering stimulus to draw rage into preeclampsia pathology. other age receptors lox-1: oxidized low density lipoprotein (lectin-like) receptor 1 (olr1) lox-1 binds, internalizes and degrades oxidized low-density lipoprotein (oxldl) [59] . this protein may play a role as a scavenger receptor. investigators have shown overexpression of lox-1 and arginase, which contribute to endothelial dysfunction and oxidative stress, in the vasculature of women with preeclampsia [59, 60] . increased arginase expression could contribute to decreased no and enhanced superoxide formation [59] . lox-1 also generates superoxide via nadph oxidase. galectin-3: lectin, galactoside-binding, soluble, 3 (lgals3) members of this protein family have an affinity for beta-galactosides. several findings suggest that the expression of galectin-3 on the extravillous trophoblast is up-regulated in preeclamptic placenta [61] . galectin-3 was up-regulated under hypoxia [62] . ligands for galectin-3 include not only ages but also other proteins such as laminin, fibronectin, tenascin, integrins, cd98 (solute carrier family 7), cytokeratins, bcl-2, and alix/aip-1 (programmed cell death 6 interacting protein) [63] . galectin-3 is therefore involved in the inhibition of apoptosis [63] . sr-a: macrophage scavenger receptor 1 (msr1) and sr-bi: scavenger receptor class b, member 1 (scarb1) these proteins mediate the endocytosis of oxldl and play a role as macrophage scavenger receptors. ages induce the gene expression of these oxldl receptors [64] . these receptors have been implicated in many macrophageassociated physiological and pathological processes including atherosclerosis, alzheimer's disease, and host defense. human extravillous cytotrophoblast cells also express oxldl receptors [65] . the pathogenesis of preeclampsia appears to overlap with those of atherosclerosis, diabetes, and alzheimer's disease. cd36: thrombospondin receptor, also known as fatty acid translocase (transporter) cd36 binds to thrombospondin, collagen, anionic phospholipids, and oxldl. this protein has an important function as a cell adhesion molecule and scavenger receptor. gene expression of cd36 was decreased in placental tissues from pregnancies complicated by preeclampsia. how cd36 is associated with preeclampsia remains unclear. feel-1: stabilin 1 (stab 1) and feel-2: stabilin 2 (stab 2) these receptors have been shown to endocytose ligands such as oxldl, gram-positive and gram-negative bacteria, and ages. these molecules may function in angiogenesis, lymphocyte homing, cell adhesion, or receptor scavenging. however, the exact role of feel-1 in this disorder and how it mediates stress and inflammation remain unclear. advanced glycation end products (ages) ages are adducts formed by glycoxidation that accumulate in metabolic disorders such as diabetes and atherosclerosis [66] . interaction of aldoses with proteins initiates a chain of nonenzymatic reactions leading to the covalent addition of ages to proteins, known as the maillard reaction. attention will be focused on the vascular endothelial cells-age interaction. ages are heterogeneous in structure, which implies that many products could be measured to estimate formation of ages. of them, pentosidine and carboxymethyl-lysine have been the best characterized [67] . it has been established that ages are directly implicated in the development of chronic complications in diabetes [68] . similar to diabetes, the mean level of serum ages in preeclamptic women was higher than those in healthy nonpregnant women or healthy pregnant women [57] . tsukahara et al. [69] showed that umbilical blood concentrations of pentosidine are high in the maternal preeclamptic condition. thus, preeclampsia is thought to be complicated with formation and production of ages. these data allow us to speculate that ages participate in the development of preeclamptic complications. high-mobility group box 1 (hmgb1) rage was the first receptor identified for extracellular hmgb1. hmgb1 acts as a danger signal ''alarmin'' to alert the innate immune system for the initiation of host defense or tissue repair [70] . this ligand recognizes not only rage but also tlr2 and tlr4, leading to production of pro-inflammatory cytokines via the nf-kappab signaling cascade. hmgb1 possesses important extranuclear functions as a potent danger signal mediating the late response to infection and inflammation [71] . a higher expression of cytoplasmic hmgb1 was found in the decidua from women with preeclampsia [72] . there are as yet no data on whether hmgb1 levels are correlated with the clinical manifestations of preeclampsia. s100 ca 2? -binding s100b protein is a rage ligand [73] . the concentrations of s100b protein were increased in the placenta under oxidative stress [73] . s100b protein up-regulates seng release from endothelial cells [73] , and thus s100b protein is thought to be associated with preeclampsia. transthyretin (ttr) rage has been shown to bind fibrillar transthyretin (ttr), triggering nf-kappab activation [74] . this protein is responsible for transporting both the thyroid hormone thyroxine and retinol-binding protein. there are several reports that the concentrations of ttr appear to be associated with preeclampsia: ttr was up-regulated in preeclampsia in comparison with normal placentas, and oxidized ttr in amniotic fluid plays a role as an early marker of preeclampsia. these data allow us to speculate that once rage is engaged in the placental tissue, a vicious cycle of ligands-rage perturbation ensues, leading to oxidative stress, chronic tissue injury, and vascular damage. this section provides new information regarding prrs and their ligands that might contribute to pathological processes involved in preeclampsia. the aim of this review is to summarize current knowledge on the pathogenesis of preeclampsia. we comprehensively analyzed the results of genome-wide gene expression profiling and proteomic studies to delineate the wide array of mediators involved in this disorder. genes and proteins that participated in response to stress, host-pathogen interactions, immune system, inflammation, lipid metabolism, carbohydrate metabolism, growth, and tissue remodeling pathways were expressed differentially in preeclampsia (fig. 1) . the most important factors are genes and proteins involved in oxidative stress and inflammation. several significant common pathways observed in preeclampsia overlap the datasets identified in tlr-and rage-dependent signaling pathways. a novel model of the ages-rage system is proposed in fig. 2 . it is focused on several main properties of tlrs and rage and their ligands and signaling pathways along with angiogenic, inflammatory, and stress-related factors. notably, up-regulation of tlrs and rages, their ligands and their downstream targets are specifically found in preeclampsia. in addition, sflt-1, seng, and angiotensin-ii receptor autoantibody may contribute to the pathogenesis of preeclampsia. these potential markers have been implicated in the regulation of vascular endothelial function. the persistent inflammatory response plays an important role in coordinating the global transcriptional regulation leading to the functional switch from the ''stress-resistant'' response to the ''stress-sensitive'' phenotype: the loss of angiogenic functions and the rise in prrs. our review suggests a progressive shift in cellular homeostasis that may underlie stress/inflammation-associated functional alteration in vascular endothelial cells. in conclusion, the present article reviews the available scientific literature related to preeclampsia. the key challenge is to move from lists of identified factors to obtaining biological information regarding their functions. this work provides new insights into and aids understanding of the pathogenesis of preeclampsia. the strong correlation of specific genes regulated in preeclampsia with the possible tlr-and rage-dependent signaling pathways suggests that placental oxidative stress and subsequent chronic inflammation are considered to be major contributors to the development of preeclampsia. new aspects in the pathophysiology of preeclampsia molecular mechanisms of preeclampsia vascular endothelial growth factor receptor 2 controls blood pressure by regulating nitric oxide synthase expression. hypertension excess placental soluble fms-like tyrosine kinase 1 (sflt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia vascular endothelial growth factor inhibition by drk6 causes endothelial apoptosis, fibrosis, and inflammation in the heart via the akt/enos axis in db/db mice tolllike receptor signaling and pre-eclampsia endogenous damage-associated molecular pattern molecules at the crossroads of inflammation and cancer toll-like receptors 2 and 4 and the cryopyrin inflammasome in normal pregnancy and pre-eclampsia microarray analysis of differentially expressed fetal genes in placental tissue derived from early and late onset severe pre-eclampsia effect of hypoxia and exogenous il-10 on the pro-inflammatory cytokine tnf-alpha and the anti-angiogenic molecule soluble flt-1 in placental villous explants managing patients with metastatic colorectal cancer on bevacizumab extra-placental expression of vascular endothelial growth factor receptor-1, (flt-1) and soluble flt-1 (sflt-1), by peripheral blood mononuclear cells (pbmcs) in normotensive and preeclamptic pregnant women decreased proportion of peripheral blood vascular endothelial growth factor-expressing t and natural killer cells in preeclampsia dysregulation of anti-angiogenic agents (sflt-1, plgf, and sendoglin) in preeclampsia-a step forward but not the definitive answer regulation of soluble vascular endothelial growth factor receptor (sflt-1/svegfr-1) expression and release in endothelial cells by human follicular fluid and granulosa cells the soluble vegf receptor sflt1 contributes to endothelial dysfunction in ckd tgf beta signalling and its role in tumour pathogenesis can the biology of vegf and haem oxygenases help solve pre-eclampsia? systemic and uteroplacental reninangiotensin system in normal and pre-eclamptic pregnancies patients with preeclampsia develop agonistic autoantibodies against the angiotensin at1 receptor autoantibody from women with preeclampsia induces soluble fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/nuclear factor of activated t-cells signaling the functional role of the renin-angiotensin system in pregnancy and preeclampsia clinical implications for vascular endothelial growth factor in the lung: friend or foe? the viva trial: vascular endothelial growth factor in ischemia for vascular angiogenesis signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family a breakthrough in diabetic nephropathy: the role of endothelial dysfunction decidual expression and maternal serum levels of heme oxygenase 1 are increased in pre-eclampsia the role of the immune system in preeclampsia systemic increase in the ratio between foxp3? and il-17-producing cd4? t cells in healthy pregnancy but not in preeclampsia th17 cells and regulatory t cells: new light on pathophysiology of preeclampsia vascular actions of nitric oxide as affected by exposure to alcohol new pathways to renal damage: role of adma in retarding renal disease progression asymmetric dimethylarginine as a mediator of vascular dysfunction and a marker of cardiovascular disease and mortality: an intriguing interaction with diabetes mellitus regional and cardiac haemodynamic effects of n g ,n g ,dimethyl-l-arginine and their reversibility by vasodilators in conscious rats elevated asymmetric dimethylarginine concentrations precede clinical preeclampsia, but not pregnancies with small-for-gestational-age infants potential markers of preeclampsiaa review prediction of preeclampsia-a workshop report genetic aspects of preeclampsia association of -2548 g/a polymorphism in the leptin gene with preeclampsia/pregnancy-induced hypertension vegf-a stimulation of leukocyte adhesion to colonic microvascular endothelium: implications for inflammatory bowel disease inhibition of vegf or tgf-{beta} signaling activates endothelium and increases leukocyte rolling perfusion of the human placenta with red blood cells and xanthine oxidase mimics preeclampsia in vitro differential placental gene expression in preeclampsia pre-eclampsia, the placenta and the maternal systemic inflammatory response-a review endothelial dysfunction in pre-eclampsia beyond the threshold: an etiological bridge between hypoxia and immunity in preeclampsia rage signaling in inflammation and arterial aging toll-like receptors in pregnancy disorders and placental dysfunction toll-like receptor 3 activation during pregnancy elicits preeclampsia-like symptoms in rats toll-like receptor-3 stimulation upregulates sflt-1 production by trophoblast cells down-regulation of flt-1 gene expression by the proteasome inhibitor mg262 novel pkc signaling is required for lps-induced soluble flt-1 expression in macrophages maternal tlr4 and nod2 gene variants, pro-inflammatory phenotype and susceptibility to early-onset preeclampsia and hellp syndrome interleukin-4-590 (c[t), toll-like receptor-2 ?2258 (g[a) and matrix metalloproteinase-9-1562 (c[t) polymorphisms in pre-eclampsia the receptor for advanced glycation endproducts (rage) and cardiovascular disease receptor for age (rage) and its ligands-cast into leading roles in diabetes and the inflammatory response accumulation of advanced glycation end products in women with preeclampsia: possible involvement of placental oxidative and nitrative stress the receptor for advanced glycation end products (rage) is elevated in women with preeclampsia evidence for increased methylglyoxal in the vasculature of women with preeclampsia: role in upregulation of lox-1 and arginase increased lectin-like oxidized low-density lipoprotein receptor-1 expression in the maternal vasculature of women with preeclampsia: role for peroxynitrite expression of galectin-1, -3 (gal-1, gal-3) and the thomsen-friedenreich (tf) antigen in normal, iugr, preeclamptic and hellp placentas proteomic analysis of hypoxia-induced responses in the syncytialization of human placental cell line bewo galectin-3 as a multifunctional protein advanced glycation end products-induced gene expression of scavenger receptors in cultured human monocyte-derived macrophages selective cholesteryl ester uptake from high density lipoprotein by human first trimester and term villous trophoblast cells isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface biologic variability in plasma glucose, hemoglobin a1c, and advanced glycation end products associated with diabetes complications molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases concentrations of pentosidine, an advanced glycation end-product, in umbilical cord blood hmgb1, an innate alarmin, in the pathogenesis of type 1 diabetes high-mobility group box 1 protein (hmgb1): nuclear weapon in the immune arsenal the novel inflammatory cytokine high mobility group box protein 1 (hmgb1) is expressed by human term placenta oxidative stress-induced s100b protein from placenta and amnion affects soluble endoglin release from endothelial cells investigation of age, their receptor and nf-kappab activation and apoptosis in patients with attr and gelsolin amyloidosis placental gene expression in a rat 'model' of placental insufficiency dysregulation of the circulating and tissue-based renin-angiotensin system in preeclampsia cytokine-related genes and oxidation-related genes detected in preeclamptic placentas differential placental gene expression in severe preeclampsia gene expression in chorionic villous samples at 11 weeks of gestation in women who develop preeclampsia later in pregnancy: implications for screening quantitative distribution of a panel of circulating mrna in preeclampsia versus controls comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology altered gene expression of caspase-10, death receptor-3 and igfbp-3 in preeclamptic placentas preeclampsia: increased expression of soluble adam 12 matrix metalloproteinase 1 in preeclampsia and fetal growth restriction: reduced gene expression in decidual tissue and protein expression in extravillous trophoblasts gene expression profiling of maternal blood in early onset severe preeclampsia: identification of novel biomarkers oxidized transthyretin in amniotic fluid as an early marker of preeclampsia acknowledgments this study was supported by kakenhi (japan society for the promotion of science (jsps) grant-in-aid no. 20591958). we thank all the study participants for their time and efforts. we thank mikiko kita for editorial assistance. no potential conflicts of interest relevant to this article were reported. key: cord-324788-echu0zmf authors: aich, palok; potter, andrew a; griebel, philip j title: modern approaches to understanding stress and disease susceptibility: a review with special emphasis on respiratory disease date: 2009-07-30 journal: int j gen med doi: nan sha: doc_id: 324788 cord_uid: echu0zmf studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. for this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. it is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. these alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. these messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. the current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. the methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. with the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. these new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels. from the preceding discussion it is clear that stress can be defi ned in many different ways depending upon the objectives or perspective of the researcher. all these defi nitions, however, share a common component of adaptive physiological responses following challenges to homeostasis. the adaptive reactions to stressors may involve mobilization of a wide variety of physiological responses including the immune response. stress responses usually include physical perturbations that can encompass either the entire body or specifi c cellular compartments. considering the volume of work in various areas of stressors and their effects the main objective of the current review is to focus on one type of stress, which includes psychological stressors. for our purposes, stress can be defi ned as a psychologically perturbing condition occurring in response to adverse external infl uences capable of affecting physical health. transportation, fear (ie, fright and fl ight response), overcrowding and weaning in the form of social reorganization are a few of the important types of psychological stressors identifi ed in the literature. these stressors have been linked to many conditions including immune suppression, disease susceptibility, hypertension and reproductive dysfunctions. [4] [5] [6] stress is a major concern because it is ubiquitous, recurring in nature and has detrimental effects on health. in this review we will primarily deal with important psychological stressors and their infl uence on respiratory disease which is one of the most widely studied models. for many years, psychological stress has been shown to signifi cantly increase disease susceptibility. 6, 7 until 20 years ago, researchers investigating the psychological factors contributing to human disease focused primarily on coronary heart disease and cancer and neglected studies on infectious diseases. 8 however, interest in this area started to shift with the publication of evidence that psychological factors infl uenced immune function. 9 furthermore, there was an increasing awareness that stress and other psychological factors played a role in the onset and progression of acquired immunodefi ciency syndrome (aids). 10 these studies demonstrated a signifi cant role for psychological stressors in compromising immunity and interest in the effects of stressors in other diseases was initiated. considerable emphasis has been placed on respiratory diseases in understanding the onset and severity of the disease as a result of psychological stress. 11 increased risk of fatal bacterial respiratory infections following a primary viral infection has been observed in a wide variety of species. this phenomenon is called viral-bacterial synergy and was fi rst established following human infl uenza epidemics when a variety of secondary bacterial respiratory infections were associated with increased mortality. 12 studies have also linked a variety of psychological stressors with an increased incidence and severity of respiratory infections in humans 13, 14 and animals. 15, 16 it is known that respiratory disease has a huge economic impact in the areas of human health care, animal welfare and the food industry. 17, 18 to focus the review, we will restrict our discussion to research related to a comparative analysis of the effects of psychological stress on respiratory disease in humans and cattle. there are reports which have shown direct connections between stress and immune system function. 19 similarly, other studies have shown that social stressors could also increase the risk for upper respiratory infection. 20 a viral challenge study provides the strongest evidence for a link between stress and susceptibility to the common cold. 21 other studies have extended these results by considering a wider range of psychosocial factors. 22 the effects of stress on health are often mediated by a number of psychological factors. social support can often act as a buffer against the effects of stress as cohen and colleagues showed that social support reduced viral replication rate and increased mucociliary clearance of infection. 11, 13 in another report, they examined the effects of stress and social support in a routine study of upper respiratory tract illnesses. 23 under low levels of stress, high levels of social support were associated with a decreased risk of infection, whereas social support had no effect when levels of stress were high. a separate study was done to examine the associations between psychosocial factors (stress, social support, fl uctuations in mood) and viral exacerbations of asthma. the study involved naturally occurring illnesses rather than experimentally-induced infections but it maintained several important features of the methodology used by cohen and colleagues. 21, 24 for example, stress was measured at the start of the study by measuring the immune responses in terms of leukocytes present in peripheral blood in order to determine the extent to which stress could predict subsequent illness. in addition, effects of personality, 25 smoking status, and alcohol consumption 21 were also studied as possible predictors of susceptibility to respiratory viral infections. before considering the effects of psychosocial factors on respiratory virus-induced exacerbations of asthma, it is essential to have strong evidence that these viruses play a direct role in asthma. until recently, it appeared to be only a weak association between asthma and upper respiratory tract infection in adults. 26, 27 the absence of a stronger association in these epidemiological studies of adult asthmatics could, at least in part, have been due to diffi culties in isolating human rhinoviruses and coronaviruses. indeed, results from a study using enzyme-linked immunosorbent assays for antibodies to human coronavirus and semi-nested reverse transcriptase polymerase chain reactions for detections of rhinovirus suggested a stronger association between these viruses and asthma in adults. 28 in summary, this study confirmed that psychosocial factors and health-related behaviors were associated with increased susceptibility to colds, which then led to an exacerbation of asthma. this conclusion was made in the context of a study where both diseases (cold and asthma) were verifi ed using objective measures. the well established buffering effect of social support was observed in the high stress group and possible confounders such as demographics, health-related behaviors or personality could not account for this effect. alcohol consumption, personality and demographic factors were also shown to be important predictors of susceptibility. in contrast smoking was related to illness severity but not disease susceptibility. these results show that one must consider a range of psychosocial factors, personality traits, demographic factors, and health-related behaviors in studies of individual differences in susceptibility and severity of upper respiratory tract infections. while studies have correlated stress-related behavior with disease susceptibility, research has also been performed to determine the immunological basis of the relationship between stress and disease. exposure to viral agents that cause upper respiratory disease provokes illness in some individuals but not others. moreover, the severity of clinical symptoms among those who develop illness can vary substantially. evidence from prospective epidemiological studies 20, 23, 29 and from experimental viral-challenge studies 21, 30 show that individuals reporting greater psychological stress have both a higher incidence and a greater severity of illness. however, past attempts to identify the behavioral patterns and biological responses linking psychological stress with upper respiratory viral illness have been unsuccessful. 20, 21, 30 other studies reported that psychological stressors acutely activate the production of interleukin-6 (il-6), a pro-inflammatory cytokine. il-6 release in response to infection is thought to be mediated by glucocorticoids, thus providing a hypothetical pathway by which stressors (via the induction of glucocorticoid production) could control cytokine release. 31 in addition, il-6 triggers additional release of glucocorticoids, possibly exacerbating the stress response through positive feedback. at least one source of il-6 is epithelial cells as evidenced by their production of il-6 in vitro and in vivo when exposed to rhinovirus. 32 because a local increase in the concentration of this pro-infl ammatory cytokine precedes the development of acute signs and symptoms of illness, it has been implicated as a mediator in the pathway for symptom expression. in fact, il-6 concentrations in nasal secretions were associated with upper respiratory tract symptoms among persons infected with infl uenza a virus (a/texas and a/kawasaki) and rhinovirus (strain hanks' and type 39). 13 cohen and colleagues addressed the hypothesis that il-6 production in response to infl uenza a virus infection represents a viable pathway through which psychological stress infl uences the severity of illness. to achieve that goal, they measured levels of psychological stress in a group of adult subjects before experimentally infecting them with influenza a virus. self-reported respiratory symptoms, mucus weights, and local il-6 concentrations were then measured on the day before and for seven days after virus exposure. the data collected supported the conclusion that the level of psychological stress predicts the severity of illness and also the magnitude of the cytokine response. the data were then examined for evidence that il-6 mediated the association between stress and illness. these analyses suggested that most of the effects of psychological stress on clinical symptoms and mucus weights could be accounted for by changes in il-6. however, it is possible that il-6 itself is not the causal link but rather just a marker (covariate) for other pro-infl ammatory cytokines which were elevated during the course of experimental infection. for example, in another related study 33 reported that interferon α and il-6 levels (but not tumor necrosis factor [tnf], il-8, il-1, or il-2) increased early in the course of infection and both correlated with viral titers, increases in body temperature, mucus production and symptom scores. there is also an issue regarding the correlational nature of the mediational analysis. although consistent with the hypothesis that the association between stress and illness was mediated by il-6, the data do not permit causal inference. for example, this pattern of data is also consistent with increases in il-6 occurring in response to tissue damage associated with illness symptoms. even with these reservations, this was the fi rst aich et al study to provide evidence consistent with the hypothetical model that psychological stress infl uences upper respiratory infections through a biological pathway. 13 there are two parts to the stress response: sympatheticadrenal-medullary (sam) and the hypothalamic-pituitaryadrenal axis (hpa). the hpa is the core stress axis in mammals and together with the sam system co-ordinates response to the diverse range of stressors from psychological to physical. there is considerable interplay between both neuronal systems especially between the noradrenergic nucleus locus ceruleus which provides central regulation of the sam and the parvocellular neurones which regulate the hpa. the sam, by triggering catecholamine release, provides the acute stress response whilst the hpa governs longer term stress defence mechanisms. together those systems regulate energy utilisation and metabolic activity throughout the body. 34 the sam system produces the immediate shock response by acting on the hypothalamus, which activates the adrenal medulla and the sympathetic autonomic nervous system (ans) (figure 1 ). the sam produces the "fi ght-or-fl ight" response which increases alertness, blood fl ow to muscles, heart rate, blood pressure, respiration rate, etc. and might decrease activity in the digestive system. the hpa system regulates release of the hormone crf to activate the anterior pituitary and uses another hormone, adrenocorticotropic hormone (acth), to activate the adrenal cortex to release a group of hormones including cortisol ( figure 2 ). cortisol and other glucocorticoid hormones have various effects such as conservation of glucose for neural tissues, elevation or stabilization of blood glucose levels, mobilization of protein reserves, conservation of salts and water, suppression of wound healing and the immune system. according to seyle's general adaptation syndrome (gas) theory the general adaptation syndrome divides the stress response into three stages: stage 1: alarm reaction (sam and hpa activity increases and result in the "fi ghtor-fl ight" response); stage 2: resistance (hpa activity takes over, bodily resources are at maximum and if the stress is experienced for short term the body returns to normalcy); stage 3: exhaustion (with very prolonged stress, bodily systems are ineffective. sympathetic ans action reappears. adrenal cortex damage causes parasympathetic action, omics approaches in understanding stress and disease susceptibility eg, energy storage failure. the immune system collapses, and stress-related diseases increase). while it has been assumed that psychological (psychogenic) and physical (neurogenic) stressors are most closely aligned with depression, the suspicion has arisen that systemic stressors, including immune alterations, may also act in such a provocative capacity. 35 communication occurs between the immune, endocrine, autonomic and central nervous systems 36 such that psychological events that affect central neurochemical processes may affect immune activity. conversely, immune activation may affect hormonal processes and the activity of central neurotransmitters. thus, by virtue of the neurochemical effects imparted, immune activation may affect behavioral outputs and may even be related to behavioral pathology such as depressive illness. 37 the hypothesis that altered immune activation may occur as a result of various stressors emerged over time. the initial theory came from hans seyles' gas, which was derived from observation and experimentation on laboratory animals. using a variety of stressors (ie, pain, thermal extremes and starvation), seyle described a common nonspecifi c stress response pathway. 38 after initial perception of a stressor, the animal mounts an emergency alarm or fi ght-or-fl ight response. this catecholamine-driven reaction results in increased cardiovascular function and an overall increase in metabolism. if the stressor persists, the resistance phase or 'conservation withdrawal reaction' is initiated, which is a physiological coping reaction to the increased demands of maintaining homeostasis. chronic stress leads to the exhaustion phase and may lead to pathology. seyle's theory unifi ed the stress phenomena because it provided a common response pathway to all the varied stressors encountered. this pathway, the hpa axis, involves perception in the brain with release of hypothalamic corticotropin-releasing factor (crf) and vasopressin which stimulates the anterior pituitary to secrete acth ( figure 2 ). circulating acth causes the adrenal cortex to produce glucocorticoids (gc). glucocorticoids cause gluconeogenesis with conversion of lipid to glucose for the central nervous system (cns) and other functions. thus, gas allowed the identifi cation of stressors (and presumably the status of well being of the animal) by measurement of gc levels. it was an attractive theory because gc levels were relatively easy to measure. 2 unfortunately the gas theory has proven to be too simplistic. mason's experiments with rhesus monkeys aich et al exposed to different types of stressors revealed a disparity in neuroendocrine responses provided by specifi c stressors. 38 for instance, monkeys subjected to emotional stress had elevated serum gc levels but those subjected to a heat-stress regime failed to show gc elevation. in addition to gc, other neuroendocrine mediators were characteristically produced in response to specifi c stressors. 38 emerging information on the response to stressors suggests that there are at least four different avenues of neuroendocrine responses. these involve the autonomic nervous system, the hpa axis, neuropeptides, neurotransmitters and neuroimmunological peptides and receptors. 2 interactions between the immune and nervous systems play an important role in modulating host susceptibility and resistance to infl ammatory disease. neuroendocrine regulation of inflammatory and immune responses as well as onset of disease can occur at multiple levels: (a) systemically through the anti-infl ammatory action of gcs released via hpa axis stimulation, (b) regionally through production of gcs within the affected tissue as well as by sympathetic enervation of immune organs such as the thymus and (c) locally at sites of infl ammation. estrogens also play an important role in immune modulation and contribute to the approximately 2-to 10-fold higher incidence of autoimmune/infl ammatory diseases in females of all mammalian species. 39 during infl ammation, cytokines from the periphery activate the central nervous system through multiple routes. this results in stimulation of the hpa axis which in turn, through the immunosuppressive effects of the glucocorticoids, generally inhibits infl ammation. recent studies indicate that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive causing a shift in patterns of cytokine production from a th1-to a th2-type pattern. interruptions of this loop at any level and through multiple mechanisms, whether genetic, or through surgical or pharmacological interventions, can render an infl ammatory resistant host susceptible to infl ammatory disease. over-activation of this axis, as occurs during stress, can also affect severity of infectious diseases through the immunosuppressive effects of the glucocorticoids. these interactions have been clearly demonstrated in many animal models using a variety of species and infectious agents. 39 the results from these models are also relevant to human infl ammatory, autoimmune and allergic illnesses including rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, allergic asthma and atopic skin disease. while many genes and environmental factors contribute to susceptibility and resistance to autoimmune/infl ammatory diseases, a full understanding of the molecular mechanisms by which a combination of neuropeptides, neurohormones and neurotransmitters can modulate immune responses is essential for effective design of future interventions. 39 it is clear from the previous discussion and review of previous research that the relationship between stress and disease susceptibility is very complex and intertwined with cascades of events. in order to understand or characterize stress-induced disease susceptibility one needs to identify various biological or functional pathways and interaction networking involved at any given time. to follow the cascade of molecular events happening over time one needs to employ methodologies which are holistic in nature and can provide global information related to the kinetics of multiple changes in gene expression and multiple biomolecular interactions. with the advent of various high-throughput genomic approaches it has become possible to explore complex biological processes and in one step obtain information about gene expression at the transcriptional (genomics) and translational (proteomics) level, as well as to identify metabolites arising from these responses (metabolomics/metabonomics). these methodologies together are often referred to as 'omics' approaches. although these methodologies are still in their infancy they have started showing promise in understanding systems' biology and various disease processes. work employing omics approaches to understand mechanism of stress and disease susceptibility is limited at this time. there are reports which describe the effects of oxidative stress on cellular apoptosis 40 but there are no reports on the effects of psychological stressor and disease susceptibility in animals using holistic methodologies such as various omics approaches. the literature in this area is very scarce because these methodologies are very new and few results are currently available. the main studies to correlate stress and disease susceptibility was done either in plant systems 41 or a correlation between oxidative stress and infectious disease. 42 the review by hirai and saito established, using liquid chromatogram based mass spectrometric proteomic and metabolomic and cdna microarray based transcriptomic analyses, that these omics studies can reveal the genomic networking involving several pathways related to oxidative stress response and key metabolic pathways. 41 bioinformatics and statistical approaches specifi c for detailed transcriptomics or cluster analyses of expressed genes have been developed and are useful for extracting information on various functional genomic responses. [59] [60] [61] it is important, however, to understand the translation of transcriptomic information into protein expression and modifi cations since it is the protein that acts as biomarkers for various disease processes or conditions. several proteomic tools have been developed. these include gel-based two-dimensional (2d) gel electrophoresis (ge), 47 2d fluorescence difference gel electrophoresis (2dige) 62 or gel-free multidimensional protein identifi cation technology (mudpit), 63,64 isotope-coded affinity tag (icat™), 65 surface-enhanced laser desorption-ionization time-of-flight (seldi-tof) ms 66,67 or isobaric tag for relative and absolute quantitation (itraq) 68 methodologies. while these technologies are being matured more appropriate biological questions can be addressed. in 2d-ge whether traditional or newer dige system, proteins are fi rst focused (fi rst dimension) in terms of their pi values followed by sds-page (second dimension). in contrast to traditional 2d-ge, dige system utilizes the fl uorescence labeling of protein samples and two-to-three different samples can then be run on a single gel which makes it superior to traditional 2d-ge in terms of eliminating gel to gel variation and comparative analysis. although 2d-ge is a very good way of separating complex protein mixtures, it is limited in terms of high-throughput analysis and sensitivity. because of low sensitivity it is mostly limited to the analysis of high abundant proteins of a system. efforts have been made to improve the sensitivity and analysis capacity of proteomics techniques. as a result, other methodologies such as 2d-hplc, mudpit, icat, and itraq have been developed and are gaining popularity. although various bioinformatic and data analyses approaches have been developed to analyze gel images and mass spectrometric based protein characterization a highthroughput methodology in this area has yet to be developed. functional genomic techniques of transcriptomics and proteomics and available bioinformatic and statistical analyses promise unparalleled global information during the analyses of complex biological responses. however, if these technologies are used in isolation, the large multivariate data sets produced are often diffi cult to interpret and have the potential to ignore key metabolic events to understand the true biology. high resolution 1 h nmr spectroscopy used in conjunction with pattern recognition provides one such tool for defi ning the dynamic phenotype of a cell, organ, or organism in terms of a metabolic phenotype. in a recent review the benefi ts of this metabonomics/metabolomics approach to problems in toxicology have been discussed. 69 one of the major benefi ts of this approach is its high-throughput nature and cost-effectiveness on a per sample basis. using such a method, the consortium for metabonomic toxicology (comet) is currently investigating approximately 150 model liver and kidney toxins. this investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the fi eld of drug metabolism. the review also included how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses such as during drug-induced fatty liver disease. by using data integration to combine metabolite analysis and gene expression profi ling, key perturbed metabolic pathways can be identifi ed. detailed omics-based bioinformatics studies, to correlate stress-dependent disease susceptibility, have yet to analyze this complex biological response. a few preliminary studies have been reported in the literature. a recent study has shown the adverse effects of using mechanical ventilators for respiratory support. 70 this acute lung injury because of mechanical stretch can lead to high mortality and this study has utilized recent advances in bioinformatic-intense candidate gene searches to correlate the lung injury and gene expression profi les in a rodent model analyzed with microarrays. 70 the authors used 2,769 mouse/rat orthologous genes identifi ed on rg_u34a and mg_u74av2 arrays and the expression profi les were simultaneously analyzed by signifi cance analysis of microarrays. this combined ortholog and signifi cance analysis of microarrays approach identifi ed 41 up-and 7 downregulated ventilator-induced stress-related candidate genes. results were validated by comparable expression levels obtained by either real-time or relative rt-pcr for 15 randomly selected genes. k-mean clustering of 48 candidate genes clustered several well-known lung injury associated genes (il-6, plasminogen activator inhibitor type 1, ccl-2, cyclooxygenase-2) with a number of stressrelated genes (myc, cyr61, socs3). the only unannotated member of this cluster (n = 14) was riken_1300002f13 est, an ortholog of the stress-related gene33/mig-6 gene. the authors speculated that the ortholog-signifi cant analysis of microarray approach is a useful, time-and resourceeffi cient tool for identifi cation of candidate genes in a variety of complex disease models such as ventilator induced lung injury. 70 using microarray-based genomic approaches work has also been initiated to identify hypertension-related genes in rat model. 71 though it is a common belief that the stressors mentioned earlier can compromise host immune responses and enhance susceptibility to various diseases, very little work has been done in this area to understand the mechanism of stress dependent disease susceptibility in mammals. kelley in 1980 identifi ed eight stressors that typically occur in modern livestock production units: heat, cold, crowding, mixing, weaning, controlled-feeding, noise and restraint. all these stressors were shown to alter components of the immune system in animals and these changes in immune function may ultimately explain the physiological basis of diseaseenvironment interactions. 72 another study in 1993 showed that neurotransmitters and neuroendocrine hormones can modify the function of immune cells. conversely, cytokines produced by immune cells can alter brain homeostasis. 73 this connection is further manifested by experimental studies showing a relationship between stress and resistance to infection. human subjects under high stress were shown to be more susceptible to infection with common cold viruses. furthermore, a diversity of experimental animal models confi rmed that laboratory stressors such as forced exercise, avoidance learning, restraint, isolation and cold exposure made animals more susceptible to primary infection with a variety of viruses and bacteria. the cellular and molecular basis for the observed modulation of host resistance is not fully understood but involves altered functioning of both t-lymphocytes and cells of the hypothalamic-pituitary adrenal axis. also involved is the altered production of cytokines and hormones produced by the immune system and brain. 73 emau and colleagues in 1987 showed that the onset of immunodepression by stress or viral infection in the pathogenesis of bovine pneumonia permits super infection of the lungs with mannheimia haemolytica (formerly called pasteurella haemolytica) which results in exudative fi brinous pneumonia. 74 although these studies provide a preliminary basis of stress-induced bovine respiratory disease (brd), there was a lack of necessary information to determine the molecular basis of such dependence, which can be found by studying changes at the level of gene expression (transcriptional and proteomic) and metabolites. a recent study compared proteomics of epithelial lining fl uid from lungs of weaned and nonweaned cattle. 75 the study was done over a shorter period of time (36 h) with a combination of stressors (weaning, transportation) and important questions such as to determine specifi city and duration (stability) of the protein biomarkers are yet to be addressed. the group also studied the proteomic profi le of bronchoalveolar lung lavage fl uid following treating the calves with dexamethasone to determine the effects of glucocorticoids, which is elevated following induction of stress. 76, 77 more studies are however needed (a) to fi nd biomarkers associated with stressors and infection, (b) to determine the duration of the biomarkers so that these are sustainable enough to identify and predict stressed animals (which might be more susceptible to infection in real life situation such as in feedlot cattle). disease models have been developed to study the molecular mechanisms underlying the viral-bacterial synergy which results in fatal brd infections. [78] [79] [80] we have confi rmed with the infectious brd model that the stress of weaning 81 significantly enhances the viral-bacterial synergy leading to fatal bacterial respiratory infection. 82 the major stressors young omics approaches in understanding stress and disease susceptibility cattle experience include maternal separation or weaning, dietary changes, transportation, social reorganization and other environmental effects (figure 3 ). with this in mind, we focused our initial studies to understand the role of weaning on the clinical response to brd. fifteen suckling calves were removed from their mothers 24 h prior to viral infection (abruptly weaned/stressed, aw). a second group of 15 calves was weaned 2 wks prior to viral infection (pre-conditioned/ control, pc); 2 wks was chosen as an appropriate interval to eliminate psychological and physiological effects associated with breaking the maternal/nutritional bond and to adapt to the dietary change and social re-organization that follows weaning ( figure 4 ). all calves were transported to vaccine and infections disease organization vido, and aerosol challenged with bovine herpesvirus-1 (bhv-1) followed by m. haemolytica; this combined viral-bacterial infection induces fatal pneumonia in 50%-70% of calves. 83 our clinical analyses revealed a signifi cant difference in brd clinical disease when comparing freshly weaned calves (80% mortality) versus pre-conditioned calves (40% mortality). 84, 85 increased mortality associated with fresh weaning was characterized by a decrease in both survival time post-infection and, interestingly, decreased lung pathology which suggested a systemic reaction. contrary to expectations, transportation induced a signifi cant cortisolemia in pre-conditioned but not freshly weaned calves ( figure 5 ). transportation is known as an important stressor which increases blood cortisol level. [86] [87] [88] [89] however, the stressor combination of weaning and transportation may have different physiological markers than commonly expected. cortisol is a potent regulator of pro-infl ammatory cytokine transcription by acting as a negative regulator of nfκb activation, and can be used as an indicator of stress. 90 fatal bacterial respiratory infection in calves was usually associated with an accelerated decline in serum cortisol levels. therefore, we hypothesized that increased respiratory disease susceptibility was associated with enhanced pro-infl ammatory responses in freshly weaned calves. elevated body temperature and interferon-gamma (ifn-γ) levels in nasal secretions during bhv-1 infection of freshly weaned calves were similar to previous reports, 82, 91 supporting this hypothesis. these immune responses were signifi cantly increased despite no signifi cant difference in virus shedding. furthermore, increased il-10 expression, during bhv-1 infection of pre-conditioned calves, was consistent with reduced pro-infl ammatory responses. 82 we conducted bovine microarray analyses of rna isolated from blood mononuclear cells to determine if changes in gene expression correlated with either stress or the severity of brd infection; results support the conclusion that differential regulation of pro-infl ammatory responses is a major mechanism contributing to increased disease susceptibility. conserved responses included an enhanced potential to aich et al respond to pathogen-associated molecular patterns through increased expression of toll-like receptors (tlrs). tlr induction of innate immune responses is mediated primarily through activation of nuclear factor kappab (nfκb). we also observed differential expression of β-defensin 5 (a host defense peptide induced by tlr4 signaling) in freshly weaned calves, consistent with reduced cortisol levels. 92 in addition, when we compared expression of select innate immune genes (eg, tlr2, tlr4, ifn-γ and 2'5' o-adenylate synthetase) between day 4 (post bhv-1 infection) and day 0 (prior to bhv-1 infection) for aw-and pc-groups, results revealed a trend of increased expression of the select genes on day 4 irrespective of the stress situation (table 1 ). this trend of activation of select immune genes clearly revealed that following bhv-1 infection the innate immune response was enhanced. this observation contradicts the current hypothesis that the primary viral enhances the secondary bacterial infection by compromising the immune system. 78, 93 our studies revealed that the anti-infl ammatory gene il-10 was upregulated on day 4 in pc groups while β-defensin was activated on day 4 in aw groups (table 1) . il-10 which acts as an anti-infl ammatory gene should be activated on day 4 to control pro-infl ammatory responses as a result of bhv-1 infection in either aw or in both groups; instead it was only over expressed in control (pc) groups. on a similar token β-defensin on day 4 should also be observed in both groups to control bhv-1 infection. these results are particularly interesting to explore further to understand the detailed mechanism of stress-induced disease correlation. although advances have been made in understanding various disease processes, successful intervention often depends upon the stage at which disease is detected. thus, identifying markers for disease or its cause, as well as understanding the mechanism of disease onset and susceptibility, are very important. systems biology approaches such as omics (genomic, proteomic, metabonomic) and multivariate analysis to understand mechanisms and to identify biomarkers provide potential tools to address these questions. 69, [94] [95] [96] it is particularly important to employ a combination of molecular approaches such as omics to understand complex processes such as stress and its correlation with disease susceptibility. with such goals in mind, we identifi ed and characterized a group of protein, metabolite and elemental biomarkers using serum samples from bhv-1 infected as well as from stressed animals. the trend of each group of biomarkers distribution was analyzed to correlate with the groupings based on stress condition or infection. 53 multivariate analysis revealed distinct differential trends in the distribution profi le of proteins, metabolites and elements following a stress response both before and after primary viral infection. a group of acute phase proteins, metabolites and elements could be specifi cally linked to either a stress response (decreased serum amyloid a and cu, increased apolipoprotein ciii, amino acids, low-density lipoprotein [ldl], p and mo) or a primary viral respiratory infection (increased apolipoprotein a1, haptoglobin, glucose, amino acids, ldl and cu, decreased lipid and p). thus, combined omics analysis of serum samples revealed that multimethod analyses could be used to discriminate between the complex biological responses to stress and viral infection. there are reports which suggest that transport can be an important stressor which alters blood leukocyte populations and sets the stage for brd. 97, 98 currently we are conducting experiments in which transportation of the animals is eliminated, such that stressors related to a new environment are removed. it was documented that abrupt weaning (separation of suckling calves from their dams) causes a prolonged psychological stress response. 81 this stress response manifests as an increase in vocalization by both calves and cows and a significant increase in time spent walking by the calf. consequently, time spent eating and resting also decreases for abruptly weaned calves. statistically, these changes in behavior return to baseline values after four to fi ve days of weaning. as is evident from the review of the literature that traditionally effects of stress have been studied by attempting to understand behavioral patterns of the subject and cellular immune response of the host. it is, however, clear that amount of data to understand such a complex condition (such as stress and stress-induced diseases) is not adequate and enough. behavioral studies may not always be correlated to a particular stressor as there might have been other parameters affecting the observations. attempts have also been made to use cortisol as a unique biomarker for stress, but cortisol is diurnal hence cannot be a reliable marker. while it is important to understand cellular immunology to establish the mechanism of stress-induced disease susceptibility, however without identifying and establishing the immune markers specifi c for stressor it will not lead into any meaningful inference. systems biology approaches using various omics and bioinformatics methodologies can identify a group of biomarkers at various levels of host immune response, gene expression and metabolism. association of the identifi ed biomarkers and the patterns in changes in their expression level above or below a determined threshold level with specifi c stressor(s) could then be used to defi ne stress situation, identif ication of individuals susceptible to stressinduced disease. moreover, establishing a pattern in changes of biomarkers is more reliable than a single biomarker. duration of identifi ed biomarkers can further strengthen the methodology for applying in real life situation. the current review established the importance of psychological stress and its effects on infectious disease severity and susceptibility with emphasis on respiratory disease. as evident from the literature, initial work focused mainly on understanding the roles of various stressors on physiology and behavior. theories emerged as a consequence of these studies which implicated the hpa axis and the sympathetic nervous system (sns) in altered immunity. however, physiological responses studied in a laboratory may not necessarily be consistent with real life phenomena, especially when attempting to analyze heterogeneous populations such as cattle or humans. experiments with human subjects cannot always be properly controlled because of ethical concerns and therefore using appropriate animal models to mimic natural disease outcomes may be more informative. with the use of high-throughput omics approaches it should be possible to extract more detailed information out of these complex biological systems and understand the complex biology and mechanisms by which stress augments disease susceptibility. more studies are needed to understand the physiological differences between acute versus chronic stress as well as the combined effects of various stressors. it is becoming more apparent that stress not only makes an individual more susceptible to a disease but can also enhance disease severity. therefore, it is very important to understand the kinetics, specifi city and stability or duration of biomarkers associated with a specifi c stressor or combination of stressors before we can predict 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vaccination against experimental bovine herpesvirus-1 and pasteurella haemolytica respiratory tract infection: onset of protection effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate infl ammation comparative approaches to the investigation of responses to stress and viral infection in cattle transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress acute phase proteins in cattle after exposure to complex stress effects of road transportation on lymphocyte subsets in calves transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls hypercortisolemia in acute stroke is related to the infl ammatory response induction of interferon activity in nasal mucus by association of levamisole by systemic route -inactivated virus by local route effect of stress on viral-bacterial synergy in bovine respiratory disease: a review of potential mechanisms immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with haemophilus somnus and bovine respiratory syncytial virus proteomic characterization of the interstitial fl uid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery unlocking the mysteries of virus-host interactions: does functional genomics hold the key? a comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in escherichia coli cells effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle effect of transportation on blood serum composition, disease incidence, and production traits in young calves. infl uence of the journey duration this project was funded by the saskatchewan agriculture and food rural revitalization (safrr), genome bc and genome prairie for the 'pathogenomics of innate immunity' research program and the ontario cattleman's association (oca). this manuscript is published with permission of the director of vaccine and infectious disease organization as article number 512. a.a.p. is the holder of an nserc senior industrial research chair. key: cord-295767-92oxdmxq authors: aga, syed sameer; khan, muhammad anwar; nissar, syed saniya; banday, mujeeb zafar title: évaluation de la santé mentale et des diverses stratégies d'adaptation dans la population générale vivant sous l'emprise de la covid à travers le monde : une étude transversal date: 2020-07-28 journal: ethics med public health doi: 10.1016/j.jemep.2020.100571 sha: doc_id: 295767 cord_uid: 92oxdmxq résumé la maladie à corona (covid-19) qui s'est déclarée en décembre 2019 dans la ville chinoise de wuhan est devenue une menace mondiale et constitue actuellement la plus grande épidémie connue de pneumonie atypique affectant tous les continents du monde avec environ 6 416 828 cas et 382 867 décès. on sait que les confinements imposés par la maladie provoquent des niveaux accrus de dépression, d'anxiété et de stress. notre étude visait à étudier l'impact immédiat du confinement imposé par la pandémie de covid-19 sur la santé mentale et la qualité de vie de la population générale âgée de 18 ans et à identifier les différentes stratégies d'adaptation utilisées dans le cadre du confinement. une enquête en ligne a été menée entre le 1er avril et le 10 mai 2020, à l'aide d'un questionnaire validé basé sur la dass-42, en utilisant une technique d'échantillonnage en boule de neige. au total, 418 réponses provenant de 16 pays différents ont été reçues. les personnes interrogées présentaient un niveau élevé de dépression et d'anxiété, qui différaient sensiblement entre les sexes. en outre, les participants des pays en développement inde et pakistan souffraient de dépression grave, tandis que les participants de l'inde, du pakistan et du royaume d'arabie saoudite souffraient d'anxiété grave. nous avons également constaté que parmi les différentes stratégies d'adaptation, a) regarder la télévision pour se divertir, b) les réseaux sociaux, c) écouter de la musique, d) dormir, e) faire des tâches ménagères banales comme le nettoyage, la lessive, etc. f) bien manger, et g) nettoyer/finir mon travail accumulé ont été classés parmi les stratégies d'adaptation les plus utilisées par tous les participants. cette étude identifie le besoin de fournir des services professionnels et psychologiques gratuits pour aider à faire face au stress pendant le confinement imposé par la maladie. abstract the 2019 corona virus disease (covid-19) which outbreak in december 2019, in the chinese city of wuhan has became a global threat and is currently the largest known outbreak of atypical pneumonia affecting every continent of the world with about 6,416,828 cases and 382,867 deaths. disease enforced lockdowns are known to cause heightened levels of depression, anxiety, and stress. our study aimed to investigate the immediate impact of the covid-19 pandemic enforced lockdown on mental health and quality of life among general population aged 18 years and to identify various coping strategies used under lockdown. an online survey was conducted between 1st of april 10th of may, 2020; using a validated questionnaire based on dass-42, employing a snowball sampling technique. a total of 418 responses from 16 different countries were received. the respondents had a high level of depression and anxiety scores, which were significantly different among genders. also, participants from developing countries – india and pakistan had severe depression while as participants from india, pakistan and kingdom of saudi arabia had severe anxiety. we also found that among the various coping strategies, a) watching television for entertainment, b) social networking, c) listening to music, d) sleeping, e) doing mundane house chores like cleaning, washing etc f) eating well, and g) clearing/finishing my piled-up work were ranked among the most utilized coping strategies by all participants. this study identifies the need to provide the free professional and psychological services to help cope with stress during the disease enforced lockdown. la maladie à corona (covid-19) qui s'est déclarée en décembre 2019 dans la ville chinoise de wuhan est devenue une menace mondiale et constitue actuellement la plus grande épidémie connue de pneumonie atypique affectant tous les continents du monde avec environ 6 416 828 cas et 382 867 décès. on sait que les confinements imposés par la maladie provoquent des niveaux accrus de dépression, d'anxiété et de stress. notre étude visait à étudier l'impact immédiat du confinement imposé par la pandémie de covid-19 sur la santé mentale et la qualité de vie de la population générale âgée de 18 ans et à identifier les différentes stratégies d'adaptation utilisées dans le cadre du confinement. une enquête en ligne a été menée entre le 1er avril et le 10 mai 2020, à l'aide d'un questionnaire validé basé sur la dass-42, en utilisant une technique d'échantillonnage en boule de neige. au total, 418 réponses provenant de 16 pays différents ont été reçues. les personnes interrogées présentaient un niveau élevé de dépression et d'anxiété, qui différaient sensiblement entre les sexes. en outre, les participants des pays en développement -inde et pakistan -souffraient de dépression grave, tandis que les participants de l'inde, du pakistan et du royaume d'arabie saoudite souffraient d'anxiété grave. nous avons également constaté que parmi les différentes stratégies d'adaptation, a) regarder la télévision pour se divertir, b) les réseaux sociaux, c) écouter de la musique, d) dormir, e) faire des tâches ménagères banales comme le nettoyage, la lessive, etc. f) bien manger, et g) nettoyer/finir mon travail accumulé ont été classés parmi les stratégies d'adaptation les plus utilisées par tous les participants. cette étude identifie le besoin de fournir des services professionnels et psychologiques gratuits pour aider à faire face au stress pendant le confinement imposé par la maladie. mots-clés : anxiété ; covid ; dass ; dépression ; dépression ; impact psychologique ; pandémie ; santé mentale ; soins de santé ; stress the 2019 corona virus disease (covid-19) started in december 2019, as a novel viral outbreak in seafood market in the chinese city of wuhan located in central hubei province [1] [2] [3] . this novel virus quickly became a global threat by spreading across the globe within three months and is currently the largest known outbreak of atypical pneumonia since the severe acute respiratory syndrome (sars) outbreak in 2003 affecting every continent of the world with about 6,416,828 cases and 382,867 deaths [4] . in order to prevent the spread and burden of disease, governments all over the world were forced to enforce the complete lockdown of their economies and life as we knew it came to a grinding halt [5] , and several countries were forced to implement the community mitigation strategies during this pandemic for the survival and management of distress [6, 7] . currently, an unprecedented lockdown is being enforced around the globe, in both developed as well as developing countries encompassing all continents [8] . life under current circumstance of covid pandemic lockdown is very challenging especially for the professionals like students, office workers and academicians and does constitute huge amounts of numerous varieties of stress [7, 9] . during these unprecedented conditions, a person (especially a health care worker) is subjected to various sources of stress emanating from numerous pressures, peer pressures, personal, emotional, work or social issues, boredom, infection fear, loss of usual routine, and reduced social and physical contact, environmental factors, financial and family issues etc [10] [11] [12] . the current lockdown and quarantine model of fighting the covid-19 does clearly have major social and psychological impact on the whole population. the lockdown has multi-faceted affects like unemployment, family separations, domestic violence, deaths of loved ones, failure of closure, social stigmatization, increased loneliness, work stress, the overabundance of (mis)information on social media and various other related factors. all of these serve as the major psychological risk factors for stress, anxiety, depression, and self-harm [7, [11] [12] [13] . most prevalent psychological disorders are depression and anxiety categorized as: "neurotic, stress-related and somatoform disorders" and "mood disorders". importance of psychological and behavioral disorders in community health is well established, as they are the most significant basis of morbidity in general care settings leading to extensive disability [14, 15] . there are numerous important factors causing emotional distress which have been described in the explanatory models of persons suffering from common psychological disorder. several rating scales purporting to assess the clinical construct of depression are developed of which dass-42 is one of the best scales to assess the levels of stress, anxiety and depression [16] [17] [18] [19] . several techniques and strategies have been identified by psychologists to master, reduce, tolerate, or minimize the effects of stressors. all these are referred to as coping strategies and usually include mindfulness-based stress reduction classes, wellness electives, informal support groups and mentoring programs [20] . coping methods often used include, effective time management, social support, positive reappraisal, and engagement in leisurely [9, 21, 22] . therefore, we designed this study to measure the mental stress of general populations and to assess their perceptions towards various stress factors, their sources, and their severity under the covid lockdown. the main aim of the study was: o to assess the severity of depression, anxiety, and stress in general population. o to identify various coping strategies used under lockdown. this study is a descriptive type of research using survey approach. the study was carried in between 1 st of april-10 th of may, 2020. the online questionnaire was distributed through social media (facebook, twitter, whatsapp and official emails) by the pi and cois to their contacts. all participants were randomly selected, and snowball sampling method was used for the inclusion. the participants were also encouraged to send the survey to their own contacts as much as possible. participants of age equal or more than 18 years, who were able to understand english and provided their informed consent were and united states of america (usa). an informed consent from all the participants was duly taken followed by the implementation of a pre-designed questionnaire based on two tools to obtain a data set composed of socio-demographic and physical characteristics. questionnaire to assess depression, stress, and anxiety. the dass questionnaire is freely available in public domain and hence requires no permission for its use [18, 19] . for this study we adopted 42-item questionnaire which constitutes three subscales including depression, anxiety, and stresseach of which is based upon fourteen items of the dass questionnaire. the participants respond to each item of dass on a 4-point likert scale (0 = did not apply to me, 1= applied to me to some degree or some of the time, 2 = applied to me to a considerable degree or for a good part of time and 3 = applied to me very much or most of the time). according to the dass-42 scoring algorithm, higher scores indicated higher depression, anxiety, and stress. total score is calculated by summing the scores for each subscale. moreover, dass scoring manual also does provide a cut-off scores for defining the severity of depression, stress and anxiety, as provided: normal (0-9 for depression, 0-7 for anxiety and 0-14 for stress), mild (10-13 for depression, 8-9 for anxiety and 15-18 for stress), moderate (14-20 for depression, 10-14 for anxiety and 19-25 for stress), severe (21-27 for depression, 15-19 for anxiety and 26-33 for stress) and extremely severe (> 28 for depression, > 20 for anxiety, > 34 for stress). tables i and ii. analysis of dass-42 scores (tool 1) revealed that male and female participants differed significantly in their overall scores for depression and anxiety (p value < 0.05) while their stress scores were almost comparable [ analysis of tool 2 questionnaire revealed that there were no marked differences in using the different coping strategies by the participants in dealing with the lockdown with males and females utilizing all strategies almost comparably; however there was slight difference on the basis of professions. all the participants used the range of strategies to manage their time in quarantine in a varying degree [ table viii ]. seven coping strategies a) watching television for entertainment, b) social networking, c) listening to music, d) sleeping, e) doing mundane house chores like cleaning, washing etc f) eating well, and g) clearing/finishing my piled-up work were ranked among the most utilized coping strategies by all participants (received highest agreement) in contrast to a) gardening/landscaping etc, b) watching television for news exclusively, c) taking up new hobby, d) participating in online classes, etc as the nonutilized or less useful coping strategy. taking solace in offering prayers and participating/playing indoor sports were chosen preferentially by 38.95 of banking professional and 36.7% students to be as strongly agreeable coping strategy. also, 53.1 % of students and 31.4% of research scientists strongly agreed to use sleeping in their lockdown quarantine hours. 27.3% of office managers and 20.4% of students strongly agreed to identifying themselves with other role models who prioritize their quality of life while ones physical limits & avoiding spending many hours without sleeping. cooking as coping strategy was strongly agreeable to 63.6% of office administrators and 44.9% students. 22.9% of doctors, 26.1% of academicians and 28.6% of students were in strong agreement to participate in online courses during the lockdown. the replies to various coping strategies by participants on the basis of 5 point likert scale are provided in the table ix. also, figures 1-18 provide the graphical representation of the likert scale scoring for various coping strategies employed by various professionals during their lockdown. covid-19 is defined as the alterative form of severe acute respiratory syndrome (sars), caused by coronavirus 2 (sars-cov-2) with a high possibility of transmission from animals to humans which has been associated with contact with a local seafood vendor in wuhan that illegally sold some wildlife animals including bats [24, 25] . the initial transmission from the reservoir (usually bats) to humans involves a zoonotic jump and then transmission between people occurs during close contact, mostly via small droplets produced by coughing, sneezing, and talking [26] . coronaviruses, belong to the family of rna viruses called coronaviridae which possess a well-developed spherical protein envelop protecting the genetic material inside it. the envelop resembles the crown ('corona' in latin), in its structure and hence their name and the rna contained in them is the largest known positive-sense rna genome in viruses [26, 27] . since the declaration of covid-19 as the pandemic by who, on 11 march 2020 the disease has presented itself as an unprecedented public health crisis because of its rapid spread and higher lethality than previous pandemic/epidemics which is compounded further by the increased international travel density and immune naivety of the worldwide population [28] . this has resulted in the huge psychological impact on the mental health of the individuals living across the globe under lockdown for limiting its spread and for their own survival [6, 11, 12] . as all pandemics are characterized by their unique set of conditions and control measures which affect the general population variedly on many factors like causality, progression of disease, casualties caused, mitigation strategies and control measures. with each day passing under lockdown the effects of quarantine and isolation become often intense and do adversely affect the mental well-being of a given population and in turn influencing their community behavior negatively [9] . as reported, epidemics have already been associated with a wide range of psychiatric comorbidities including anxiety, panic, depression, and trauma-related disorders [29] and this psychosocial impact has been found to be even higher during quarantine/lockdown measures [7] . lockdown and quarantine have also been associated with high stress levels, depression, irritability, and insomnia [9, [30] [31] [32] . hence, to answer these burning questions we designed this study to attempt to evaluate the depression, anxiety and stress of the general people living under the covid enforced lockdown in various cities of the world and also to understand how they are utilizing their lockdown time to cope with such an unprecedented isolation and quarantine. to the best of our knowledge, this study was among one of the first multicentric collaborative studies to investigate the immediate impact of the covid-19 lockdown on the mental health and coping strategies of the general public across the world in five main cities lockdown cities. in our current study, we found that there was a significant level of depression and anxiety in our participants and it differed in between male and females (p value < 0.05). the results were in concordance with the earlier reports from china, india and italy [9,11,24,] . rossi et al., italian general population reported that depression, anxiety, insomnia, high perceived stress and adjustment disorder were 37%, 17.3%, 20.8%, 7.3%, 21.8% and 22.9%, respectively among the 18,147 respondents of the survey [30] . in connection with the covid-19 pandemic enforced lockdown, a study by wang et al. [33] , in china, with 1210 respondents found rates of 30% of anxiety and 17% of depression among participants. in another survey-based study by qiu et al. [34] , with more than 50.000 chinese participants 35% of the participants reported trauma-related distress symptoms, with women and young adults showing significantly higher psychological distress. in their study, zhang et al [25] reported that nearly 85% of respondents reported their worries about covid-19, and over 20% reported at least one form of mental distress in line with previous acute emergencies. also, covid-19 outbreak significantly reduced the young adults' sleep quality and thereby increased their global negative emotions, especially stress and anxiety. our results also tell the similar story of increased dass scores reflecting increased mental distress among the participants especially indians and pakistanis. also, in our study majority of the participates strongly agreed to using sleep as one of the best coping strategy in the current lockdown time, which was consistent with a previous study revealing that individuals with better sleep quality showed reduced morbidity rates of post-traumatic stress disorder [25, 34, 35] . furthermore, we also found that the participants used the range of strategies to manage their time in quarantine with a varying degree of agreement depending upon their personal choices and likings. among the top coping strategies that were used for spending time and coping the stress during the lockdown with highest levels of agreements were: a) watching television for entertainment, b) social is the first kind of study to report on the coping strategies. please see the figure 1-18 and table?? for more in-depth information. sleeping and eating well was utilized by most of the professionals as the strategy to cope with the stress. 63.6% of office administrators 44.9% of students, and 36.4% of engineers etc strongly agreed with utilizing eating well as the strategy to be used in lockdown. also, 53.1% of students, and 31.4% of academicians strongly agreed with proper sleeping as strategy to cope with stress. these results are in tune with the study by zang et al. [25] , in chinese population. also, taking solace in prayers was also regraded as coping strategy by students (40.8% = strongly agreed) and banking professionals (38.9% = strongly agreed). the depression and stress scoring of the participants was very high especially among indian and pakistani nationals. therefore, we recommend that during the lockdown and quarantine period it is imperative for the government to provide the free professional and psychological services to help cope with stress during the disease enforced lockdown and to make general people aware about the coping strategies which can be utilized to relive the stress and make effective use of spare time. this is the first study that has evaluated the mental health of respondents during covid lockdown using dass-42 across general population. however, this study has an important limitation in sample collection as we included only the well-educated and smart phone savvy participants who are well versed with the use of social media. also, the sample from different countries were not equal and stratified which might skew the results. also, this was a self-reported and voluntary study that could introduce a systematic bias as well. i would like to express their deep gratitude towards all participants who pro-actively participated in this study. my sincere gratitude to dr. zafrina majeed for helping in data collection in uk. i also acknowledge the help of many of my friends who voluntary dispensed the survey to various friends and colleagues for the collection of data. a novel coronavirus outbreak of global health concern the 2019 novel coronavirus disease (covid-19) pandemic: a review of the current evidence first case of 2019 novel coronavirus in the united states lockdown is the world's biggest psychological experiment -and we will pay the price covid-19 and community mitigation strategies in a pandemic the psychological impact of quarantine and how to reduce it: rapid review of the evidence nearly 3 billion people around the globe under covid-19 lockdowns study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic mass gathering events and reducing further global spread of covid-19: a political and public health dilemma mental health outcomes among frontline and second-line health care workers during the coronavirus disease 2019 (covid-19) pandemic in italy covid-19 pandemic and its impact on mental health of healthcare professionals public mental health crisis during covid-19 pandemic the icd-10 classification of mental and behavioural disorders. 1992. world health organization common mental disorders and disability across cultures the exploratory models of mental health amongst low-income women and health care practitioners in lusaka. zambia somatic and psychological models of common mental disorders in india the structure of negative emotional states: comparison of the depression anxiety stress scales (dass) with the beck depression and anxiety inventories manual for the depression anxiety stress scales test anxiety, depression and academic performance: assessment and management using relaxation and cognitive restructuring techniques kaplan and sadock's synopsis of psychiatry: behavioral sciences/clinical psychiatry perception of stress and coping strategies by medical students at king saud university stress coping strategies among medical students and trainees in saudi arabia: a qualitative study impact of the covid-19 pandemic on mental health and quality of life among local residents in liaoning province, china: a cross-sectional study mental health problems during the covid-19 pandemics and the mitigation effects of exercise: a longitudinal study of college students in china zoonotic origins of human coronaviruses fenner and white's medical virology a new threat from an old enemy: re-emergence of coronavirus (review) the forgotten plague: psychiatric manifestations of ebola, zika, and emerging infectious diseases covid-19 pandemic and lockdown 1 measures impact on mental health among the general population in italy. an n=18147 web-based survey sars control and psychological effects of quarantine factors influencing compliance with quarantine in toronto during the 2003 sars outbreak immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china a nationwide survey of psychological distress among chinese people in the covid-19 epidemic: implications and policy recommendations prevalence and predictors of ptss during covid-19 outbreak in china hardest-hit areas: gender differences matter key: cord-294945-hcf7gsv8 authors: lin, k.h.; chen, l.f.o.; li, s.d.; lo, h.f. title: comparative proteomic analysis of cauliflower under high temperature and flooding stresses date: 2015-02-12 journal: sci hortic (amsterdam) doi: 10.1016/j.scienta.2014.12.013 sha: doc_id: 294945 cord_uid: hcf7gsv8 high-temperature and waterlogging are major abiotic stresses that affect the yield and quality of cauliflower. cauliflower cultivars ‘h41’ and ‘h69’ are tolerant to high temperature and flooding, respectively; however, ‘h71’ is sensitive to both stresses. the objectives of this study were to identify the proteins that were differentially regulated and the physiological changes that occurred during different time periods in ‘h41’, ‘h69’, and ‘h71’ when responding to treatments of flooding, 40 °c, and both stresses combined. changes in the leaf proteome were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof-ms) and identified by mascot peptide mass fingerprint (pmf) and database searching. stress treatments caused significant reductions in electrolyte leakage, chlorophyll fluorescence fv/fm, chlorophyll content, and water potential as stress times were prolonged. by the comparative proteomic analysis, 85 protein peaks that were differentially expressed in response to combination treatments at 0, 6, and 24 h, 69 (33 in ‘h41’, 29 in ‘h69’, and 9 in ‘h71’) were identified, of which were cultivar specific. differentially regulated proteins predominantly functioned in photosynthesis and to a lesser extent in energy metabolism, cellular homeostasis, transcription and translation, signal transduction, and protein biosynthesis. this is the first report that utilizes proteomics to discover changes in the protein expression profile of cauliflower in response to heat and flooding. cauliflower (brassica oleracea var. botrytis), a member of the brassicaceae family, is an economically and nutritionally important cole crop. the optimum mean temperature range for growing cauliflower is 18-25 • c. high temperatures cause cauliflower to form uneven and loose heads, puffy buds, yellow eyes and leaves in the head, narrow leaves, reduced leaf growth, delayed initiation of heading, and increased petiole-to-blade ratio, all of which lower the quality of or even make the heads unmarketable (wurr et al., 1996; nowbuth and pearson, 1998) . however, heat-tolerant cultivars are able to form heads at mean temperatures higher than 25 • c. to expand the area of food production, crops are grown under stressful environments that likely lead to lower yields. the main contributing factors in the reduction of yield and quality in these areas are variations in climatic conditions such as flooding caused by rains (lin et al., 2010) . heavy rainstorms and standing water can leave soils saturated for days before draining, making waterlogging a problem in many parts of the world. air pockets in the soil become filled with water during saturation, thus creating hypoxic conditions followed by anoxia. flooding causes oxygen starvation, which is a consequence of the relatively slow diffusion of gases in water and from oxygen consumption by plant roots (takeshi and julia, 2004) . rapid leaf chlorosis is seen when cauliflower is subjected to waterlogging (shih et al., 2013) . flooding from large rainfall events is a major risk to fresh-market cauliflower production in taiwan, and most cauliflower cultivars are unable to tolerate flooding during typhoon-caused heavy summer rains. waterlogging and increasing temperatures associated with global warming are a growing concern, as they limit plant growth and productivity, especially in temperate species. over the past decade in the tropics and subtropics, there have been considerable increases in production because of the availability of new, tropically adapted cultivars, resulting in increased farmer incomes. in the field, the co-occurrence of several abiotic stresses rather than individual stresses are most damaging to crop production (mittler, 2006) . many physiological changes occur during high temperature and flood stressing that result in increased heat and flood http://dx.doi.org/10.1016/j.scienta.2014.12.013 0304-4238/© 2014 elsevier b.v. all rights reserved. (hf) tolerance. the physiological mechanisms of hf tolerance are extensively studied in various plant species; however, our current understanding of the molecular biology of acquired tolerances to high temperature and waterlogging are still limited, and relatively little is known about proteins that are critical for controlling these dual mechanisms (wahid et al., 2007) . understanding these processes requires the identification and analysis of major proteins that underlie stress-regulatory networks. plant adaptations to environmental stresses depend on the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of stress-related proteins. knowledge of hf-responsive proteins is critical for further understanding of the molecular mechanisms of stress tolerance. plants respond to stress in part by modulating protein regulation, which eventually leads to restoration of cellular homeostasis, neutralization of toxins, and recovery of growth. however, there are no reports on the effect of hf stresses on the functioning of cauliflower. proteins are the direct effectors of the response of plants to stress. they not only include enzymes that mediate changes in the levels of metabolites but also serve as components of the transcription and translation machinery. in recent years, methods for analyzing the proteome have advanced considerably, and together with emerging sequence information in crops, plant proteomics has become increasingly useful for understanding gene functioning and networks in response to environmental stimuli. proteomics is a powerful tool for the quantitative analyses of different biochemical pathways including plant stress-related responses. comparative proteomic analyses of plants subjected to specific stress conditions allow the exploration of various defense-related mechanisms. for instance, proteomics approaches for the comparative analysis of protein abundance between untreated and stress-treated or tolerant and intolerant rice plants have greatly facilitated the study of plant cellular stress responses (komatsu et al., 2003) . therefore, identifying novel proteins and studying their differential display patterns in response to hf stresses will provide the molecular and physiological bases for improving tolerance to hf by cauliflowers. understanding the basis of hf stress signaling and tolerance mechanisms in cauliflowers is required for engineering local cauliflower genotypes that are more tolerant to heat and flood stressing. this goal can be achieved by deciphering the physiological and proteomic responses of cauliflower genotypes, heat-tolerant 'h41', flood-tolerant 'h69', and hf-sensitive 'h71' to flood stress at 40 • c. the objective of this study was to identify the leaf proteins that are differentially regulated in response to hf stress using a pro-teomelab pf-2d aided by improved databases. our hypothesis was that both hf stresses trigger plant responses that result in quantifiable changes in the proteins of 'h41', 'h69', and 'h71'. proteomic characterization and functional analysis should facilitate a better understanding of the hf response mechanisms in brassica so that effective strategies for the genetic improvement of hf-tolerant plant cultivars can be established. to the best of our knowledge, there are no published reports addressing the identification of hfresponsive proteins in brassica using a proteomic approach. 2.1. plant materials, culturing, and heat-and flood-stress treatments seeds of cauliflower (b. oleracea var. botrytis) 'h41', 'h69', and 'h71' were obtained from chin-long seed co. (tainan, taiwan). 'h41' is a heat-tolerant cultivar used especially in warm-subtropical regions such as southern taiwan, where average day temperatures reach as high as 40 • c during the summer (june-august). 'h69' is a popular cultivar grown in taiwan and is flood-tolerant during the rainy season, particularly in rain-fed lowlands. 'h71' is a heat-and flood-sensitive cultivar, and mostly grown during winter in taiwan due to its cold tolerance. seeds were sterilized with 1.5% (v/v) sodium hypochlorite, rinsed with distilled-deionized (dd) h 2 o, and sown in a commercial potting soil mixture, and germinated seedlings were transplanted into 12.7-cm diameter plastic pots and raised in a growth chamber under 300 mol m −2 s −1 light with a 14 h photoperiod and 22/18 • c day/night temperatures at a relative humidity (rh) of 80%. plants were watered three times a week and fertilizer (17:36:39, n:p:k) applied once a week to maintain optimal growth for 30 days before the imposition of stress treatments. pots containing 'h41' and 'h69' plants were subjected to four treatments: non-flooding at 20 • c (nfc, as control), flooding at 20 • c (fc, control temperature), non-flooding at 40 • c (nfh, high temperature), and flooding at 40 • c (fh), for periods of 0, 6, 12, 24, 48, 72, and 96 h in four growth chambers having a 14 h photoperiod at 300 mol m −2 s −1 radiation and 80% rh (lin et al., 2010) . in the case of flooding treatments, pots were randomly placed in 28 cm × 14 cm × 14 cm plastic buckets and subjected to flooding by filling the buckets with tap water to 5 cm above the soil surface. pots were removed from the buckets at different times following flooding, and plants were removed and their leaves from each plant clipped, frozen in liquid nitrogen, and stored at −80 • c in an ultrafreezer until used. three replicates of each time period for the four treatments were randomly placed in a growth chamber. the experiment was performed twice independently in a randomized design for growth environment, sampling day, and physiological analyses. 2.2. determination of electrolyte leakage (el), chlorophyll fluorescence (cf), chlorophyll content (cc), and water potential (wp) cell membrane stability was estimated by measuring leaf ion leakage according to the method of huang and guo (2005) . leaves were excised and immersed in 15 ml of distilled water in test tubes overnight at room temperature. the initial conductivity of the water was determined using a conductivity meter (model cdm 210, radiometer, cedex, france). tubes were placed in boiling water for 15 min and then cooled to room temperature, and conductivity was again determined. the relative el (%) was calculated as the ratio of conductivity before boiling to that after boiling. cf components were quantified with a portable modulated fluorometer (mini-pam photosynthesis yield analyzer, walz, effeltrich, germany). the measurement of variable fluorescence to (fv)/maximum fluorescence level in light-adapted leaves (fv/fm) was previously described (lin et al., 2007) . relative cc per unit leaf area was determined using a spad (soil plant analysis development) analyzer (spad-502 chlorophyll meter, konica minolta, tokyo, japan). wp (in bars) was measured on the third leaf from the top of each plant using a pressure chamber (plant water system, skye skpm 1400, tokyo, japan) (sairam et al., 1998) . the data shown in tables 1-4 represent the means of at least two independent sets of experiments with similar results. measurements of physiological parameters were analyzed by a three-factor completely randomized anova that compared cultivars, treatments, and time periods. for significant values, means were separated by a least significant difference (lsd) test at p ≤ 0.05 using pc sas 8.2 (sas institute, cary, nc, usa). because the negative effects of flood stressing on hf-sensitive 'h71' at 40 • c were observed after 48 h of stress treatments (see section 4), 'h41', 'h69', and 'h71' plant leaves subjected to hf conditions for 0, 6, and 24 h were used for subsequent proteomics analysis. proteins were extracted according to a previously published method (yan et al., 2013) with some modifications. briefly, two grams of plant leaves were ground in liquid nitrogen and crude protein extracts solubilized in 7 ml of extraction buffer containing 0.5% sds (sodium dodecyl sulfate), 5% ␤-mercaptoethanol, 10% glycerol, 0.2% polyvinylpyrrolidone, and 50 mm tris-hcl, ph 8. after 30 min of incubation at 4 • c, samples were centrifuged for 30 min at 15,000 × g at 4 • c; these two steps were done twice. the extraction buffer was further replaced by 25 ml of start buffer (50 mm tris-hcl, ph 8) with a pd-10 column (ge healthcare) according to manufacturer (proteomelab pf-2d kit, beckman coulter) protocols. protein concentrations in the samples were determined using the bradford assay (bio-rad, hercules, ca, usa) and bovine serum albumin (bsa) was used to generate a standard curve. a 200 g aliquot of the total protein sample was passed through a 0.45 m filter before injection into the chromatography column. the high performance chromatofocusing (hpcf) column was treated according to manufacturer instructions (proteomelab pf-2d protein fractionation system, beckman coulter, fullerton, ca, usa). briefly, the column was washed with 10 volumes of water at a flow rate of 0.2 ml/min for 45 min and then equilibrated with 30 volumes of start buffer for 130 min at 0.2 ml/min. after equilibration, each sample was introduced with a manual injector into the column and absorbance of the column effluent was monitored at 280 nm. in the first dimension, proteins were bound to a strong anion exchanger and eluted with a continuously decreasing ph from 8.0 to 4.0. fractions were collected at ph intervals of 0.3 in a 96 deepwell plate. proteins eluted in the gradient were then separated in the second dimension using high performance reversed phase (hprp) chromatography. fractions were separated from the second dimension with an rp-hplc column using two solvents: 0.1% trifluroracetic acid (tfa) in hplc water (solvent a) and 0.08% tfa in acetonitrile (acn) (solvent b) (lee et al., 2008) . separation was performed at 50 • c with a flow rate of 0.75 ml/min and protein fractions were detected by uv absorbance at 214 nm. equilibration was achieved with solvent a for 10 min followed by solvent b for 5 min prior to each injection. from the selected first dimension fractions, 0.2 ml were injected, run for 2 min, and the column eluted with a linear gradient of 0-100% solvent b for 25 min. thereafter, solvent b was continued for 5 min, followed by re-equilibration with 100% solvent a for 10 min (irar et al., 2010) . fractions from the second dimension were analyzed with karat tm version 7.0 (beckman coulter). protein extraction and two-dimensional gel electrophoresis (2-de) were carried out on three independent technical replicas of the bulk samples. proteins were in-gel digested with trypsin in the automatic investigator progest robot of genomic solutions. briefly, excised gel bands were washed sequentially with 50 mm ammonium bicarbonate (nh 4 hco 3 ) buffer and acn. proteins were reduced and alkylated, respectively, by treatment with 10 mm dithiothreitol (dtt) solution for 1 h at 60 • c and treatment with a 50 mm solution of iodine acetamide. after sequential washings with buffer and acn, proteins were digested overnight at 37 • c with 120 g/ml of trypsin. tryptic peptides were extracted from the gel matrix with 10% formic acid and acn, and extracts were then pooled and dried in a vacuum centrifuge. digested peptide solutions were desalted with zip-tip pipet tips (nikkyo technos, tokyo, japan) and redissolved in 10 l of 0.1% tfa in 50% acn according to manufacturer instructions. proteins excised from two-dimensional gels were analyzed in matrix-assisted laser desorption ionization time-of-flight (maldi-tof, 4700 proteomics analyzer, applied biosystems) mass spectrometers. a 1 l aliquot was mixed with the same volume of a matrix solution (5 mg/ml ␣-cyano-4-hydroxycinnamic acid in 0.1% tfa in 50% acn) and spotted on a maldi plate. ms spectra were acquired in the positive reflector mode, with 500 shots per spectrum being accumulated. three major peaks were selected for further characterization by ms/ms analysis. ms spectra were acquired using collision-induced dissociation (cid) with atmospheric air as the collision gas, the ms 1 kv positive mode being used. the peak lists for ms/ms spectra from same-replicate samples were merged into a single file prior to database searching. the pkl and mgf files were searched against the swiss-prot database using mascot (http://www.matrixscience.com; london, uk). search parameters were set as follows: 1 missed cleavage, fixed modification, peptide charge ±1, and variable modifications were carbamidomethyl of cysteine and oxidation of methionine. trypsin was specified as the proteolytic enzyme. peptide tolerance and ms/ms mass tolerance were 50 ppm and 0.25 da, respectively. peptide mass fingerprinting (pmf) match confidence was based on the mowse score and confirmed by accurate overlapping of matched peptides with mass spectrum major peaks. scores greater than 67 (p < 0.05) were considered positive. peaks with multiple proteins detected by ms were not considered. only significant hits, as defined by mascot probability analysis (p < 0.05), were accepted. characteristic physiological responses of plants to different temperature and waterlogging treatments were evaluated. table 1 illustrates the comparison of fv/fm values under four treatments at seven different times in leaves of three genotypes of cauliflower. levels of fv/fm in all plants progressively decreased as flood stress and heat stress (nfh and fh) durations were extended. fv/fm in 'h41' plants showed significantly lower values after 6 h with table 5 identification of differentially expressed proteins found in cauliflower 'h41' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 6 h. peak ( table 4 lists the different points in time that waterlogging and high temperature treatments were monitored by measuring the changes in wp. when different treatments across time were compared, wp with stress treatments was significantly lower (more negative) than those with nfc treatment from 12 to 96 h for all genotypes, indicating that high temperature and/or flooding induced a decrease in leaf water level, subsequently affecting leaf wp. the trends in wp differed in all stressing treatments from 12 to 96 h in all plants. thus, the wp of the different genotypes responded differently to specific stresses. the lowest value in wp (−2.27 mpa) was detected at 48 h with exposure to nfh treatment in 'h71'. proteome alterations in the three genotypes in response to short-term exposures to flood and high-temperature conditions were compared relative to treatments and controls. the proteome-lab system uses two-dimensional liquid chromatography based on high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension, and has become available for sample fractionation and more resolution at extreme ph values (soldi et al., 2005; pirondini et al., 2006) . protein peaks were identified and their expression patterns analyzed. protein fractions were separated according to isoelectric point (pi) and hydrophobicity and detected in a ph range of 8.0-4.0 by proteomelab pf-2d. proteins were analyzed by maldi-tof-ms and identified via pmf and database searching by their calculated molecular weight, pi score, and percent coverage, and their database accession numbers and alteration values among stress treatments are represented in tables 5-8 . changes in the protein levels of 'h41' exposed to fh stresses at 0 and 6 h and their differentially expressed protein peaks are listed in table 5 . compared to nfc control treatment, two protein peaks (291 of phosphoglucosamine mutase and 292 of rotidine 5 -phosphate decarboxylase) under fc treatment for 6 h were up-regulated (supplementary fig. s1a) . furthermore, the expression level of s-adenosylmethionine synthetase (peak 294) was increased during high-temperature stressing over 6 h (supplementary fig. s1b ). two identified peaks, 301 of 16 kda calcium-binding protein and 302 of acetyl-coenzyme a carboxylase carboxyl transferase subunit beta, were also up-regulated when 'h41' plants were treated with fc for 6 h in comparison to nfc treatment at 0 h (supplementary fig. s1c) . nine protein expression patterns were detected in 'h71' plants under high-temperature and flood stress at 6 h, and the dynamic changes in the level of each protein are displayed in table 6 . compared to nfc treatment at 0 h, nfh treatment for 6 h showed that the abundances of peaks 271 (phosphoserine aminotransferase), 272 (imidazole glycerol phosphate synthase subunit hisf), table 6 identification of differentially expressed proteins found in cauliflower 'h71' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 6 h. peak ( 273 (phosphoribosyl formyl glycin amidine synthase 1), and 295 (probably hydrogenase nickel incorporation protein hypa) were up-regulated (supplementary fig. s2a) . moreover, the abundance of peak 296 (putative holliday junction resolvase) in 'h71' plants was also up-regulated during 6 h of fc treatment as compared to nfc treatment at 0 h (supplementary fig. s2b ). the last comparison includes four proteins (peaks 281, 282, 297, and 304, namely trna-methyltransferase, arginyl-trna synthetase, elongation factor g, and methionyl-trna formyltransferase, respectively) whose abundances were consistently up-regulated during flood stressing for 6 h at 40 • c. interestingly, all peaks were up-regulated after all treatments for 0 h and 6 h in both 'h41' (table 5) and 'h71' (table 6) plants. the identified proteins showed altered expressions by upregulation or down-regulation in 'h41' plants at the 0 h and 24 h time points when combination treatments were compared (table 7) . only six protein peaks (231, 232, 241, 251, 252, and 253 ; supplementary fig. s3 ) from comparative proteomic analyses between fh and nfc for 24 h were down-regulated, all others being up-regulated. there were 18 peaks found in fh vs nfc for 24 h, with the remaining 12 peaks being among other comparisons, including fh vs nfh, fc vs nfc, nfh vs nfc, nfh vs fc, and fh at 24 h vs nfc at 0 h. the carbohydrate metabolism related protein, fructose-1,6-bisphosphatase (peak 232), was down-regulated under fh treatment compared to nfc treatment. nevertheless, the photosynthetic-related proteins ribulose bisphosphate carboxylase (rubisco) small chain (peak 236) and large chain (peak 255) were increased in abundance in 'h41' under flooding for 24 h. additionally, dna mismatch repair protein muts was found in peak 253 with down-regulation (fh vs nfc) and peak 254 with up-regulation (fc vs nfc) in protein abundance through a homologue search. differentially expressed proteins identified in 'h69' plants at 0 h and 24 h under stressing are shown in table 8 . the protein abundances of eight peaks (258, 259, 2511, 2512, 2513, 264, 265 , and 266 of ribose import atp-binding protein, phenylalanyl-trna synthetase, rubisco large subunit, gmp synthase, atp synthase subunit, and phosphoribosylformyl glycinamidine synthase) were consistently down-regulated during the fh condition when compared to nfc treatment for 24 h, but the other 33 peaks were up-regulated in protein abundance. mitochondrial inner membrane protease (peak 221) and rubisco small subunit (peaks 222 and 223) were up-regulated in 'h69' upon fc treatment compared to nfc after 24 h ( supplementary fig. s4a ). peaks 2514 and 2515, respectively identified as uncharacterized 37.2 kda and sec14 cytosolic factor, were increased under heat stress at 24 h ( supplementary fig. s4b) . meanwhile, rubisco small subunit (peak 268) was also increased in 'h69' under fh at 24 h as compared to nfc at 0 h ( supplementary fig. s4c) . notably, the majority of peaks found in 'h69' at 24 h represented rubisco down-regulation (peaks 2511, 264, and 266) and up-regulation (peaks 2312, 2314, 2318, 222, 223, 2310, 2311, 246, 247, and 268) in protein abundance. because flood and heat often co-occur in stress-prone environments, this study compared the combined effects of flood and heat with those of the single stresses on plant physiology. in general, the changes in plant physiology were greater under the combined treatment than under heating or flooding alone. the differences in responses to flood and heat in fv/fm, cc, el, and wp seen between cultivars suggested that the three genotypes have unique mechanisms for coping with environmental stresses. the heat-tolerant 'h41' genotype showed significantly higher fv/fm, cc, and wp values (tables 1, 2 and 4) and lower el% (table 3 ) than the sensitive 'h71' genotype when heated for 24 h under nfh treatment, which reflects its tolerant nature. the exposure to flooding for at least 6 h under fc treatment provoked greater reductions in fv/fm and cc values in 'h71' plants than 'h69' plants (tables 1 and 2 ). in addition, flooding also significantly decreased the el in 'h69' plants relative to 'h71' plants after 48-96 h ( table 3 ), indicating that 'h69' is flood-tolerant. similar losses of fv/fm, cc, and wp and an increase in el were observed in 'h71' under flooding and high temperature stresses (fh), but responded at different levels over time (tables 1-4) . table 7 identification of differentially expressed proteins found in cauliflower 'h41' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 24 h. peak ( a soil water potential of approximately −2 mpa was used in greenhouse experiments to simulate field situations under severe stress conditions (ashoub et al., 2013) . the leaf wp of 'h71' plants under high-temperature treatment was significantly lower than in 'h41' and 'h69' plants after 12 h, indicating that 'h71' plants suffered from heat exposure. a lower osmotic potential is responsible for maintaining turgor when water loss occurs due to high temperature. thus, high electrical conductivity due to a high concentration of electrolytes in the leaf sap of 'h71' plants should lead to alterations in membrane permeability and a reduced ability to retain solutes and water during high temperature and waterlogging. however, the ability of 'h41' and 'h69' plants to tolerate heat and flood stress, respectively, strongly depends on adjustments in water balance and ionic leakage. a decline in wp and an increase in el during heat and flood stress were associated with leaf water deficits as well as increases in ionic leakage. at the physiological level, the many effects of flooding and heat stresses indicate the importance of protecting plants from oxidative damage caused by the overproduction of ros that is elicited by increased ion leakage (kangasjarvi et al., 2008) . the chlorophyll fluorescence emission parameter, fv/fm, which is widely used as a proxy for the maximum quantum efficiency of ps ii photochemistry, was correlated with flooding and high temperature tolerance. in healthy leaves, the fv/fm value is close to 0.8, which is a typical value for uninhibited plants. a lower table 8 identification of differentially expressed proteins found in cauliflower 'h69' plants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) in comparison to combination treatments for 0 and 24 h. peak ( value indicates that some proportion of the psii reaction centers are damaged, which is often observed in plants under stress (camejo et al., 2006) . flooding and high temperatures inhibit photosynthetic co 2 fixation and damages photosynthetic electron transport at the site of psii where there exists a very sensitive photosynthesis apparatus. this reduction in the co 2 assimilation rate observed in 'h71' plants was generated by effects on the calvin cycle and also on psii functioning. exposure of 'h71' plants to flooding and high temperature may lead to reductions in net photosynthetic rate, stomatal closure, and cell activities that damage photosynthetic membranes (jiang and huang, 2001) . these results suggested that the chlorophyll fluorescence parameters were stress specific and were not expressed solely in response to an increasing excess of photon energy. chloroplast development in 'h71' plants may be particularly sensitive to high temperature and flooding. alternatively, the pigments of the sensitive cultivar might have been destroyed because of a high sensitivity to oxidative stress. 'h71' plants suffered greater cc losses than 'h41' and 'h69' plants after 24-96 h of exposure. flooding and heat may induce stomatal closure and consequently reduce cc levels and wp as well. typically, the amount of cc is reduced by stress. this is consistent with our observations that chlorophyll loss was more pronounced in waterlog-and heat-sensitive 'h71' plants in accordance with the more pronounced and increased visible symptoms of leaf injury. leaf curling or folding were the initial and most obvious changes observed. most 'h71' leaves progressively became necrotic, epinastic, or wilted over the course of time; however, most 'h41' and 'h69' leaves visually appeared to be green and healthy after 72 h of flooding and high temperature (photos not shown). along with visual symptoms, reduced cc could be used to monitor flooding and heat damage in green or senescent leaves. attempts have been made to breed for increased flooding tolerance and modify brassica cultivation or management practices to avoid injury from flooding and heat. environmental stresses represent the most limiting conditions for horticultural productivity and plant exploitation worldwide. important factors among them are water and temperature. flooding and high temperature are major abiotic stresses resulting in serious problems for the growth and yield of flood-and heat-sensitive plants. it is necessary to identify the physiological characteristics that reflect their complex underlying genetic make-up. these easily measured physiological biomarkers could be used as generic tools to develop a reliable method for selecting cauliflower cultivars having flood-and heat-stress resistance. tables 1-4 demonstrate that the chlorophyll losses in 'h71' plants were in concert with increasing el accumulations after 6 h of flooding and heat treatments, which is an index of oxidative damages to cell constituents in general. this suggests that fv/fm and cc can be effectively used as reporter signals in screening for flooding and high temperature tolerances. these easily measured physiological biomarkers could be used as generic tools for developing a reliable method to select 'h41' and 'h69' cultivars having flooding and heat-stress resistances. these findings are important for farming in high-temperature areas and wetlands or other areas subject to short and intense rainfall events. proteomics techniques are becoming indispensable for understanding how plants adapt to abiotic stresses, and have promoted the identification of numerous stress-related proteins and the pathways in which they function (kosová et al., 2011) . changes in protein accumulation under stress are directly related to the physiological phenotypic responses of plants to stress. the proteomic responses to high-temperature or flood stresses are known for several plants, including rice (lin et al., 2005) , wheat (majoul et al., 2004) , barley (süle et al., 2004) , soybean (komatsu et al., 2013) , radish (zhang et al., 2013) , arabidopsis thaliana (palmblad et al., 2008) , agave americana (shakeel et al., 2013) , and populus euphratica (ferreira et al., 2006) . in the present study, we analyzed physiological and proteomics data in different genotypes of cauliflower under conditions of high-temperature and waterlogging stresses over different time periods. approximately 250 peaks were detected at the tested time points, whereas 85 peaks were successfully observed to be differentially regulated (14 down-regulated, 71 up-regulated) and expressed at a minimum of one time point. among them, 33, 29, and 9 proteins were identified in 'h41', 'h69', and 'h71' plants, respectively, indicating that they were regulated in a genotype-specific manner ( supplementary fig. s5 ). rubisco small and large chains, 16 kda calcium-binding protein, molybdenum cofactor biosynthesis protein a, and phosphoserine aminotransferase were observed in both 'h41' and 'h69' plants. phosphoribosylformyl glycinamidine synthase and arginyl-trna synthetase were both found in 'h71' and 'h69' plants ( supplementary fig. s5 ). moreover, in 'h41' plants, peaks 253 and 254 were both identified as dna mismatch repair protein muts, while 291 and 263 were identified as phosphoglucosamine mutase (tables 5 and 7 ). these sister peaks may represent close homologues, but this could not be resolved based on mass spectrometric data and might instead be isoforms resulting from differential post-translational modifications (rollins et al., 2013) . as stress times increased, additional responsive proteins continued to be identified. for instance, 5 (table 5 ) and 30 (table 7) peaks were observed in 'h41' under stress treatments for 6 h and 24 h, respectively. in this study, some of the proteins identified were well characterized in terms of response to stressing, while others were not. the biological functions of differentially regulated proteins include roles in photosynthesis, energy metabolism, cellular homeostasis, response to stimuli, transcription and translation, protein biosynthesis, mediation of signal transduction, and other functions. for survival, plants must respond to flood and heat stresses in a different manner from regulating protein expressions for biochemical and physiological adaptations. photosynthesis is one of the systems that is most sensitive to high-temperature stress. changes in environmental temperatures are primarily reflected in photosynthesis, triggering a response that is aimed toward the best possible performance under new conditions. for this, a balance is sought between the energy of absorbed light, carbon assimilation, and consumption by metabolic sinks. several studies have shown that high-temperature stress can significantly inhibit the rate of photosynthesis (yan et al., 2013; salvucci and crafts-brandner, 2004) . in our study, rubisco units (small and large chain) related to photosynthesis were differentially expressed and regulated by combination treatments and among genotypes. for example, the up-regulation of rubisco was detected in 'h41' (peaks 236 and 255, table 7 ) and 'h69' (peaks 2312 (peaks , 2314 (peaks , 2818 (peaks , 222,223, 2310 (peaks , 2311 . however, the level of expression of the rubisco large subunit was down-regulated in 'h69' during fh treatment for 24 h (peaks 2511, 264, and 266, table 8 ). rubisco proteins resist high-temperature and flood stresses by inhibiting photosynthesis and other nonessential metabolic processes. in contrast, to increase energy metabolism, the expression of proteins related to redox homeostasis and response to stimuli were up-regulated, thereby maintaining physiological balance during stress. rubisco catalyzes the first major step of carbon fixation in photosynthesis. affinity of rubisco for co 2 decreases with increasing temperature (yan et al., 2013) . the diminished capacity of rubisco and its low affinity for co 2 suggest that carbon fixation or assimilation was highly inhibited when 'h69' plants were exposed to stress, especially under the high temperature and flooding stresses ( table 8 ) that lead to reduced rates of photosynthetic co 2 assimilation. photorespiration serves as an energy sink, preventing the over-reduction of the photosynthetic electron transport chain and photoinhibition. lee et al. (2007) reported that high temperature over 2 h significantly inhibits the activity and quantity of rubisco subunits in rice seedlings. the effects of drought stress on rubisco represent reductions in rubisco activity (parry et al., 2002) . exposure to salt stress brings about a reduction in the overall growth and productivity of plants by disturbing the function of vital components of photosynthesis, such as psii and rubisco (ghaffaria et al., 2014) . consequently, a strong reduction in fv/fm value (table 1 ) and cc content ( table 2 ) while maintaining photosynthesis under stress after 24 h was seen in all plants. some researchers report its up-regulation (ge et al., 2012; budak et al., 2013) , whereas others find downregulation (gaoa et al., 2011) or even both (guoa et al., 2012) in response to abiotic stresses. as a result, an up-regulation in rubisco levels may also indicate an increase in the photorespiration rate. molecular chaperones, including heat shock proteins (hsps), are key components of innate immunity in plants. they have major roles in protein folding and signal-transduction networks, cellcycle control, protein degradation, and protein trafficking (wang et al., 2004) . hsp chaperone pathways require energy in the form of atp hydrolysis in order to function. the atp-dependent clp proteases (hsp100) represent a class of hsp. in addition to their function as molecular chaperones, they function in protein disaggregation and protein degradation when removal of potentially harmful polypeptides arising from misfolding, denaturation, or aggregation are important for the maintenance of cellular homeostasis. the mechanism for rescuing proteins from aggregation is proposed to include the cooperation hsp100 (kregel, 2002; sarkar et al., 2011) . hsps are typically induced when cells are exposed to various types of environmental stresses. the synthesis of hsps under high temperatures is reported to be related to thermotolerance in plants, and plants with a decreased expression of hsps show compromised tolerance to acquired thermo-tolerance (charng et al., 2006) . hsps are closely related to the acquisition of heat resistance in plants. choi et al. (2012) identify several high molecular weight hsps whose expression was up-regulated significantly in pyropia tenera under high-temperature stress for 1 h and 3 h. furthermore, lee et al. (2007) report that several hsps are expressed in rice leaves during heat stress. high temperature might increase the potential risk of protein misfolding, and an active protein quality control system inside cells plays an important role in plant tolerance to heat stress. hsps are also increased by drought stress in the sugar beet (hajheidari et al., 2005) , wheat (demirevska et al., 2008) , and sugarcane (jangpromma et al., 2010) . in our study, peak 261 of atp-dependent clp protease proteolytic subunit 1 was up-regulated in response to heat stress for 24 h in 'h41' plants (table 7) . this suggests that the increased level of hsp100 under high-temperature stress allowed the heat-tolerant 'h41' to produce more energy through atp hydrolysis and the degradation or reactivation of damaged proteins and prevented protein misfolding, resulting in reestablishing normal protein conformations and cellular homeostasis. s-adenosylmethionine (sam) synthetase catalyzes the synthesis of sam that serves as a methyl group donor in transmethylation of proteins, nucleic acids, and a precursor in the biosynthesis of polyamines, biotin, and nicotianamine in plants (roeder et al., 2009) . sam in shoots underwent increased protein synthesis in a heat-tolerant barley cultivar and showed a reduced trend in an abiotic stress-susceptible cultivar (süle et al., 2004) . in our study, the expression of sam synthetase was significantly up-regulated (peak 294, table 5 ) in heat stressed 'h41' over 6 h. therefore, it is most likely that an increased amount of sam synthetase (as a result of the increased formation of sam methyl donors) in 'h41' contributes to its stronger heat tolerance compared to controls. in addition, peak 238 of adenylate kinase (adk) was observed in 'h69' under heat stress for 24 h (table 8) . adk catalyzes the salvage synthesis of adenine monophosphate from adenosine and atp (wang et al., 2010) . heat and flood treatments markedly increased the abundance of two elongation factors (efs): (1) elongation factor g was increased in 'h71' (peak 297) under flooding at 6 h ( table 6) and (2) transcription elongation factor grea was increased in 'h69' (peak 2816) under fh at 24 h ( table 8 ). the elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukaryotes. efs are the critical regulators of protein synthesis, which is influenced by high temperature and drought stress in arabidopsis (rizhsky et al., 2004) and rapeseed (mohammadi et al., 2012) . regulation of the transcriptional and translational machinery is considered to be an important component of cellular stress response. the increased expression of ef grea in 'h69' and ef g in 'h71' likely led to increased protein synthesis, which served to reduce the effects of treatment stress. plants under stress can respond by sensing and transferring stress signals through signal transduction networks. the response to stress is likely mediated through signaling pathways that regulate the expression levels of a range of proteins (hirayama and shinozaki, 2010) . in our study, gtp-binding protein lepa (peak 233, table 7 ) involved in signal transduction was identified. the up-regulation of the lepa protein in response to flooding at 40 • c might reflect the role of this protein in cell signaling under stress. ca 2+ -binding protein is mainly resident in the endoplasmic reticulum where it serves as a calcium modulator and chaperone of newly synthesized glycoproteins (nam et al., 2012) . the latter have functions in plant growth and development as well as biotic and abiotic stress responses. aghaei et al. (2008) indicated that ca 2+ -binding protein was up-regulated by salt stress in potato leaves. salinity induces ca 2+ accumulation in the cytosol that starts the signaling pathway. the modulation of intracellular ca 2+ levels is regulated by calcium-binding proteins, which, after activation, induce specific kinases (capriotti et al., 2014) . a 16 kda calcium-binding protein was identified as an up-regulated peak (301) under flooding (table 1) , suggesting the involvement of intracellular calcium homeostasis and signal transduction in 'h41' during waterlogging. atp synthase is a large multi-subunit transmembrane enzyme complex. the catalytic site for atp synthesis is localized primarily on the beta subunit. atp synthase is known to be involved in energy metabolism and was found in 'h69' plants upon fh stressing for 24 h (table 8) . down-regulation of the catalytic subunit of atp synthase subunit beta (peak 2513) suggested that the energy production systems of 'h69' were highly affected by heat and flood stresses. huseynova et al. (2007) also found that the atp synthase complex was less accumulated in drought-sensitive wheat 'giymatli-2/17' than in drought-tolerant azamatli-95 under water stress. in addition, impaired atp synthesis under drought is regarded as a major constraint to photosynthesis (flexas and medrano, 2002) . fructose-1,6-bisphosphatase (fbpase) is involved in carbohydrate metabolism during gluconeogenesis, and also an important enzyme in the calvin cycle that plays a crucial role in plant responses to some abiotic stimuli (fan et al., 2009) . the downregulation of fbpase in 'h41' under fh at 24 h (peak 232, table 7) indicated that gluconeogenesis was inhibited under stressing. an increase in ribosomal protein is a candidate for initiating a translation site, and the phosphorylation of ribosomal proteins is important in the regulation of protein synthesis (fatehi et al., 2012) . the 50s ribosomal subunit catalyzes the peptidyl transfer reaction of mrna-directed protein biosynthesis (sobhanian et al., 2010) . up-regulation of the 50s ribosomal protein l11p (# 2914) was observed under high temperature stress at 24 h in 'h69' (table 8) , indicating the resistance of 'h69' plants to the inhibitory effect of high temperature on protein biosynthesis. in addition, pentatricopeptide repeat (ppr) proteins are characterized by tandem arrays of degenerate 35 amino acid motifs (o'toole et al., 2008) and are thought to be rna binding proteins involved in posttranscriptional processes in mitochondria and chloroplasts (schmitz-linneweber et al., 2006) . meanwhile, ppr proteins are also essential for rna editing in chloroplasts (emi et al., 2005) . peak 2313 (pentatricopeptide repeat containing protein) of 'h69' was up-regulated under fh within 24 h ( table 8 ), suggesting that 'h69' was able to increase rna maturation and protein function in chloroplasts under fh. taken together, genetic differences are supported by the high number of proteins differentially regulated between genotypes. our data suggest that the early response of cauliflower to flooding and high temperature might be an important stress adaptation for survival following not only hypoxia and heat, but also direct damage to cells by flooding and high temperature. all of the identified proteins likely work cooperatively to reestablish cellular homeostasis under stress. the long term goal of our work is to help breed a competitively higher flood-and heat-tolerant cauliflower to be grown in lowlands during summer. the identification of the unique stress-responsive proteins will allow further dissection of the genetic basis of this transgressive performance in offspring. our results not only provide information for selecting lines having better tolerance to waterlogging and heat stresses, but also provide a basis for understanding cauliflower metabolic pathways and their cross-talk under stress. further verification of the correlative stress-responsive protein by gene expression analyses of these stressed-responsive genes, such as rubisco, efs, hsp100, fbpase, adk, atp synthase, sam synthetase, ppr protein, gtp-binding protein lepa, ca +2 -binding protein, and ribosomal protein l11p, would facilitate our understanding of the heat-and flood-response mechanism in cauliflowers. this study provides information on mechanisms underlying the contrasting responses to hf stresses. different genotypes showed differing physiological responses to combinations of stress treatments. all dynamics of the peaks were analyzed across all treatments, and a comparative analysis identified 85 stressresponsive proteins in cauliflower under short-term stressing. the majority of these stressed-responsive proteins were genotype specific. these identified proteins emerged as key participants in stress tolerance. most of the changes in the expression levels of these proteins in response to stressing were involved in photosynthesis. 'h69' plants resisted high-temperature stressing by inhibiting photosynthesis and other unnecessary metabolic processes. energy metabolism was increased and up-regulation of the expression of proteins related to cellular homeostasis, as well as those that respond to stimuli, served to maintain physiological balance during stress. concurrently, the expression levels of proteins related to transcription, translation, and signal transduction were also up-regulated to promote survival under hf stressing. the genetic variations identified in the proteomes, plant growth, and photosynthetic performance in response to flood and heat represent stress adaption mechanisms to be exploited in future cauliflower breeding efforts. proteome analysis of potato under salt stress comparative analysis of barley leaf proteome as affected by drought stress proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanolc-esi-ms/ms changes in photosynthetic 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of soybean leaves, hypocotyls and roots under salt stress proteome profile of human urine with two-dimensional liquid phase fractionation proteomic analysis of small heat shock protein isoforms in barley shoots plant responses to hypoxia -is survival a balancing act? heat tolerance in plants: an overview role of plant heat-shock proteins and molecular chaperones in the abiotic stress response proteomic alternations of brassica napus root in response to boron deficiency investigating trends in vegetable crop response to increasing temperature associated with climate change proteomic profile analysis of pyropia haitanensis in response to high-temperature stress proteomic analysis of heat stress response in leaves of radish (raphanus sativus l.) this research was supported by grants (nsc 97,98 -2313-b-002 -047 -my3) from national science council, taiwan, roc. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta. 2014.12.013. key: cord-304208-jwlc8fxj authors: vagni, monia; maiorano, tiziana; giostra, valeria; pajardi, daniela title: coping with covid-19: emergency stress, secondary trauma and self-efficacy in healthcare and emergency workers in italy date: 2020-09-03 journal: front psychol doi: 10.3389/fpsyg.2020.566912 sha: doc_id: 304208 cord_uid: jwlc8fxj coping with the coronavirus disease (covid-19) is a significant risk factor for the psychological distress of health workers. hence, this study explores the relationship between coping strategies used by healthcare and emergency workers in italy to manage the stress factors related to the covid-19 emergency, which may result in the risk of developing secondary trauma. we study differences between healthcare (n = 121) and emergency workers (n = 89) in terms of their coping strategies, emergency stress, and secondary trauma, as well as the relationships of these differences to demographic variables and other stress factors (instructions and equipment). for this purpose, we collected data from participants through the following questionnaires online: secondary traumatic stress scale – italian version, the coping self-efficacy scale – short form, an original questionnaire on stressors, and the emergency stress questionnaire (to assess organizational–relational, physical, decisional inefficacy, emotional, cognitive, and covid-19 stress). we performed a t-test, correlational analysis, and hierarchical regression. the analyses reveal that compared with the emergency worker group, the health worker group has greater levels of emergency stress and arousal and is more willing to use problem-focused coping. healthcare workers involved in the treatment of covid-19 are exposed to a large degree of stress and could experience secondary trauma; hence, it is essential to plan prevention strategies for future pandemic situations. moreover, individual efficacy in stopping negative emotions and thoughts could be a protective strategy against stress and secondary trauma. the coronavirus disease , or the acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), began spreading in china at the end of 2019 and, to date, represents an international health emergency without precedents in terms of its health, economic, and organizational effects on people's lives (world health organization, 2020) . after china, italy was the first country to be affected by this epidemic, with the first deaths on february 20, 2020, and a rapid increase in the spread of infection and mortality. covid-19 was first detected in northern italy, and it then spread, although at different rates of incidence, to the other regions. it was immediately evident that healthcare and emergency workers were at great risk of contagion and that protection and intervention protocols needed to be introduced in the absence of adequate points of reference because of the exceptional nature of the epidemic, the rate of spread of the infection, the seriousness of patients' health condition, and the mortality index. the extreme conditions in which health workers have had to work, especially in the most affected regions in northern italy, are indicated by the following data from the italian national institute of health (2020): over 150 doctors died and 25,000 other health workers were infected within the general context of the population of 30,000 deaths and 220,000 infections in a span of 11 weeks. it was also clear that the medical staff would experience serious psychological repercussions because of the working conditions as well as the difficulty of having scientific points of reference on care and intervention procedures. to this must be added the increase in workload, the extension of working hours and, for health workers, the frequent exposure to the suffering and death of their patients. therefore, healthcare and emergency workers were subjected to serious psychological as well as physical stress. hence, the aim of this study, which was also the aim of a previous study (vagni et al., 2020) , is to focus on the similarities and the differences in the stress management of two professional groups-healthcare and emergency workersduring the acute phase of the pandemic. both groups have had to deal with covid patients as frontline responders and have been exposed to the related risks of infection and psychological consequences, which, to date, have not been examined in detail through a comparative analysis. as regards the stress that they experience, the literature clearly explains that healthcare and emergency workers who intervene in emergency situations are exposed to the risk of developing dysfunctional reactions that can be identified at different levelsphysical and/or physiological (e.g., psychosomatic disorders, sleep/wake cycle alterations, and sense of tiredness); emotional (e.g., irritability, nervousness, agitation, anger, low self-esteem, and guilt); cognitive (e.g., distractibility, sense of ineffectiveness, and negative anticipation of events); and relational (e.g., increase in conflicts within emergency teams and/or with their organization/institution, and social withdrawal)-and may also develop reactions from secondary trauma (del missier et al., 2008; sbattella, 2009; argentero and setti, 2011; fraccaroli and balducci, 2011; bellelli and di schiena, 2012; walton et al., 2020) . faced with stressful events regarding which they lack previous experience and specific, necessary knowledge, and which cause tension owing to the need for rapid decision timings and a sense of responsibility, emergency workers may experience a sense of decision ineffectiveness. in fact, emergency situations are characterized by high levels of decisional and operational uncertainty with associated regret and guilt (del missier et al., 2008) . several studies have highlighted that insufficient instructions and a lack of personal protective equipment (ppe) are important predictors of stress for healthcare and emergency workers in large-scale emergencies (oh et al., 2017; du et al., 2020; el-hage et al., 2020; walton et al., 2020) . oh et al. (2017) highlighted that nurses involved in managing the middle east respiratory syndrome (mers) experienced lower levels of stress when the levels of goods supply and hospital training were higher. some studies have highlighted that frontline healthcare workers had lower secondary traumatization scores than non-frontline health workers and the general public in contrast to the findings of previous research on the sars outbreak in the same area in singapore (chan and huak, 2004) . according to barleycorn (2019) and tan et al. (2020) , these results may be due to the dedicated training and psychological support given to healthcare workers after the sars outbreak and demonstrate the validity of policy strategies for prevention of stress in the psychological health field. an analysis of 14 studies published from january to march 2020 aimed at investigating the stress experience of healthcare workers in facing covid-19 shows that health workers experienced symptoms of depression and anxiety related to this stressful experience. moreover, the severity of their symptoms was influenced by their age, gender, role, specialization, type of activity performed, and exposure to patients with covid-19; however, prevention, resilience, and social support interventions mediated their response to stress (bohlken et al., 2020) . in a review of the literature, spoorthy (2020) underlined that sociodemographic variables, such as age, gender, profession, and workplace, and psychological variables, such as poor social support and self-efficacy, affect the stress level experienced by health workers. in addition, covid-19 emerged as an independent stress risk factor. xiao et al. (2020) found that social support plays a role in reducing the anxiety levels in medical staff and increases their sense of self-efficacy. according to walton et al. (2020) , the specific stressors that health workers face in the covid-19 emergency are related to the organizational context. the challenges for medical staff include not only an increased workload but also a fear of infection, the need to work with new protocols that change frequently, and the use of ppe. in uncontrollable situations such as a pandemic, when specific action protocols are absent and limited resources are available, health workers must make individual decisions with a heavy burden of responsibility that may be contrary to their moral principles. for example, in the case of covid-19, they may have to choose which patients to save because only a few places are available in intensive care. in this regard, cai et al. (2020) showed that for a sample of 534 healthcare professionals who worked closely with covid-19 patients in hubei, the most stressful factors were the lack of protocols for the treatment of covid-19, the scarcity of ppe, the exhausting work shifts, their concern about the risk of infection, and their exposure to the death and suffering of their patients. they also found that the support of superiors proved to be one of the most important motivational factors for medical staff, and the presence of clear guidelines and effective safety protocols were protective factors against the development of stress, in particular, for females. further, walton et al. (2020) identified the organizational stressors as the changes in work shifts, the prevalence of night shifts, an excessive workload, staff roles, autonomy, the lack of support from superiors, and the absence of adequate information and clear instructions. on the basis of these stressors, they estimated that 10% of the medical staff working on the front line of this pandemic are at risk of developing posttraumatic stress disorder (ptsd). in addition, limited resources, longer shifts, decreased hours of rest, and the occupational risks associated with covid-19 exposure have increased the physical and mental fatigue, stress, anxiety, and burnout of these staff members (sasangohar et al., 2020) . the loss of a social support network, which can be an important resilience factor, is another risk factor (ozbay et al., 2007) . in the covid-19 emergency, healthcare and emergency workers have often experienced a separation from their affective links, either because of the restrictions on social contacts imposed by the lockdown or the fear of spreading the infection to their family members. to this must be added that although, at first, health workers received unanimous encouragement from the population, later, they also experienced demonstrations of stigma and isolation. some studies have shown that being able to resort to their own social support network is a significant protective factor for health workers dealing with this emergency . as favretto (2005) stated, when individuals experience situations that go beyond their coping strategies, their vulnerability to, and risk of developing, psychopathological reactions increases. studies conducted during previous epidemics, such as the sars, mers, and ebola epidemics, converge in detecting how healthcare and emergency workers may experience extremely high levels of stress and even develop secondary traumatic stress or vicarious trauma. this trauma is defined as an experience of symptoms similar to those found in people with ptsd, such as in emergency nurses working with traumatized patients (beck, 2011) . figley (1995) defined it as a form of stress that derives from the feelings of empathy experienced when helping traumatized people. the symptoms may include intrusive recurring thoughts, disturbed sleep, fatigue, physical symptoms, hyperarousal, increased stress response, anxiety, depression, and feeling emotional (adriaenssens et al., 2012) . wolf et al. (2016) described how nurses may feel "overwhelmed, " and this condition becomes a source of moral distress that triggers feelings of powerlessness, guilt, fear, anger, and frustration. the sense of frustration and impotence felt by nurses when they are unable to treat and save a patient has been highlighted as a risk factor for secondary traumatic stress in several studies (missouridou, 2017) . avoidance and emotional numbing can become tools for self-protection from intrusive symptoms that exceed the personal tolerance level (coetzee and klopper, 2010; mealer and jones, 2013) . their frustration obviously intensifies on a patient's death. the onset of ptsd in the health workers involved in treating mers was also detected after the acute phase of the emergency was over, highlighting a risk not only in the immediate period but also in the medium-term period (lee et al., 2018) . in reference to covid-19, updated studies conducted on chinese health workers have already highlighted the strong impact of the epidemic on the psychological health of doctors and nurses. some studies have found that healthcare workers have high levels of anxiety, depression, insomnia, and distress (lai et al., 2020; li et al., 2020; zhu et al., 2020) . in particular, female professionals with more than 10 years of experience and previous psychiatric pathology present more risk factors of developing the symptoms of stress, anxiety, and depression (lai et al., 2020; zhu et al., 2020) . huang j. z. et al. (2020) studied stress levels during the covid-19 emergency in a sample of medical staff. they found that females showed higher levels of anxiety and ptsd than males did and that the levels were higher for nurses than for doctors. moreover, li et al. (2020) found that nurses had developed higher levels of vicarious trauma than those of the general population and that nurses who did not work closely with covid-19 patients showed a more severe symptomatology, both physical and psychological, compared with their colleagues working on the frontline emergency services. in italy, a study conducted on healthcare workers found that doctors and nurses developed high levels of stress and anxiety, greater than those developed by the general population, and that healthcare workers operating in the north, the area of italy most affected by the virus, showed a more severe symptomatology (simione and gnagnarella, 2020) . this study also confirmed that females tend to have a greater perception of the risk of infection, which increases their risk of developing the symptoms of anxiety and distress. because of their long, intense exposure to various stressors, it is important to note the nature of the coping strategies used by these healthcare and emergency workers in these situations and their effectiveness in terms of reducing and effectively coping with stress. indeed, the effective management of stress levels in the acute/emergency phase could reduce the risk of developing long-term ptsd or other pathologies, such as anxiety and depression (fullerton et al., 2004; slottje et al., 2005; argentero and setti, 2011; sakuma et al., 2015; birinci and erden, 2016; li et al., 2017) . coping may be defined as a series of cognitive and behavioral efforts to manage specific internal or external issues that test or exceed individual resources (lazarus and folkman, 1984) . a distinction can be made between problem-focused and emotion-focused coping strategies. the former is aimed at modifying and solving the stressful situation through active interventions. by contrast, emotion-focused coping is aimed at managing the emotions connected to the stressful event and regulating affective reactions, such as anxiety and the tension of response to stress, for example, by trying to avoid the threat (denial) or re-evaluating it (reappraisal). the choice of coping strategies is influenced by the individual's cognitive evaluation of the event, termed secondary evaluation, which involves estimating the resources available and the most effective strategies to deal with the situation (lazarus and folkman, 1984) . a key element of this assessment is the extent to which the individual can maintain control over the outcome of the situation. the literature indicates that individuals apply dysfunctional coping when they face an uncontrollable event by responding primarily with a coping strategy focused on the problem, and conversely, when they face a controllable situation, they respond with coping strategies focused on emotions (strentz and auerbach, 1988; vitaliano et al., 1990) . a coping strategy may be defined as adaptive when the controllability of the stressful event corresponds with the choice of coping strategy: in this case, the subject will experience fewer symptoms related to stress (park et al., 2001) . the strategies used to cope with trauma may differ among individuals, but they can also vary according to the profession and the features of the traumatic event (nydegger et al., 2011) . individuals differ in their choice of coping strategies (connor-smith and flachsbart, 2007) , and factors related to the situation can also have a decisive influence on such choice (brown et al., 2002) . a few studies have considered the ways in which gender influences the perception of stress in emergency situations and the choice of coping strategy. these studies highlight that females tend to perceive events as more negative and uncontrollable and to resort more to coping strategies focused on emotions and avoidance, whereas males tend to resort more to applying problem-focused coping and to inhibiting emotions (matud, 2004; matud et al., 2015; matud and garcia, 2019) . the literature on the relationship between coping strategies and the stress levels of emergency workers has shown that the use of coping strategies focused on the problem usually tends to correlate with lower stress levels, both in healthcare workers (watson et al., 2008; howlett et al., 2015) and in other emergency workers, such as firefighters (brown et al., 2002) . however, a coping strategy frequently used by emergency workers is that of avoidance and minimization, and this strategy is associated with higher levels of stress (brown et al., 2002; chang et al., 2003; kerai et al., 2017; witt et al., 2018; theleritis et al., 2020) . loo et al. (2016) found that in a group of emergency workers, avoidance as well as coping strategies focused on emotions were associated with the development of post-traumatic symptomatology. rodríguez-rey et al. (2019) revealed that among health workers working in a pediatric emergency department, approximately 30% of the variance in ptsd was explained by the frequent use of coping strategies focused on emotions and the infrequent use of those focused on the problem. in addition, kucmin et al. (2018) , who considered a sample of 440 paramedics, highlighted that the risk of developing ptsd symptoms was predicted by the use of coping strategies focused on emotions. however, the literature does not offer unanimous results. chamberlin and green (2010) found that in a group of firefighters, all coping strategies actually correlated with high levels of stress: the authors explained this finding by suggesting that it is not the individual coping strategies that are maladaptive in themselves, but that greater effort is needed to adjust in stressful situations. by contrast, young et al. (2014) indicated that firefighters use problem-focused coping strategies more often at the beginning of the operation and emotion-focused coping strategies more commonly in the phase of breakdown and fatigue. however, after the incident, they use both strategies (young et al., 2014) . a meta-analysis by shin et al. (2014) highlighted that different coping strategies have different effects on work burnout: in particular, emotional stress and depersonalization are associated with the use of emotion-focused coping strategies, whereas professional ineffectiveness is associated with the use of problem-focused strategies. further, a few studies have investigated the coping strategies that emergency workers can use during health emergencies similar to covid-19. maunder et al. (2006) revealed that healthcare professionals who tended to apply dysfunctional coping strategies, based on avoidance, hostile comparison, or self-blame, tended to develop higher stress levels. wong et al. (2005) highlighted that during the sars epidemic, doctors and nurses tended to use different coping strategies. the doctors tended to turn more to action planning, but this strategy did not affect their stress level. instead, their stress level was positively correlated with their use of coping strategies based on emotional outlets. by contrast, the nursing staff tended to resort more to behavioral disengagement and distraction strategies, which, however, correlated with higher levels of stress among them. in this regard, during the mers epidemic, hospital staff tended to adopt coping strategies related to the use of ppe and the adoption of all prevention measures, as well as social support, whereas the coping strategy that they adopted the least was that based on an emotional outlet (khalid et al., 2016) . a recent study on healthcare workers in hubei, china, during the covid-19 epidemic , yielded similar results: to reduce stress, the medical staff tended to rely on active coping strategies, such as using security protocols, practicing social isolation measures, and seeking support from family and friends, but they did not find it necessary to discuss their emotions with a professional. huang l. et al. (2020) found that a sample of nurses working during the covid-19 emergency presented greater emotional reactions and turned more to problem-focused coping compared with university nursing students. emergency workers must have sufficient self-efficacy in terms of their coping skills to be able to manage and cope with stress levels. self-efficacy in coping appears to be an effective protective factor in relation to stress levels and maladaptive responses (chesney et al., 2006) . self-efficacy to cope with traumatic events has been effective in reducing the risk of developing ptsd (bosmans et al., 2015) . the main objective of this study is to identify the coping strategies activated by healthcare and emergency workers to deal with stress factors related to the covid-19 emergency that may be associated with the risk of developing vicarious or secondary trauma. few studies have considered both groups simultaneously when analyzing the strategies they have adopted to manage stress during the covid-19 emergency. hence, in this study, we are interested in detecting the similarities and differences in the approaches they adopted to manage their stress during the acute phase of the current pandemic according to walton et al. (2020) , the main acute stress reactions of emergency workers to emergency medical situations are emotional, cognitive, physical, and social reactions. therefore, these factors were included in the questionnaire used in the present study. moreover, reactions linked to stress factors for difficulties due to ineffective decision-making and dealing with stress were also considered (chesney et al., 2006) . in addition, fears regarding contracting the virus and infecting their own families because of covid-19 were specifically considered (du et al., 2020; huang j. z. et al., 2020; ornell et al., 2020; walton et al., 2020) . based on results found in the literature, the specific objectives of this study are as follows: (1) to examine the relationships between coping strategies, emergency stress, and secondary trauma in healthcare and emergency workers. (2) to identify significant differences in stress factors, coping strategies, and secondary trauma between two groupshealth workers and emergency workers. participants were selected on a voluntary basis through a trasversal sampling in order to take a picture of the situation caused by the pandemic emergency. we used an internet platform to conduct the study and approached the participants using social media, dedicated mailing lists, and forums. participants from all italian regions completed the questionnaire online. the sample consists of 210 participants-90 males (42.9%) and 120 females (57.1%)-whose average age was 42.53 years (sd = 10.97; min 22 -max 67). further, 52.9% of the sample were married, 10.6% were separated, and the remaining 36.5% were single. we selected various professional figures who had directly worked in various sectors during the covid-19 emergency and who could be divided into two main groups. the first, the "health group, " consists of 121 participants (57.6%) who were healthcare workers: 57 doctors (50%), 47 nurses (37.3%), 9 psychologists (7.14%), and seven healthcare assistants (5.56%). their average age was 42.13 years (sd = 11.35), and their average years of active professional service was 14.60 (sd = 11.56). the second, the "emergency group, " consists of 89 participants (42.4%): 48 emergency workers (53.9%), 21 firefighters (23.6%), and 20 civil protection staff (22.5%), whose average age was 45.43 years (sd = 10.19) and average years of service was 14.41 (sd = 11.89). there was an age difference between the two groups (t = −2.170; p <0.05), and the distribution of the gender variable differed between the two groups, with 41 males and 80 females in the health group and 49 males and 40 females in the emergency group (χ 2 9.38; p < 0.01). the study involved participants from the entire national territory, and their workplace could be divided as follows: 38, 36, and 26% were from north, central, and south italy, respectively. further, 59% of the sample worked directly with covid-19 patients and 24.8% worked in specific covid-19 departments. among the healthcare workers, 73% had worked in direct contact with covid-19 patients, whereas among the emergency workers, only 33% had assisted these patients (χ 2 36.251; p < 0.01). in the present study, we included two variables, lack of necessary instructions and lack of ppe, in accordance with the findings in the literature on their impact on the stress reactions of healthcare and emergency workers during the covid-19 pandemic. among the participants, 62 and 45% of healthcare and emergency workers, respectively, did not have sufficient instructions to intervene (χ 2 2.441; p n.s.), and 57 and 52% of healthcare and emergency workers, respectively, lacked adequate ppe when working (χ 2 2.857; p n.s.). this study used an online questionnaire and was conducted during the lockdown period owing to the covid-19 pandemic. the questionnaire had three parts: one each to collect online informed consent and baseline sociodemographic information, and one with an online series of questionnaires, as described in the next section. participants' anonymity was maintained in collecting the data. the institutional ethics committee approved all the procedures. we administered a series of questionnaires to evaluate the psychological stress and coping style of each participant. we included the following questionnaires. in detail, the arousal items describe situations characterized by anxiety, confusion, physical and psychological complaints, and agitation. intrusion refers to the re-experiencing of the traumatic event-even if not directly suffered-through internal images and memories. instructions for the stss-i indicated that respondents should specify how frequently an item was true for them in the previous 4 weeks. the statements are evaluated on a 5-point scale (1 = never; 5 = very often) that provides scores for intrusion (example items: "i thought about my work with victims when i didn't intend to"; "reminders of my work with clients upset me") and arousal (example items: "i had trouble concentrating"; "i was easily annoyed"; "i expected something bad to happen"; "i felt jumpy"). the reliability coefficients of the instrument are 0.87 and 0.81 for arousal and intrusion, respectively. the coping self-efficacy scale -short form (cses-sf; chesney et al., 2006) this is a 13-item measure of perceived self-efficacy for coping with challenges and threats. this measure focuses on the changes in individuals' confidence in their ability to cope effectively, based on the self-efficacy theory (bandura, 1997; chesney et al., 2006) . participants were asked, "when things aren't going well for you, or when you're having problems, how confident or certain are you that you can do the following." then, they were asked to rate on an 11-point scale the extent to which they believed they could perform important behaviors for adaptive coping. the instrument yields three subscale scores: "problem-focused coping" (α = 0.91), "stop unpleasant emotions and thoughts" (α = 0.91), and "support" (α = 0.80). anchor points on the scale are 0 ("cannot do at all"), 5 ("moderately certain can do"), and 10 ("certain can do"). we constructed an ad hoc 7-item questionnaire that included yes/no questions to detect stress factors identified by the literature, such as the availability of suitable equipment and the receipt of clear instructions during the covid-19 coping experience. in this study, we present the results related to two of these items: "instructions, " which refers to having received the necessary instructions to intervene, and "equipment, " which refers to having ppe. predictions of these factors have also been made in other studies (du et al., 2020; walton et al., 2020) . in light of the relevance and specificity of the lack of clear information or instructions and adequate ppe in the management of covid-19 in the italian context, as well as the findings in other studies, we decided to focus attention on these two risk factors. our analysis of the literature revealed that in situations in which they have to cope with a pandemic, several factors may affect the stress of medical staff and emergency healthcare workers and that covid-19 represents an independent specific stressor (spoorthy, 2020) . these stress factors have been identified as frequently affecting healthcare and emergency workers in emergency situations and leading to physical, emotional, cognitive, decision-making, relational, and organizational stress (del missier et al., 2008; sbattella, 2009; argentero and setti, 2011; fraccaroli and balducci, 2011; bellelli and di schiena, 2012; du et al., 2020; walton et al., 2020) . focusing on the specificity of the covid-19 epidemic, items have been constructed regarding the fears of contracting the infection and of infecting colleagues or family members (walton et al., 2020) , since covid-19 represents a factor of independent stress (spoorthy, 2020) that has great impact (huang j. z. et al., 2020) . consequently, we constructed the esq consisting of 33 items assessed on a 5point likert scale, with scores ranging from 0 (not at all) to 4 (very much), grouped into six scales. the participants were asked to indicate how often they experienced certain emotions and thoughts while performing intervention and emergency activities during the covid-19 pandemic. the scales correspond to the factors identified and confirmed by factorial analysis through an analysis of the main components with orthogonal rotation of factors (varimax). the number of factors to be extracted was initially verified through the unit's largest eigenvalue criterion and, subsequently, by the scree test. the esq is based on six scales: (1) organizational-relational stress: measures the stress levels related to the organizational context, relationships with colleagues, and social support (consisting of eight items: 7, 10, 13, 14, 15, 16, 19, and 23); (2) physical stress: composed of five items describing symptoms of physical fatigue (11, 12, 18, 20, and 32); (3) inefficacy decisional stress: consists of five items that analyze decision-making aspects and the possibility to act, which are related to the level of self-efficacy (22, 25, 27, 28, and 29); (4) emotional stress: comprises six items that indicate the participant's emotional reactions (1, 2, 3, 4, 6, and 26); (5) cognitive stress: consists of four items on the cognitive aspects of stress (5, 17, 21, and 24); (6) covid-19 stress: comprises five items regarding worries related to the covid-19 emergency (8, 9, 30, 31, and 33) . first, we performed pearson's correlation analyses to identify the associations between the variables for the two groups that we considered in this study. subsequently, we checked for significant differences between the two groups as their stress levels, coping strategies, and secondary trauma. we used hierarchical linear regression models to verify the predictive effect of the risk factors (lack of adequate information and ppe) on the different stress levels (in step 1). then, we verified the protective effect of the coping strategies (in step 2). the models were controlled for age, gender, and group. lastly, we used hierarchical regression models to verify the predictive effect of stress factors on the components of secondary trauma. the models were controlled for age, gender, and group. first, we conducted correlational analyses and comparisons of averages on the reference sample. table 1 shows the correlations between the scales of the esq and the other instruments. preliminary comparisons were made through the student's t-test between the health group and the emergency group in relation to the esq, cses-sf, and stss-i scores. table 2 shows the comparison between the two groups. as shown in table 2 , significant differences emerged between the two groups in relation to their stress and arousal levels. the results indicate higher levels of both for the health group, and that emergency workers turn more to the stop unpleasant emotions and thoughts strategy. further, we performed comparisons with reference to the gender variable to detect differences in the levels of stress factors, coping strategies, and secondary trauma. the emergency group, there were no differences in levels of stress and secondary trauma or coping strategies. moreover, we found similar correlations between the two groups for the stop unpleasant emotions and thoughts strategy and the stress factors, whereas for the other two coping strategies, we found a different association, particularly for the emergency group. however, the t-test comparisons highlight differences only at the level of the stop unpleasant emotions and thoughts strategy. given the findings of the preliminary analyses, we considered it necessary to include the age, gender, and group variables to test the predictiveness of the coping strategies on the participants' stress levels. to test the predictive effect of the coping strategies on various levels of stress, hierarchical regression was conducted. considering the age and gender differences within the groups, we included these variables in all models together with the group variable (health vs. emergency) and the "instructions" and "equipment" variables. the models generated by assuming the esq scales as dependent variables are shown in table 3 . regarding the coping strategies, we observed an important effect of the stop unpleasant emotions and thoughts coping strategy on all the stress scales, except for physical stress where the effect of the focused problem coping strategy is recorded. as shown in table 1 , significant negative associations between stressors and secondary trauma were found for both groups. the hierarchical regression models of stress scales were analyzed for the arousal and intrusion levels of secondary trauma. the models included the age, gender, health/emergency group variables, and the esq scales. the results are shown in table 4 . .041*** f = 9.273*** f = 7.352*** f = 15.346*** f = 13.634*** f = 6.596*** *p < 0.05, **p < 0.01, ***p < 0. 001; 1 gender (1 = male; 2 = female); 2 instructions (1 = yes; 2 = no); 3 equipment (1 = yes; 2 = no); cses-sf scales, focused problem; stop emotion_thought; support. the same regression models were generated by including coping strategies as predictors and were analyzed by age, gender, and health/emergency group. compared with arousal, the results of this study show that healthcare and emergency workers both experienced high stressors during the covid-19 epidemic, exposing them to the risk of developing secondary trauma (dominguez-gomez and rutledge, 2009; argentero and setti, 2011; adriaenssens et al., 2012; duffy et al., 2015; aisling et al., 2016; morrison and joy, 2016; wolf et al., 2016; roden-foreman et al., 2017; lai et al., 2020; li et al., 2020; zhu et al., 2020) . we found significant differences between the two groups regarding their reactions and their levels of organizational, physical, and relational stress, their sense of decision-making, and their emotional and cognitive ineffectiveness. compared with emergency workers, healthcare workers had higher stress levels, leading them to perceive more serious tensions and difficulties in teamwork, physical fatigue, somatic illnesses, irritability, and difficulty in maintaining control over the situation, in taking decisions, and in predicting the consequences of their actions. higher levels of stress have been reported related to the fears of contracting covid-19 and of infecting family members. in line with other studies, we found that the covid-19 emergency led health workers, in particular, to perceive specific stress factors that affected the organizational area, with consequences in terms of tension in teamwork and a sense of ineffectiveness since they had to intervene without sufficient tools and resources. they also experienced deep emotional reactions of anger, powerlessness, and frustration with inevitable cognitive stress, in terms of increased arousal levels. many of the healthcare workers also developed physical stress, due not only to the lack of sleep but also to the possible forms of somatization of the psychoemotional tension they perceived (sasangohar et al., 2020; walton et al., 2020) . the differences recorded between the two groups in stress levels may be explained by taking into account, for example, the fact that the emergency group perceived their intervention with a greater sense of continuity in their usual procedures compared with the health group. the former performed their usual activities on the organizational, cognitive, and procedural levels, although with greater levels of safety and self-protection and a greater frequency of interventions. conversely, the health group had to reorganize aspects such as departments, teams, and shifts to cope with the emergency, which thus involved making radical changes. in addition, the health group helplessly witnessed a large number of deaths of their patients and had to make decisions in conflict with their moral sense and in situations of insecurity and unpredictability regarding the consequences of their actions walton et al., 2020) . however, in terms of physical stress, there was no predictive effect of the group, which indicates that the health and emergency groups were both exposed to very similar physical stressors. it is important to consider the significant impact of the gender variable. according to other studies, females developed a greater reaction of physical and emotional stress and the sense of decision-making ineffectiveness than did males (lai et al., 2020; zhu et al., 2020) . in fact, females apparently tend to perceive events as more negative and uncontrollable, and thus suffer higher levels of stress. further, females tend to resort to coping strategies focused on emotions, which tend to be less effective in emergency situations (matud, 2004; matud et al., 2015; matud and garcia, 2019) . however, in the present study, these gender differences did not have an impact in terms of psychopathological or specific maladaptive consequences, and coping strategies. in fact, females and males perceived a similar sense of efficacy/ineffectiveness in dealing with stressful situations and had similar scores on the secondary trauma scale. the results shown in table 3 also indicate that predictive impact is also assumed by the lack of adequate instructions and knowledge about the emergency and the lack of necessary ppe. in particular, for the health group, the lack of necessary instructions on how to conduct quick interventions affected almost all stressors, leading to tensions or conflicts within the team, difficulty in making decisions, irritability, anger, and frustration. above all, the lack of ppe affected the sense of making the right decisions, the emotional sphere and, most importantly, the fear of contracting the virus or of transmitting it to their families. these results converge with those of other studies that have highlighted that the lack of adequate and specific information and of equipment for healthcare staff in dealing with covid-19 affected their self-efficacy and the factors protecting them from stress, thus increasing their fear of contracting an infectious disease and causing them greater emotional, decisional, and physical stress. conversely, the professionals who were provided with the necessary knowledge and equipment were more resilient during the emergency response (du et al., 2020; huang j. z. et al., 2020; ornell et al., 2020; walton et al., 2020) . the lack of specific equipment and instruments in emergency situations along with the risk of infection increases the feeling of poor control, leading to cognitive and emotional stress and a sense of ineffectiveness (placentino and scarcella, 2001; walton et al., 2020) . higher levels of stress were found in the health group than in the emergency group because of the absence of ppe, the risk of infection from the virus, and the lack of necessary instructions or prompt information . the incidence of these variables is contained and limited by the use of coping strategies. the coping strategy that assumes a predictive effect, reducing stress levels, is to block those negative or unpleasant emotions and thoughts associated with the risk of developing secondary trauma. in fact, the use of the stop unpleasant emotions and thoughts strategy reduces the arousal and intrusion levels of the secondary trauma. the effectiveness of this strategy in reducing the arousal levels appeared to be greater in the health group. as fraccaroli and balducci (2011) suggested, in situations of high emergency stress, healthcare workers and emergency workers may have a deficit in the cognitive process of emotions, thus failing to identify their emotional reactions, which tends to be associated with maladaptive behaviors. the lack of a complete recognition of one's unpleasant emotions, which tends to be denied and dismissed as a coping strategy, would explain the greater predictive impact of cognitive stress and physical stress on post-traumatic arousal compared with emotional stress. further, the results of this study highlight that the stop unpleasant emotions and thoughts strategy has an inhibitory and therefore effective and highly significant impact on the stress levels and the components of secondary trauma, unlike the problem-focused and social support strategies. the literature points out that the avoidant matrix coping strategies tend to present themselves when healthcare and emergency workers experience a condition of fatigue and exhaustion, and this would explain the presence of the greater acute stress responses in healthcare workers (maunder et al., 2006; young et al., 2014) . the results of this study show that the problem-focused coping strategy (the strategy most frequently used in the health group in line with the finding of huang l. et al., 2020) in this emergency situation did not appear to demonstrate protective efficacy. this is likely to be because the workers were dealing with an emergency that was not yet fully understood and the therapeutic and treatment procedures were not fully known. moreover, the supply of ppe was scarce, especially in the first few weeks of the covid-19 emergency in italy, in all hospitals (e.g., a lack of respirators and insufficient number of resuscitation beds), which meant that the level of protective efficacy of this strategy may have been lower than the stress levels. in other words, emergency workers, although task-oriented, were faced with a problem that was not fully understood, and in the absence of ppe, perceived poor self-efficacy in terms of trust and belief in their ability to organize and make effective decisions. the strategy that ensured optimal levels of self-efficacy was the one that allowed negative thoughts and emotions associated with the epidemic to be removed from consciousness, which was also found to have a protective function against the risk of developing traumatic symptoms. the government lockdown and the consequent restriction of visits outside the working environment limited the use of coping strategies involving social support, family, and friends, implying a greater use of emotional and cognitive avoidance methods to deal with anguished thoughts, intrusive memories, and the constant vision of corpses or the seriously ill. in this regard, the health group appears to have developed a greater secondary trauma arousal than the emergency group. by contrast, the latter appears to have developed more aspects of intrusiveness related to secondary or vicarious trauma than the health group (see table 2 ). since they were interviewed during the covid-19 emergency, the healthcare and emergency workers who participated in the present study do not appear to have developed a complete secondary trauma. this may explain the prediction of the stress factors on arousal and not on intrusion. in other words, these individuals were interviewed while the emergency was still in the acute phase and before a structuring of answers in a psychopathological sense could be performed. therefore, performing a follow-up study would be interesting. ptsd can take several months to fully emerge, and its stabilization can depend on the individual's internal as well as external factors. because they blocked negative emotions and unpleasant memories, the healthcare and emergency workers' arousal appears to be mainly due to, at least in the full phase during the epidemiological emergency, the factors of a cognitive matrix, linked with the difficulty of focusing on and identifying the most appropriate intervention strategies, leading them to experience regret, disappointment, and both physical and relational tension. the health workers apparently blocked the emotional aspects related to pain, impotence, and guilt, which allowed them to continue their work. in an emergency phase that is still active, and a few weeks after the start of the pandemic, it is possible to detect high arousal and a lower level of intrusiveness of stressful or traumatic events. this condition may be more likely if the blocking of negative emotions and intrusive thoughts linked to one's personal experience intervenes as a coping strategy. low perceptions of self-efficacy regarding coping has been found to be a predictor of ptsd in other studies (benight and harper, 2002; bosmans et al., 2015) . in emergency situations, high stress can cause emergency workers to experience impotence, breathlessness, cognitive difficulties, and difficulties in decision-making and managing emotional reactions along with a prevalence of feelings of anger, as recorded in this study. if the lack of adequate knowledge and of ppe are added to these factors, even professional experts may perceive a loss of self-efficacy in coping and, simultaneously, experience an inability to orient their skills more effectively, thus developing maladaptive responses. this study has several limitations. the first is the limited sample size. the second is that our study involved participants in the very midst of the covid-19 emergency, which means that the level of stress in healthcare workers may have been more severe and acute. moreover, the long-term psychological implications for the healthcare and emergency population should be investigated for the presence of a full secondary trauma. therefore, a large-sized longitudinal study is called for to further explore the pathogenesis of vicarious traumatization. the third is that participants were not selected based on whether they had existing psychological problems. in proposing the hypothesis of this study, we anticipated that we would be able to discover the relationships between coping strategies, emergency-related stress, and secondary trauma in healthcare and emergency workers and commenced our investigation by assuming that the impact of stress can provoke psychological consequences in emergency situations. in future work, this assumption could be tested to verify whether an emergency situation has a different impact on workers who have already experienced psychological problems. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. the studies involving human participants were reviewed and approved by comitato etico per la sperimentazione umana -cesu of the university of urbino. the patients/participants provided their written informed consent to participate in this study. mv, tm, vg, and dp: conceptualization, writing -original draft preparation, and writing -review and editing. mv, tm, and vg: methodology and investigation. mv and tm: formal analysis and data curation. tm and vg: visualization. mv, tm, and dp: project administration. all authors contributed to the article and approved the submitted version. we would like to thank all the participants involved for the contribution they made to this study during this serious emergency. the impact of traumatic events on emergency room nurses: findings from a questionnaire survey an assessment of psychological need in emergency medical staff in the northern health and social care trust area diagnostic and statistical manual of mental disorders (dsm-5) engagement and vicarious traumatization in rescue workers self-efficacy: the exercise of control awareness of secondary traumatic stress in emergency nursing secondary traumatic stress in nurses: a systematic review emozioni e decisioni. come la psicologia spiega il conflitto tra ragione e sentiment coping self-efficacy perceptions as a mediator between acute stress response and long-term distress following natural disasters yardım çalışanlarında üstlenilmiş travma, ikincil travmatik stres ve 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acute respiratory syndrome outbreak on healthcare workers in emergency departments and how they cope the effects of social support on sleep quality of medical staff treating patients with coronavirus disease 2019 (covid-19) in january and february 2020 in china stressors and coping strategies of u.k. firefighters during on-duty incidents covid-19 in wuhan: immediate psychological impact on 5062 health workers key: cord-272268-8vrcwwll authors: kedersha, nancy; anderson, paul title: chapter 4 regulation of translation by stress granules and processing bodies date: 2009-10-27 journal: prog mol biol transl sci doi: 10.1016/s1877-1173(09)90004-7 sha: doc_id: 272268 cord_uid: 8vrcwwll stress necessitates rapid reprogramming of translation in order to facilitate an adaptive response and promote survival. cytoplasmic stress granules (sgs) and processing bodies (pbs) are dynamic structures that form in response to stress-induced translational arrest. pbs are linked to mrna silencing and decay, while sgs are more closely linked to translation and the sorting of specific mrnas for different fates. while they share some components and can interact physically, sgs and pbs are regulated independently, house separate functions, and contain unique markers. sg formation is associated with numerous disease states, and the expanding list of sg-associated proteins integrates sg formation with other processes such as transcription, splicing, and survival. growing evidence suggests that sg assembly is initiated by translational arrest, and mediates cross talk with many other signaling pathways. nuclear dna is packaged into chromatin, organized into active and inactive domains, and its availability is regulated by the many factors that control its access to polymerases, splicing factors, and the nuclear periphery. in the cytoplasm, rna is similarly structured, organized, and regulated by interactions with membranes, the cytoskeleton, different organelles, and by a host of aggregation-prone rna-binding proteins. localized gradients of specific mrna transcripts regulate the organization and the timing of gene expression that occurs during development. within individual somatic and germ cells, discrete mrna granules are transiently assembled as mrna transcripts move into and out of polysomes. the recruitment, retention, and removal of specific transcripts into and out of rna granules is orchestrated by numerous mrna binding proteins, which regulate the fate of specific transcripts. consequently, biochemical analysis of protein translation in cell lysates cannot completely recapitulate the translational regulatory pathways that occur in live cells. critical components of the ''cell biology'' of protein translation are mrnp granules known as processing bodies (pbs) and stress granules (sgs). these transient cytoplasmic ''structures'' are actively assembled from untranslated mrna by a host of rna-binding proteins, which determine whether specific transcripts will be reinitiated, degraded, or stored. the mrna within pbs and sgs is in a dynamic equilibrium with polysomes, and this equilibrium is shifted by changes in environmental conditions. specific rna-binding proteins promote the translation of some transcripts and inhibit the translation of others; silenced transcripts are assembled into sgs or pbs, whereas preferential translation occurs outside these bodies. in stressed cells, the selective translational repression of ''housekeeping'' genes conserves anabolic energy for the repair of stress-induced molecular damage. at the same time, enhanced translation of repair enzymes directly repairs this damage while promoting an adaptive response to the altered conditions. thus, shifting specific transcripts between polysomes and rna granules tailors translation to changes in environmental conditions. sgs and pbs are dynamic structures that are continuously assembled/ disassembled from the flux of mrnps that comprise them. drugs that arrest translational elongation and thereby stabilize polysomes promote the disassembly of sgs and pbs, whereas drugs that enhance polysome disassembly promote (but are not sufficient to cause) sg/pb assembly. numerous photobleaching studies indicate that the mrnps that comprise sgs and pbs are in very rapid flux (seconds), although sgs and pbs persist for minutes to hours. a useful analogy holds that mrnps are to sg/pbs as water is to a river; rapidly moving water creates a large stable river, but the water is always in flux. similarly, moving streams of mrnps create sgs and pbs. when examining the functional properties of sg/pbs, it is useful to consider that the functional properties of rivers (e.g., erosion, navigation, and hydroelectric power) are different from those of water alone. similarly, the discrete domains (sgs and pbs) created from relocalized mrnps alter the trafficking of other proteins and have important secondary effects (alternative splicing, transcription, nucleocytoplasmic transport) on overall cell metabolism. in 1999, it was noted that stress-induced translational arrest causes untranslated mrnps to assemble into large cytoplasmic ''sgs,'' whose formation is triggered by, and dependent upon, the phosphorylation of eif2a. 1 these granules contain a large proportion of cytoplasmic poly(a) mrna and pabp, suggesting that most of the cytoplasmic polya(þ) mrna is assembled into the granules upon stress. remarkably, the normally nuclear rna-binding protein tia-1 (t-cell internal antigen 1) rapidly colocalizes in these cytoplasmic granules, and a truncation mutant of tia-1 blocks their formation. as similar rnacontaining ''heat-shock granules'' had been described in plant cells, 2 it seemed likely that mammalian sgs were related to plant ''heat-shock granules.'' only recently 3 was it shown that rna-containing sgs and plant heat-shock granules are in fact distinct: plant heat-shock granules do not contain mrna as originally reported, although plant cells do indeed form bona fide sgs and pbs in addition to heat-shock granules. thus in hindsight, and despite the frequent use of the phrase ''it has long been known'' to describe rna-containing sgs, in actual fact these sgs were first described only a decade ago. sgs were then demonstrated to be dynamic sorting centers rather than stable repositories of mrna, as their apparent identity with plant heat-shock granules had predicted. 4 pharmacological data revealed that sg assembly is blocked by agents that arrest translation elongation (e.g., emetine and cycloheximide), but not by agents that promote translation termination (e.g., puromycin), indicating that polysome disassembly is required for sg assembly. importantly, preassembled sgs are forcibly disassembled by emetine even in the continued presence of phospho-eif2a suggesting that they do not represent static storage depots of mrnps but instead contain mrnps in equilibrium with polysomes. direct measurements using frap (fluorescence recovery after photobleaching) confirmed that both tia-1 and pabp rapidly shuttle into and out of sgs, with tia-1 moving more rapidly than pabp. the half-lives revealed by frap were on the order of 2-10 s, in marked contrast with the much slower kinetics of sg assembly (15-30 min) . the first movies of sg assembly and disassembly revealed that sgs form synchronously throughout the cell and progressively fuse to become larger and fewer. as cells recover from stress, the large ''mature'' sgs disassemble synchronously within individual cells, in a time frame of 4-6 min. these data established that sgs are dynamic microdomains into which pabp and tia-1 rapidly shuttle, and which are in equilibrium with polysomes. pabp is a translational enhancer, and tia-1 is a translational silencer, 5 hence we proposed that sgs constitute dynamic mrna triage domains, in which mrna processing, sorting, and remodeling events regulate the expression of specific transcripts. 6, 7 a more detailed in situ analysis of sgs 8 revealed that they contain small but not large ribosomal subunits, and the initiation factors eif3, eif4e, and eif4g. however, eif5 and eif2 are not prominent components of sgs, despite the fact that phospho-eif2a drives sg assembly. contemporary models of translation initiation suggested that eif5 normally links eif2/gtp/trna met i to eif3 and the small ribosomal subunit to form the 43s complex, which is thought to form prior to the recruitment of eif4f, pabp, and the mrna to form the 48s complex. as sgs contain most components of the 48s complex but lack eif5 and eif2, sgs appear to contain aggregates of aberrant 48s complexes, lacking eif5/eif2/gtp/trna met i but containing instead the tia proteins. in sucrose gradients, these tia-1-containing complexes migrate with small mrnps near the top of the gradient rather than with polysomes. interestingly, phospho-eif2a is detected in late sgs, but not newly formed ones, supporting a model in which lack of ternary complexes drives sg formation via the formation of aberrant 48s complexes. another report 9 partly confirmed these results, but differed in finding eif2a and eif2b in arsenite-induced sgs: whether this reflects differences between antibodies or cell lines remains unresolved. this study showed for the first time that thapsigargin, a specific activator of perk, induces sgs that are prevented by a kinase-dead form of perk. together, these studies established that sg formation is a result of stalled initiation and confirmed a central role for phospho-eif2a in their assembly. in 2003, it was shown that another rna-binding protein, ras-gap binding protein 3 (g3bp), is a sg component whose overexpression nucleates sg assembly, and that this ability is linked to the phosphorylation of a specific g3bp serine residue. 10 g3bp is a multifunctional protein linked to numerous pathways, 11 and its ability to nucleate dynamic (e.g., cycloheximide-reversible) sgs has subsequently been widely exploited in the field to study sg composition. although originally described as an endonuclease, no such activity has been demonstrated in vivo, so the functional consequences of g3bp-nucleated sgs are unknown. however, the au-rich mrna decay factor tristetraprolin (ttp) similarly nucleates sgs which are prevented by its phosphorylation and interactions with 14-3-3 proteins, 12 providing the first example of a protein that moves to sgs under some conditions but not others. a flood of proteins too numerous to cite individually were subsequently identified as sg-associated (summarized in table i ). some of these are considered below. importantly, a natural product isolated from new zealand sponge, pateamine a, induces sgs compositionally similar to those induced by arsenite: these sgs contain eif3, eif4e, eif4g, and tia-1 but are assembled in the absence of eif2a phosphorylation. pull-down studies established that the molecular target of pateamine a is eif4a, 13 the dead-box helicase necessary for cap-dependent translation requiring 48s scanning. toe-printing studies and polysome profiles on in vitro translated material revealed that pateamine a inactivates scanning, apparently by stabilizing interactions between eif4a and eif4b. this study added eif4a and eif4b to the growing list of initiation factors found in sgs. a similar compound named hippuristanol, found in the coral isis hippuris, also blocks eif4a scanning activity by preventing its binding to mrna. 14 remarkably, both pateamine a and hippuristanol induce sgs in mutant eif2a-s51a mefs, which contain only a non-phosphorylatable form of eif2a, 15, 16 and the sgs induced by these drugs contain eif2 and eif5. these drugs have been widely used in subsequent studies to determine whether a given protein is recruited to sgs or not, and as such have been invaluable tools. however, these drugs circumvent the physiological stress response that activates one or some of the multiple kinases that target eif2a (and perhaps other proteins: pkr targets rna helicase a; ref. 17) as well as other kinase cascades. moreover, eif4a is present in multiple isoforms and has other functions (e.g., nmd), and these drugs have other targets in addition to eif4a (e.g., pateamine a binds unrip; ref. 13) . the discovery that yeast 18 and human cells 19 contain dynamically organized sites of mrna decay enzymes and proteins later recognized as part of the microrna silencing machinery (reviewed in refs. 20-23) occurred somewhat later than the first sg studies. pbs exhibit similar dynamics and share some proteins with sgs, but are associated with mrna decay rather than translation. while regulated mrna decay is perhaps the ultimate form of translational control, this subject is beyond the scope of this chapter. sgs are absent from the cytoplasm of normally growing cells, but they are rapidly assembled in cells exposed to sudden changes in environmental conditions. in most cases, sg assembly results from stress-induced phosphorylation of eif2a, a component of the eif2/gtp/trna met i ternary complex that directs trna i met to the 40s ribosomal subunit. phospho-eif2a is a competitive inhibitor of eif2b, a gdp-gtp exchange factor that charges the eif2/gtp/trna met i ternary complex. thus, phosphorylation of eif2a effectively depletes active ternary complex resulting in translational repression. this translation control pathway is initiated by a family of eif2a kinases that are activated by different types of stress granules and processing bodies environmental stress. protein kinase r (pkr) senses heat, uv irradiation, and viral infection. 24, 25 perk/pek (pkr-like er kinase) senses endoplasmic reticular stress accompanying excess production of secretory proteins; gcn2 (general control non-derepressible 2) senses nutrient stress (e.g., amino acid starvation); hri (heme-regulated inhibitor) senses heme levels in erythroid progenitor cells to balance the synthesis of globin and heme, as well as arsenite-induced oxidative stress. lack of ternary complex triggers sg assembly by stalling initiation while allowing elongation to proceed normally, resulting in polysome disassembly, necessary but not sufficient for both sg and pb assembly. notably, pb formation is independent of phospho-eif2a, although it still requires free mrnps. the specificity of stress-induced translational repression correlates with sg assembly. whereas mrnas encoding ''housekeeping'' transcripts are concentrated at sgs, mrnas encoding molecular chaperones that refold stressdenatured proteins (hsp70) or alter the conformation of signaling proteins (hsp90) are selectively excluded from sgs. this has been proposed to account, in part, for the ability of certain transcripts to be selectively translated during stress. although the mechanism by which hsp70 transcripts are excluded from sgs is not known, it is likely that mrnas transcribed during stress acquire or lack distinct protein ''marks'' that determine their functional fate in the cytoplasm. hsp70 transcripts are unusual in several ways: they lack introns, and thereby lack proteins deposited at splicing junctions, one of which (mln51) is selectively recruited to sgs 26 -whether this allows a selective recruitment of spliced mrnas into sgs remains to be determined. in addition, hsp70 mrna possesses a long structured 5 0 -untranslated region (5 0 -utr) that allows translation initiation to occur via a shunting mechanism, 27 which would preclude reliance on eif4a, whose inactivation causes sg assembly. 15, 16 as certain viral 5 0 -utrs exempt transcripts from sg recruitment and translational silencing, 28 the hsp70 5 0 -utr may function similarly. a number of viral mrnas elude or suppress sg assembly by various mechanisms. stress-activated translation of some transcripts is a consequence of upstream open reading frames (orfs) found in the 5 0 -utr. under normal conditions, translation is initiated at the start codon of these orfs, preventing ribosomes from initiating translation at the protein-encoding orf. when stress-induced phosphorylation of eif2a depletes the levels of ternary complex, initiation at the upstream orfs becomes inefficient, allowing some initiation complexes to scan to the cryptic productive initiation codon. this mechanism allows stress-activated translation of atf4, a key transcription factor that regulates the integrated stress response. thus, eif2a-induced sg formation correlates with enhanced translation of this class of transcripts. most environmental stimuli which induce sgs (arsenite, clotrimazole, heat shock, osmotic shock, thapsigargin, uv) increase levels of phospho-eif2a, usually by activating a kinase and/or inactivating an eif2a phosphatase. these stimuli do not induce sgs in mutant mefs (s51a) expressing a nonphosphorylatable form of eif2a, 29,29a,30 which have been invaluable in discriminating between phospho-eif2a-dependent and phospho-eif2a-independent mechanisms of sg induction. eif2a-independent sgs can be induced through inactivation of eif4a helicase activity (pateamine a, hippuristanol, the lipid anti-inflammatory mediator 15-pgj2) which disrupts mrna scanning (table ii) . sgs can be nucleated by ectopic expression of a wide range of sg-associated proteins (e.g., caprin1, g3bp1, fmrp/fxr1, tia-1/tiar; see table i ). these proteins induce the formation of compositionally typical sgs (containing mrna, pabp, and initiation factors) and exhibit dynamic behavior: they are disassembled in response to cycloheximide or emetine, but not puromycin. to date, all tested nucleator proteins (g3bp, tia-1/tiar, ttp, fastk, fmrp/fxr1) fail to nucleate sgs in the s51a mutant mefs, so this type of sg induction is phospho-eif2a dependent. cotransfection of a pkr inhibitor usually prevents sg nucleation in wild-type cells, implicating pkr as the responsible kinase 28 . most sg nucleators bind mrna directly, and possess self-aggregation domains. in many cases, overexpression of the rna-binding domains alone has no effect on sg dynamics, whereas overexpression of the aggregation domains can dominantly inhibit sg assembly. deletion or knockdown of individual sg-nucleating proteins can have no effect, limit sg size, or delay sg assembly, or prolong sgs during recovery (table i) . much of our understanding of pbs comes from pioneering studies performed in saccharomyces cerevisiae, 21 but the situation in metazoans is complicated by (1) additional decapping proteins such as hedls/ge-1, which are lacking in yeast 23 ; (2) the fact that metazoan pbs also house the microrna machinery and associated noncoding rnas which are absent in budding yeast 20 ; and (3) metazoan pbs contain many proteins (4et, fast, apobe-c3ag) also lacking in yeast. moreover, yeast display a range of different dynamic granules that appear related, but may not be identical, to those in metazoans. glucose starvation triggers the formation of yeast ''egp'' bodies (containing eif4e, eif4g, and pabp) that exhibit dynamic behavior but lack eif3 and 40s subunits. 31, 32 these bodies contain other mrna-binding proteins related to mammalian sg-associated proteins, and were referred to as ''yeast sgs,'' 33 despite their lack of eif3/40s and nonreliance on phospho-eif2a for their formation. recently, eif3a/40s-containing ''yeast sgs'' have been described in s. cereviseae 34 exposed to robust heat shock, that require energy for their assembly but not phospho-eif2a, and appear to contain the pb marker dcp2. further studies and consistent definitions will be required to classify the yeast sgs/egpbs/pbs before we can understand their function(s) and relevance to metazoan systems, especially given the historical confusion between plant heat-shock granules and metazoan sgs. unlike sgs, mammalian pbs are found in normally growing cells, in a cellcycle-dependent manner. whereas sgs are highly variable in size and shape, pbs are uniform, spherical structures that may exhibit directed movements within the cytoplasm. the signature components of pbs are deadenylases (ccr4), decapping enzymes (dcp1 and dcp2), decapping enhancers (hedls/ge-1), and exonucleases (xrn1) that are required for 5 0 -3 0 mrna decay. pbs increase in size and number in cells lacking one or more components of the mrna decay machinery, 19 suggesting that mrna accumulates in pbs while awaiting decay. the number of pbs found in the cytoplasm of normally growing cells varies among different cell lines: hela and cos exhibit higher basal levels of pbs than u2os cells, most of which lack ''resting'' pbs entirely. pb number and size is increased several fold in response to some types of environmental stress (e.g., oxidative stress), however, in mammalian cells some stresses (heat shock, clotrimazole, glucose deprivation, etc.) induce sgs without inducing pbs, 35 providing additional evidence that other independent signaling pathways regulate sg and pb assembly, beyond their dependence (sgs) or independence (pbs) on phospho-eif2a. complicating the picture is the fact that mammalian pbs are usually, but not invariably, coincident with ''gw bodies,'' defined by the scaffolding proteins gw182, and housing components of the microrna machinery, notably argonaute-2 (ago-2). ago2 is recruited to sgs (here defined as containing eif3), whereas gw182 appears largely restricted to pbs (defined as containing dcp1a or hedls, which have not been reported to enter sgs). whether the decapping machinery and the microrna machinery share a common unidentified molecular link, or whether they are linked to some common cellular membrane or structure is not yet clear. two nuclear structures linked to mrnp maturation and processing, cajal bodies and gems, are coincident in many but not all cell types, so there is precedent for this duality. 36 this spatial confluence is ascribed to coupled mrna processing or remodeling steps that tether these structures together. factors regulating pb/gwb fusion have not been identified, although there are reports of pb heterogeneity. interactions between sgs and pbs are more clearly documented. 35, 37 some conditions (arsenite, overexpression of ttp or cpeb) similarly cause sgs and pbs to juxtapose and fuse, whereas under other conditions they are largely independent. these regulated interactions between sgs and pbs suggest that mrnp components move from one type of granule to the other. this could indicate the degradation of untranslated transcripts passing through sgs into pbs, or the rescue of decay-bound mrnps from pbs. an evolving model of sg assembly is shown in fig. 1 . it posits a series of reversible aggregation-driven stages, which allow for the rapid assembly and disassembly of sgs, as well as the inclusion or export of specific mrnps to pbs, such as those containing ttp. sg initiation requires sudden polysome disassembly caused by stalled or abortive initiation. metabolic labeling experiments indicate that translation must be severely affected (>90%) before sgs form in response to arsenite 38 (fig. 1a) . primary nucleation occurs when the stalled 48s mrnps, suddenly stripped of ribosomes, create localized regions in which the free mrna concentration suddenly spikes, favoring the binding of all locally available rna-binding proteins, regardless of sequence. many of these proteins (table i) normally shuttle between the nucleus and the cytoplasm, but are trapped by their affinity for the suddenly high concentrations of free mrna in the cytoplasm. this binding in turn localizes the rna-binding proteins, many of which (sg nucleators) self-aggregate. small mrnp aggregates rapidly form in an energy-independent manner, evenly dispersed throughout the cytoplasm (primary aggregation). within the small sg aggregates, competition and exchange occur, as high-affinity sequence-specific interactions exchange with low-affinity, sequence-independent ones. as most mrna transcripts bind multiple rna-binding proteins, crosslinking between mrnps (secondary aggregation) occurs just as readily as selfaggregation. in particular, the c-terminal domain of pabp is notoriously insoluble, and as pabp is present on most transcripts, it facilitates this stage. moreover, knockdown of pabp severely impairs sg assembly, as does overexpression of a c-terminal truncation of pabp (kedersha, tisdale, and anderson, unpublished data). small ribosomal subunits likely contribute to the aggregation, as o-glcnac modification of ribosomal subunits appears necessary to promote sg assembly downstream of polysome disassembly, but has less effect on p-body assembly. 39 in the second phase of sg assembly, small aggregates fuse to form larger aggregates (growth/fusion). this stage is facilitated by microtubules and motors, 40 model depicting the graded stages of sg assembly. stalled initiation allows elongating ribosomes to leave polysomes, resulting in stalled 48s mrnps which recruit available cytoplasmic rna-binding proteins (blue region), many of which would normally shuttle into the nucleus (pink area). locally high concentrations of mrnps promote aggregation into small complexes, which progressively fuse into larger aggregates, assisted by microtubule-dependent motors. sorting ensues as the higher affinity interactions prevail, and as other signaling molecules are recruited. subsets of mrnps may be removed from the sgs pending phosphorylation and 14-3-3 binding and shunted to pbs for decay (e.g., ttp). other mrnps may be exported for other fates. proteins and more proximal, core sg proteins that are highly concentrated in small foci. within the larger sgs as well as in the smaller ones, mrnp sorting and remodeling occur, as the most stable mrnps form, containing partners of the highest mutual affinity. the protein component of each mrnp could then determine the fate of its transcript. some mrnas might be reinitiated (left branch, yellow), others detached from the eif3/40s complex and transferred to the decapping and decay machinery within pbs (e.g., those bound to ttp, bottom, dark blue), while others packaged into more long-lived storage granules (right branch, light blue). sg disassembly occurs when translation returns to normal (or polysomes are artificially stabilized via drugs). note that the ''new normal'' following stress likely contains a different spectrum of transcripts, and that lack of proteins required to achieve this new normal state (zbp1, staufen) will prolong the time it takes for sgs to disperse. this model accounts for the following observations: (1) sg assembly is diminished, but not completely abolished, by knockdown of any individual sg-nucleating protein. (2) the abundance of a particular nucleator and its mrna targets is proportional to the effects of its loss on sg assembly rate or size. (3) proteins recruited to sgs by piggyback interactions are blocked from sg recruitment if their particular target protein is blocked or missing. as many of these are signaling proteins (ccar1, traf2, src3, rsk2), this could have important functional effects on cell survival or growth. (4) individual sg nucleators can exit sgs along with their associated mrnas, without disassembly of the sg. for example, ttp leaves sgs but not pbs upon phosphorylation and 14-3-3-binding, 12 whereas phosphorylated hmex3b leaves pbs but not sgs when bound to 14-3-3. 43 a large number of sg-associated proteins (dbpa, fastk, fmrp/fxr1, hnrnpa1, hur, mbnl1, mln51, sam68, tia-1/tiar) are nuclear shuttling proteins which regulate alternative splicing, a regulated process usually studied by overexpression/depletion experiments. although direct data are lacking, rerouting of these shuttling proteins to sgs should result in the altered splicing of genes transcribed during periods when sgs are present. this has been proposed to occur with hnrnpa1, a sg-associated multifunctional protein that regulates splicing (as well as microrna processing). its recruitment to sgs is predicated on its nuclear export, prior to its cytoplasmic phosphorylation which allows its targeting to sgs in an rna-binding-dependent manner. core components of sgs are stalled 48s preinitiation complexes. one approach to determine whether sg assembly is pro-or antisurvival is to eliminate a specific eif2a kinase and determine whether its loss sensitizes or promotes resistance to its activating stress. for example, hri kinase mediates arsenite-induced sg assembly, 30, 39 and hri knockout mefs and u2os cells in which hri is depleted by sirna fail to respond to phospho-eif2a or assemble sgs in response to arsenite, but exhibit unimpaired sg assembly in response to other stresses. both hri knockout mefs and mice are resistant to arsenite, 30 linking sg assembly to apoptosis. by sequestering specific proteins that regulate cell survival, sgs may influence different signaling pathways that determine whether a stressed cell repairs its damage and lives, or dies by apoptosis. deletion studies have shown that some sg-associated proteins regulate ''relative viability'' as assessed by atp levels following stress (hnrnpa1, 44 ; mln51, 26 ), whereas other proteins regulate survival/apoptosis in a localization-specific manner (rack1, 45 ; rsk2, 46 ). for example, rack1 is a scaffold protein required for the activation of the mapkkk. when rack1 is sequestered at sgs, this stress kinase cascade is inactive and stress-induced survival is enhanced. similarly, the recruitment of rsk2 to sgs and to the nucleus requires its binding to tia-1, which affects both its ability to induce cyclin d1 and promote survival following arsenite stress. 46 an important factor in modeling sg/pb dynamics must account for the contribution of nucleocytoplasmic shuttling proteins in sg assembly. as shown in table i , most sg-associated proteins other than initiation factors are nuclear shuttling proteins, including many (g3bp, fxr1/fmrp, tia-1/tiar) whose overexpression nucleates sgs. knockdown of any one of these proteins delays sg formation or reduces the size of sgs, but does not abolish sg formation altogether. this suggests that these shuttling proteins contribute to the size of sgs, but act downstream of polysome disassembly. the relocalization of these normally nuclear proteins into cytoplasmic sgs must prevent their nuclear function(s) such as alternative splicing, transcription, mrna processing, etc., thus allowing a mechanism whereby sg assembly can alter nuclear events. one nucleolar protein (sgnp) has been shown to accumulate in sgs, suggesting a link between sgs and ribosome assembly. after acting as a nuclear transcriptional coactivator of nfkb-induced inflammatory cytokine genes, src-3 (steroid receptor co-activator-3) moves to the cytoplasm where it binds tia-1 and promotes the translational silencing of tnfa mrna. this enables it to turn off expression of the same genes that it turns on early in the inflammatory process. 47 it requires binding to tia-1 for its localization into sgs, and provides a clear molecular link between nuclear transcription and cytoplasmic translational silencing. whether it also interacts with the tia proteins to regulate cytokine splicing remains to be determined. in addition to src-3, the transcriptional coactivator ccar1 (cell division cycle and apoptosis regulator 1) is recruited to sgs via interactions with g3bp/caprin1; the functional implications of ccar1 at sgs remain to be determined. 48 sg assembly is linked to membrane-associated signaling events as well as nuclear ones. traf2, a cytoplasmic signaling molecule that mediates tnfa signaling, is recruited to sgs upon heat shock, corresponding to its shift into an insoluble form due to its binding to eif4g. 49 its sequestration in sgs prevents its association with the tnf receptor, suggesting a mechanism whereby feverinduced sgs could mute the immune response. plakophilin-3 is recruited to sgs as part of a complex containing g3bp, 49 linking sg formation with cell adhesion. sg assembly is associated with a number of posttranslational modifications, among them phosphorylation, arginine methylation (cirp), 49 o-glcnac modification, 39 ubiquitinylation (tudor-3, hdac6), 41, 50, 51 and acetylation. it is also linked to the molecular chaperones hsp70 (which prevent aggregation of the prion-like domain of tia-1) and hsp90, required for ago-2 targeting to both sgs and pbs. 52 phosphorylation-driven binding to the chaperone 14-3-3 inhibits ttp interactions with sgs but not pbs, but is required for hmex3b/ago complexes to dock at pbs, yet has no effect on their targeting to sgs. polysome disassembly is not sufficient to induce sgs or pbs: whereas puromycin promotes rapid (30 min) polysome disassembly, sg assembly is not observed for several hours. 28 however, brief puromycin exposure lowers the threshold at which other sg/pb promoting drugs induce pb assembly, 4 implicating the activation of pathways downstream of termination in the sg assembly process. a single species of mrna can be assembled into a sg or a pb, even within the same cell. 35 while the factors that influence sorting into distinct types of granules are not well understood, there is evidence that the mode of polysome disassembly affects the type of rna granule that is assembled, leading to the following model. when polysome disassembly is initiated by deadenylation, depletion of pabp disrupts the link between the 5 0 -and 3 0ends of the transcript and allows the recruitment of decapping enzymes. these linearized transcripts recruit the decay machinery, and are thus assembled into pbs. when polysome disassembly is mediated by stalled initiation on a circularized mrna, ribosome run-off leaves a circular mrnp that is assembled into sgs, thus accounting for the fact that eif4e is present in both sgs and pbs, whereas only sgs contain eif3, eif4g, eif4a, and pabp, while pbs contain instead the eif4e binding protein 4-et. new information on sg/pb dynamics comes from an sirna-based screen for genes that are required for sg/pb assembly in response to arsenite. 39 strikingly, five different subunits of eif3 (eif3c, d, e, g, and i) are required for sg but not pb assembly, while eif3b is required for both sg and pb assembly. notably, eif3e mediates the interaction of eif3 with eif4g and stabilizes the circular form of the mrna; 53 its deletion would be predicted to destabilize circular mrnps that might be preferentially routed to sgs rather than pbs. not detected in the screen was eif3j, a protein required for ribosome recycling 54 suggesting that impairing recycling might prevent sg assembly. moreover, erf1 (eukaryotic translation release factor 1) was found to be required for pb, but not sg formation, also suggesting that the mode of polysome disassembly may influence whether sgs or pbs result. the importance of these rna granules in the regulation of protein expression is underscored by their involvement in several aspects of disease pathogenesis. our understanding of their roles in cellular physiology may allow us to exploit these regulatory pathways for the development of new classes of drugs for the treatment of disease. studies of the translation of viral rnas have provided general insights into mechanisms of protein translation. the finding that many viruses interact, functionally or physically, with sgs and pbs highlights their relevance to mrna translation and decay. the different ways that viruses interact with rna granules is summarized below. poliovirus is a plus-strand rna virus that encodes proteinases that cleave essential host proteins to allow preferential transcription and translation of viral rna. poliovirus infection results in the transient assembly of sgs. the disassembly of sgs correlates with the cleavage of g3bp by viral proteinase 3c. expression of a noncleavable g3bp mutant prevents the disassembly of sgs and inhibits virus production, suggesting that sgs function in host defense against virus infection. 55, 56 semliki forest virus is another plus-strand rna virus that shuts down host protein synthesis prior to initiating the synthesis of virus proteins. this is accomplished, in part, by the activation of a stress response program that phosphorylates eif2a to repress translation initiation. in mefs expressing a nonphosphorylatable eif2a mutant, host protein shutoff is markedly impaired. phosphorylation of eif2a results in the assembly of sgs during the early stages of virus infection, but sgs are disassembled as viral protein synthesis is initiated, and thereafter sgs cannot be induced by arsenite. infection of tia-1 à/à mefs that exhibit impaired sg assembly results in a significant delay in host protein synthesis shutoff. thus, tia-1 and sgs appear to facilitate host protein synthesis shutoff, and their disassembly is required for efficient translation of viral proteins. 57 reovirus is a double-stranded rna virus that also activates a stress response program in infected cells. this involves the activation of pkr, phosphorylation of eif2a, and transcription of atf4. individual strains of reovirus differ in the extent to which host protein synthesis is turned off following infection. the phospho-eif2a induced assembly of sgs parallels the extent of host protein shut off in different reovirus strains. thus, reovirus appears to utilize sgs to promote the preferential translation of virus proteins. 58, 59 west nile virus is a plus-strand rna virus that also induces host protein shut down in infected cells. tia-1 and tiar, related proteins that nucleate sg assembly, bind to a 3 0 -terminal stem loop in the minus strand viral rna. during virus infection, tia-1 and tiar are concentrated at perinuclear sites of viral replication, suggesting that these proteins play a role in virus replication. consistent with this possibility, virus replication is severely impaired in tiar à/à mefs. the sequestration of tia-1 and tiar at sites of virus replication has been proposed to contribute to the impaired assembly of sgs and pbs observed in virally infected cells. 59 sendai virus is a minus strand rna virus that induces the assembly of sgs during virus infection. sg assembly is regulated by a short rna transcribed from the 3 0 -end of plus-strand viral rnas. this rna binds and sequesters tiar to impair sg assembly. sequestration of tiar also inhibits virus-induced apoptosis, a phenomenon that may promote cell survival to allow optimal virus production. 60 rotavirus is a double-stranded rna virus that also promotes the phosphorylation of eif2a in infected cells. unlike sindbis virus, reovirus and west nile virus-infected cells, phospho-eif2a does not induce sg assembly in rotavirusinfected cells. moreover, the growth of rotavirus is not impaired in fibroblasts expressing a nonphosphorylatable eif2a mutant. it is possible that rotavirus encodes a factor that prevents phospho-eif2a-mediated translational repression and/or the aggregation of untranslated mrnps. mouse hepatitis coronavirus is a plus-strand rna virus that induces the phosphorylation of eif2a and shuts down host protein synthesis in infected cells. virus infection also induces the assembly of sgs and pbs suggesting that rna granules play a role in reprogramming mrna translation/decay during viral infection. 61 brome mosaic virus (bmv) is a plus-strand rna virus that replicates in yeast. the translation and replication of bmv rnas is dependent upon the pb components pat1p, dhh1p, and lsm1p-7p. moreover, bmv rnas are concentrated in pbs suggesting that pbs facilitate viral replication. 62 retroviruses and retrotransposons share the ability to reverse transcribe dna copies for insertion into the genome. apobec3g and apobec3f are antiviral proteins that deaminate cytidines in retroviral or retrotransposonencoded rnas. although both of these proteins are concentrated at sgs and pbs, the functional significance of this localization remains to be determined. 63 similarly, tsn (tudor staphylococcal nuclease) is a risc-associated nuclease 64 that specifically cleaves inosine-modified dsrna, and associates with sgs. 65 line-1 transposon, still active in the human genome, encodes line1p, a protein required for tranposition that nucleates sgs 66 and coaggregates the line-1 mrna, 67 suggesting that sg formation may be a manifestation of the host antiviral response. mutations in the fragile mental retardation protein (fmrp) are associated with an x-linked form of mental retardation. fmrp is an rna-binding protein that is expressed in dendrites and at synapses. it has been proposed to function as a translational repressor that dampens the expression of synaptic proteins to impair neuronal function. 68 in normal cells, fmrp is found in association with polysomes suggesting a possible role in protein translation. in cells subjected to oxidative stress, or in hippocampal neurons perturbed by electrode insertion, fmrp accompanies untranslated mrnas to sgs. 69 in cells lacking fmrp or expressing an fmrp mutant associated with fxs, sg assembly is markedly impaired 70 suggesting that fmrp actively contributes to sg assembly. impaired sg assembly may prevent the reprogramming of protein translation that is required to protect cells from the adverse effects of environmental stress. it is possible that mental retardation in fxs resulting from neuronal cell death is caused by impaired sg assembly. sg assembly has been shown to regulate several aspects of immune cell function and inflammation. sgs are assembled following t cell receptormediated activation of cd4þ t cells. in these cells, il-4 and il-13 mrnas are transcribed and held in a translationally repressed state. t cell receptormediated restimulation releases these transcripts from translational repression allowing cytokine secretion. lentiviral expression of a dominant-negative mutant of the sg nucleator tia-1 releases the activation-induced translational silencing of these transcripts, implicating sgs in the regulation of t cell activation. 71 roquin is an e3 ligase that regulates the expression of icos, a costimulatory molecule that promotes t cell activation. 72 mutant mice expressing mutant roquin overexpress icos and develop a severe autoinflammatory disease. roquin appears to promote the mirna-dependent repression of icos expression. as roquin resides at sgs and pbs, these rna granules may be involved in this autoimmune syndrome. sg and pb partitioning may also regulate the expression of inflammatory proteins in activated macrophages. ttp, an mrna destabilizing protein that represses the expression of inflammatory mediators (e.g., tnfa, il-1b, il-6), plays a major role in regulating interactions between sgs and pbs. 35 in activated macrophages, ttp is phosphorylated and complexed with 14-3-3 proteins, modifications that prevent it from delivering its associated mrnas to the degradation machinery. 73 because phosphorylation of ttp causes sg: pb conjugates to dissociate, inhibition of mrna decay may be a consequence of altered sg:pb interactions. reduced blood flow caused by transient occlusion of the carotid artery or by reduced cardiac output confers ischemic injury to susceptible neurons. resumption of blood flow after ischemia can potentiate this process by a number of different mechanisms. in postischemic neurons, protein translation is turned off to conserve anabolic energy for the repair of stress-induced damage. in neurons that are particularly susceptible to ischemic injury (e.g., hippocampal cornu ammonis 1; ca1), translational arrest is prolonged and translational reprogramming that allows the preferential translation of repair proteins (e.g., heat-shock proteins) is impaired. in neurons that are relatively resistant to ischemia, sgs are transiently assembled. in ca1 neurons, sgs persist for prolonged periods. the persistence of sgs correlates with the prolonged translational arrest observed in these cells. 74 in addition to sgs, a related rna granule characterized by the presence of hur and the absence of tia-1 is observed in ischemic ca1 neurons. besides exhibiting prolonged translational arrest, these cells fail to synthesize protective heat-shock proteins in response to ischemic insults. 75 thus, alterations in the assembly and function of different types of rna granules may impair translational reprogramming in the susceptible ca1 neuron leading to increased vulnerability to ischemic injury. sg and pb formation is a regulated consequence of translational arrest, but there appears to be many levels to the story. monitoring sg formation may be of diagnostic use in assessing viral infection or hypoxia, as it affords us a window on the translational status of individual cells within tissues. however, while sgs and pbs are primarily sites of remodeling, packaging, and sorting of mrna, their assembly is linked to other cellular processes and pathways via a growing number of specific proteins. sgs secondarily recruit proteins involved in splicing, transcription, and signaling as part of an adaptive process. defining the specific molecules that link sg formation to other pathways, and selectively targeting them may be of therapeutic value. the impact of pb/gwb formation on other pathways is presently less clear, but the ongoing river of data will doubtlessly flush out new ideas as these exciting young fields mature. rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2a to the assembly of mammalian stress granules cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mrnas plant stress granules and mrna processing bodies are distinct from heat stress granules mammalian stress granules: highly dynamic sites of mrna triage during stress induced translational arrest tia-1 is a translational silencer that selectively regulates the expression of tnf-alpha visibly stressed: the role of eif2, tia-1, and stress granules in protein translation stressful initiations evidence that ternary complex (eif2-gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules mammalian stress granules represent sites of accumulation of stalled translation initiation complexes the rasgapassociated endoribonuclease g3bp assembles stress granules rasputin, more promiscuous than ever: a review of g3bp mk2-induced tristetraprolin: 14-3-3 complexes prevent stress granule association and are-mrna decay inhibition of eukaryotic translation initiation by the marine natural product pateamine a rnamediated sequestration of the rna helicase eif4a by pateamine a inhibits translation initiation eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine a inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation an antiviral response directed by pkr phosphorylation of the rna helicase a decapping and decay of messenger rna occur in cytoplasmic processing bodies cytoplasmic foci are sites of mrna decay in human cells p bodies: at the crossroads of post-transcriptional pathways p bodies and the control of mrna translation and degradation the highways and byways of mrna decay the control of mrna decapping and p-body formation perk is essential for translational regulation and cell survival during the unfolded protein response signal integration via pkr the exonjunction-complex-component metastatic lymph node 51 functions in stress-granule assembly distinctive properties of the 5'-untranslated region of human hsp70 mrna mammalian stress granules and processing bodies translational control is required for the unfolded protein response and in vivo glucose homeostasis metazoan stress granule assembly is mediated by p-eif2 {alpha}-dependent and -independent mechanisms heme-regulated inhibitor (hri) kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 (eif2) inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure stress-dependent relocalization of translationally primed mrnps to cytoplasmic granules that are kinetically and spatially distinct from p-bodies accumulation of polyadenylated mrna, pab1p, eif4e, and eif4g with p-bodies in saccharomyces cerevisiae p bodies promote stress granule assembly in saccharomyces cerevisiae robust heat shock induces eif2{alpha}-phosphorylation-independent assembly of stress granules containing eif3 and 40s ribosomal subunits in budding yeast, saccharomyces cerevisiae stress granules and processing bodies are dynamically linked sites of mrnp remodeling subnuclear organelles: new insights into form and function the translational regulator cpeb1 provides a link between dcp1 bodies and stress granules dynamic shuttling of tia-1 accompanies the recruitment of mrna to mammalian stress granules a functional rnai screen links o-glcnac modification of ribosomal proteins to stress granule and processing body assembly dynein motor contributes to stress granule dynamics in primary neurons the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation interaction with 14-3-3 adaptors regulates the sorting of hmex-3b rna-binding protein to distinct classes of rna granules hnrnp a1 relocalization to the stress granules reflects a role in the stress response formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways codependent functions of rsk2 and the apoptosis-promoting factor tia-1 in stress granule assembly and cell survival an essential function of the src-3 coactivator in suppression of cytokine mrna translation and inflammatory response microtubuledependent association of akap350a and ccar1 with rna stress granules sequestration of traf2 into stress granules interrupts tumor necrosis factor signaling under stress conditions tdrd3 is a novel stress granule-associated protein interacting with the fragile-x syndrome protein fmrp tdrd3, a novel tudor domain-containing protein, localizes to cytoplasmic stress granules hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and p-bodies translation initiation factor eif4g-1 binds to eif3 through the eif3e subunit recycling of eukaryotic posttermination ribosomal complexes inhibition of cytoplasmic mrna stress granule formation by a viral proteinase how viruses avoid stress importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation reovirus induces and benefits from an integrated cellular stress response interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies interactions between brome mosaic virus rnas and cytoplasmic processing bodies antiviral protein apobec3g localizes to ribonucleoprotein complexes found in p bodies and stress granules a micrococcal nuclease homologue in rnai effector complexes inosine-containing dsrna binds a stress-granule-like complex and downregulates gene expression in trans line-1 orf1 protein localizes in stress granules with other rna-binding proteins, including components of rnai risc retrotransposons revisited: the restraint and rehabilitation of parasites fragile x syndrome: loss of local mrna regulation alters synaptic development and function fragile x mental retardation protein shifts between polyribosomes and stress granules after neuronal injury by arsenite stress or in vivo hippocampal electrode insertion cells lacking the fragile x mental retardation protein (fmrp) have normal risc activity but exhibit altered stress granule assembly activation of the integrated stress response during t helper cell differentiation a ring-type ubiquitin ligase family member required to repress follicular helper t cells and autoimmunity mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay prolonged translation arrest in reperfused hippocampal cornu ammonis 1 is mediated by stress granules persistent redistribution of poly-adenylated mrnas correlates with translation arrest and cell death following global brain ischemia and reperfusion argonaute 2/risc resides in sites of mammalian mrna decay known as cytoplasmic bodies quantitative analysis of argonaute protein reveals microrna-dependent localization to stress granules the anti-hiv-1 editing enzyme apobec3g binds hiv-1 rna and messenger rnas that shuttle between polysomes and stress granules human retroviral host restriction factors apobec3g and apobec3f localize to mrna processing bodies ataxin-2 interacts with the dead/h-box rna helicase ddx6 and interferes with p-bodies and stress granules post-translational arginylation of calreticulin: a new isospecies of calreticulin component of stress granules distinct structural features of caprin-1 mediate its interaction with g3bp-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mrnas identification of fuse-binding proteins as interacting partners of tia proteins the cold-inducible rnabinding protein migrates from the nucleus to cytoplasmic stress granules by a methylationdependent mechanism and acts as a translational repressor the eif4g-homolog p97 can activate translation independent of caspase cleavage mbnl1 associates with yb-1 in cytoplasmic stress granules the dead-box rna helicase ddx3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control recruitment of the rna helicase rhau to stress granules via a unique rna-binding domain a functional link between disrupted-in-schizophrenia 1 and the eukaryotic translation initiation factor 3 the enhancer of decapping proteins, edc1p and edc2p, bind rna and stimulate the activity of the decapping enzyme multiple processing body factors and the are binding protein ttp activate mrna decapping a role for the eif4e-binding protein 4e-t in p-body formation and mrna decay regulation of stress granule dynamics by grb7 and fak signalling pathway trapping of messenger rna by fragile x mental retardation protein into cytoplasmic granules induces translation repression the multifunctional fus, ews and taf15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response functional dissection of the human tnrc6 (gw182-related) family of proteins ge-1 is a central component of the mammalian cytoplasmic mrna processing body nuclear rna export factor 7 is localized in processing bodies and neuronal rna granules through interactions with shuttling hnrnps hnrnp k interacts with rna binding motif protein 42 and functions in the maintenance of cellular atp level during stress conditions human hnrnp q re-localizes to cytoplasmic granules upon pma, thapsigargin, arsenite and heat-shock treatments stress granule assembly is mediated by prion-like aggregation of tia-1 hur binding to cytoplasmic mrna is perturbed by heat shock importin 8 is a gene silencing factor that targets argonaute proteins to distinct mrnas intracellular localization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase localization of the developmental timing regulator lin28 to mrnp complexes, p-bodies and stress granules the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci identification of novel argonaute-associated proteins neural rna-binding protein musashi1 inhibits translation initiation by competing with eif4g for pabp inhibition of g(1) to s phase progression by a novel zinc finger protein p58(tfl) at p-bodies identification of patl1, a human homolog to yeast p body component pat1 selective localization of pcbp2 to cytoplasmic processing bodies identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing rna-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules polysome-bound endonuclease pmr1 is targeted to stress granules via stress-specific binding to tia-1 dendritic localization of the translational repressor pumilio 2 and its contribution to dendritic stress granules rna-associated protein 55 (rap55) localizes to mrna processing bodies and stress granules rpp 20 interacts with smn and is re-distributed into smn granules in response to stress the rna polymerase ii subunit rpb4p mediates decay of a specific class of mrnas sam68 functions in nuclear export and translation of hiv-1 rna sgnp: an essential stress granule/nucleolar protein potentially involved in 5.8s rrna processing/transport mammalian smaug is a translational repressor that forms cytoplasmic foci similar to stress granules staufen recruitment into stress granules does not affect early mrna transport in oligodendrocytes mammalian staufen 1 is recruited to stress granules and impairs their assembly survival motor neuron protein facilitates assembly of stress granules probing the mrna processing body using protein macroarrays and "autoantigenomics zbp1 regulates mrna stability during cellular stress a pkr-like eukaryotic initiation factor 2alpha kinase from zebrafish contains z-dna binding domains instead of dsrna binding domains key: cord-267907-zbsbqj9o authors: brown, samantha m.; doom, jenalee r.; lechuga-peña, stephanie; watamura, sarah enos; koppels, tiffany title: stress and parenting during the global covid-19 pandemic date: 2020-08-20 journal: child abuse negl doi: 10.1016/j.chiabu.2020.104699 sha: doc_id: 267907 cord_uid: zbsbqj9o background: stress and compromised parenting often place children at risk of abuse and neglect. child maltreatment has generally been viewed as a highly individualistic problem by focusing on stressors and parenting behaviors that impact individual families. however, because of the global coronavirus disease 2019 (covid-19), families across the world are experiencing a new range of stressors that threaten their health, safety, and economic well-being. objective: this study examined the effects of the covid-19 pandemic in relation to parental perceived stress and child abuse potential. participants and setting: participants included parents (n = 183) with a child under the age of 18 years in the western united states. method: tests of group differences and hierarchical multiple regression analyses were employed to assess the relationships among demographic characteristics, covid-19 related stressors, mental health risk, protective factors, parental perceived stress, and child abuse potential. results: greater covid-19 related stressors and high anxiety and depressive symptoms are associated with higher parental perceived stress and child abuse potential. conversely, greater parental support and perceived control during the pandemic may have a protective effect against perceived stress and child abuse potential. results also indicate racial and ethnic differences in covid-19 related stressors, but not in mental health risk, protective factors, perceived stress, or child abuse potential. conclusion: findings suggest that although families experience elevated stressors from covid-19, providing parental support and increasing perceived control may be promising intervention targets. it is well-established that exposure to stressors play an important role in child maltreatment potential (rodriguez-jenkins & marcenko, 2014; whipple & webster-stratton, 1991) . exposure to stressors can lead to cognitive, emotional, and physical fatigue, which may in turn place undue strain on the parent-child relationship (deater-deckard, 2004) . indeed, as parental stress levels rise, parents may be more likely to engage in harsh parenting (beckerman et al., 2017) , thereby increasing the risk of child maltreatment (martorell & bugental, 2006; rodriguez & green, 1997) . however, certain factors, such as perceived control over stressful events or supportive family environments, may act as buffers to decrease distress and the risk of child maltreatment (frazier et al., 2011; li et al., 2011) . despite ample evidence linking individual and family level stressors with poor parenting and child maltreatment, families across the world are collectively experiencing a new range of stressors that threaten their health, safety, and economic well-being due to the global coronavirus disease 2019 (covid-19) pandemic. although is not the only global public health challenge to threaten society, it will likely have long-term negative impacts on today's children and families. the greater awareness of the impact of stress as we all experience a global pandemic offers an opportunity to better understand how stressors external to the family increase risk of maltreatment. as such, this study examined the influence of risk and protective factors on parents' perceptions of stress and risk of child abuse potential during the covid-19 pandemic. stress can accumulate as a result of a broad range of factors. in the context of child maltreatment, a large body of literature focuses on stressors at the individual and family levels (i.e., ontogenic and microsystem levels, respectively; cicchetti & lynch, 1993; cicchetti & rizley 1981 ). that is, child maltreatment may occur from stressors that result from economic hardship, low education, single parenthood, or a large number of dependent children (centers for j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 3 disease control and prevention, n.d.; sedlak et al., 2010) , among others, but that might not otherwise impact persons outside of the family. research indicates that there is not a single factor that is related solely to child maltreatment; instead, the accumulation of risk factors is a more robust indicator of adverse outcomes (patwardhan et al., 2017; sameroff, 1979) . moreover, the extent to which co-occurring stressors affect adverse outcomes, such as poor parenting behaviors, may be due, in part, to the ways through which individuals perceive these events as stressful. in other words, the degree to which situations in one's life are appraised as stressful (i.e., perceived stress) can influence adaptive versus maladaptive outcomes (cohen et al., 1983; lazarus, 1977) . consequently, parents experiencing elevated levels of cumulative stress show more rigid and abusive parenting behaviors (hutchison et al., 2016; liu & merritt, 2018; yang, 2015) . the global covid-19 pandemic is a stressor that originated outside of the family system but given the novelty and uncertainty concerning this disease, it is likely to be perceived as a significant stressor for many parents and children. in fact, emerging research has shown that parents' perceived impact of covid-19 is associated with increased parenting stress and, in turn, increased risk of harsh parenting (chung et al., 2020) . even for families who have not been directly exposed to the virus, they are likely to experience indirect effects of the covid-19 pandemic (van bavel et al., 2020) . for example, in countries outside of the united states, covid-19 has led to serious mental health burden (e.g., marazziti et al., 2020; pierce et al., 2020) , with the prevalence of anxiety, depression, and sleep problems ranging from 18% to 35%, especially among younger individuals who are preoccupied with thoughts regarding the disease (huang & zhao, 2020) . given that there are already mental health implications associated with the covid-19 health crisis, children in families with more risk factors may be more vulnerable j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 4 to child maltreatment. indeed, parents with elevated stress and co-occurring anxiety and depressive symptoms have been shown to be less responsive to their children's needs, which in turn is a strong predictor of child abuse potential (mcpherson et al., 2008) . although several public health efforts were taken to mitigate transmission of covid-19 after its emergence, these efforts have had unintended consequences that could further impact parent-perceived stress and poor parenting. specifically, actions included recommendations to increase physical distancing as well as to close schools, childcare agencies, and many customer service businesses. as a result, families are more likely to experience increased social isolation, the inability to access supportive and educational services, and economic difficulties, which may exacerbate stress in many households. in fact, social isolation increases susceptibility to stress and may have harmful effects on both mental and physical health (hawkley & cacioppo, 2010) . parents who are faced with competing demands of limiting social interactions and remaining at home with their children may be particularly vulnerable during this time; research shows that continual close contact under stress is a risk factor for aggressive behaviors and violence (brooks et al., 2020; greenaway et al., 2014; reynolds et al., 2008) . furthermore, some families are experiencing other challenges, such as working from home while also caring for and educating their children. given that school and childcare professionals are central to identifying concerns of abuse and neglect (fitzpatrick et al., 2020; u.s. dhhs, 2020) , children who may have once been identified as at risk in these settings may be more vulnerable to maltreatment as they spend most of their time at home. some families are also experiencing more financial strain as a result of the changing economy and increasing unemployment. although the stresses of poverty have long been associated with increased risk of child maltreatment, several specific economic indicators may be particularly implicated in abuse potential. for example, the rise in j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 5 unemployment and foreclosure rates are associated with an increased likelihood of investigated and substantiated maltreatment (frioux et al., 2014) . in addition to changes in the economy, other social conditions may worsen the impact of covid-19 related stressors on overall perceptions of stress and parenting, particularly among minoritized populations. the centers for disease control and prevention (2020) reported that covid-19 disease burden is disproportionately high among racial and ethnic minority groups, such that black/ african american and latinx individuals are more likely than their white counterparts to acquire the illness, be hospitalized, and die from covid-19 (centers for disease control and prevention, 2020) . systemic inequities contribute to disparities in health outcomes among minoritized populations. for example, families of racial and ethnic minorities experience racism and discrimination (e.g., brondolo et al., 2009) and may be less able to social distance due to increased segregation into residential housing, (iceland et al., 2010; popescu et al., 2018) , limited paid sick leave (bartel et al., 2019) , and inadequate access to health insurance and health care phillips et al., 2000) . these economic and social inequities may in turn place minoritized families at greater risk for increased stress and disparate outcomes during the covid-19 pandemic. however, not all parents experiencing cumulative stressors from covid-19 may be at risk of higher perceived stress or poor parenting, suggesting that protective factors may mitigate the impact of covid-19 on parental stress and child abuse potential. specifically, adaptive coping strategies and supportive family environments may serve as protective factors for families experiencing stress and may differentially influence abuse potential. for example, perceived control is a key construct in understanding stress and coping (dijkstra & homan, 2016) . indeed, the belief that one has influence over life events is related to different psychological and j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 6 behavioral outcomes. perceived control over present events is associated with decreases in overall stress, anxiety, and depression as well as better adjustment across situations (ballash et al., 2006; frazier et al., 2004; grote et al., 2007) . similarly, other coping strategies, such as acceptance, or the ability to accept negative thoughts or experiences without judging them, are strongly and negatively associated with perceived stress (donald & atkins, 2016) . in addition to adaptive coping strategies, supportive family environments may also be promotive in the context of parenting. previous research shows that mother's perceptions of family support are associated with less parenting stress; thus, parents with more support are better able to engage in positive parenting (deater-deckard, 1998; sanders et al., 2014) . consistent with the ecologicaltransactional model of child maltreatment (cicchetti & lynch, 1993; cicchetti & rizley 1981) , risk and protective factors compete at each level of the ecology, namely the macrosystem (cultural level factors), exosystem (community level factors), microsystem (family level factors), and ontogeny (individual level factors) to affect maltreatment and developmental outcomes of children. experiencing multiple risk factors across levels of the ecology may increase risk of child abuse potential in an additive manner. importantly, however, certain factors may also protect a family from the accumulation of stress, thereby acting as main effects in decreasing the risk of maltreatment. despite a robust literature on myriad risk and protective factors implicated in parentperceived stress and child maltreatment, little research has been done to examine the extent to which cumulative stressors from global pandemics shape overall perceptions of stress and impact parenting. indeed, very few articles have been published to date regarding covid-19 in relation to stress and family violence (e.g., campbell, 2020; prime et al., 2020) , particularly risk of child abuse potential. the current study offers exploratory evidence to address the following research procedure families were recruited from child-and family-serving agencies and educational settings in the rocky mountain region of the united states. agency staff shared the study with families they served. families were also contacted about the current study by phone or email if they had participated in previous studies conducted by the research team. parents, aged 18 years or older, with a child under the age of 18 years were invited to participate in the study and provided an online survey link that was administered via qualtrics. the survey was available from april 21, 2020 to may 9, 2020 and took approximately 20 minutes to complete. participants were compensated with a $10 gift card after completion of the survey. two hundred and sixteen participants began the survey; however, 33 participants were not included in final analyses for the following reasons: (1) they did not correctly complete a study validation question to ensure they were not completing questions at random (n = 21), (2) they indicated that another caregiver in the household completed the survey (n = 4); these participants were removed to eliminate interdependent data, and (3) they did not complete at least half of the survey (n = 8); participants could progress through the survey and select 'prefer not to answer' to questions they did not wish to answer in order to indicate study completeness. therefore, the final sample included 183 j o u r n a l p r e -p r o o f demographic characteristics. basic demographic characteristics included parent age, gender, race/ ethnicity, education, relationship status, receipt of financial assistance, and number of adults and children in the household. the following variables were recoded for analyses: parent gender (0 = male, 1 = female), relationship status (0 = single or partner not living in the home, 1 = married or partner living in the home), and financial assistance (0 = not receiving financial assistance, 1 = receiving any financial assistance). in addition, because few participants identified as american indian or alaska native (n = 1), asian (n = 4), or other race/ ethnicity (n = 1), racial and ethnic categories were recoded as follows: 1 = american indian or alaska native, asian, mixed race/ ethnicity, or other race/ ethnicity ("other or mixed race/ ethnicity"), 2 = black/ african american, 3 = latinx, and 4 = non-latinx white, with non-latinx white as the reference category. because no or few participants had no schooling (n = 0) or indicated the highest education completed was 1 st -8 th grade (n = 3) or trade school (n = 7), education categories were recoded as follows: 1 = less than high school, 2 = high school graduate/ ged, 3 = some college, 4 = associates degree or trade school, 5 = four-year college degree, and 6 = post graduate degree. for each demographic characteristic, participants could also select "prefer not to answer"; these responses were subsequently recoded as missing. covid-19 stressors were assessed using investigator developed questions. using a checklist, parents were asked to indicate whether they or their children experienced any stressors as a result of covid-19 stay-at-home restrictions and school and childcare closures in the following domains: 1) parent mood or stress, 2) parent physical j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 9 health, 3) parent's relationship/ interactions with partner, 4) parent's relationship/ interactions with child(ren), 5) child(ren)'s physical health, and 6) child(ren)'s academics/ learning. given prior research on cumulative risk in relation to child maltreatment (e.g., lamela & figueiredo, 2015) , which postulates that poor outcomes are due to the accumulation of individual risk factors, items were dichotomously coded to indicate whether stressors in that category occurred (0 = no, 1 = yes); these items were subsequently summed to create a total covid-19 risk score. parents were also asked whether or not they knew someone who tested positive for covid-19 (0 = no, 1 = yes) or who died from covid-19 (0 = no, 1 = yes); these responses could include a partner or spouse, child, family member, friend, or someone else they know. finally, parents were asked an open-ended qualitative question, "in general, how has your life been affected (e.g., mental, physical, financial health, relationship difficulties, etc.) by covid-19 and recommendations to isolate?" mental health risk factors. parent anxiety, depression, and sleep were assessed as possible mental health risk factors. parental anxiety was measured by a single item from the general anxiety disorder-7 scale (gad-7; spitzer et al., 2006) : "during the past two weeks, i felt nervous, anxious, or on edge" (0 = not at all, 1 = several days, 2 = more than half the days, 3 = nearly every day, 4 = prefer not to answer). the item was recoded into a dichotomous anxiety indicator where "not at all" was coded as 0 and "several days" to "nearly every day" were coded as 1. "prefer not to answer" responses were coded as missing. parental depression was measured by a single item from the center for epidemiologic studies depression scale (cesd-r; radloff, 1977) : "during the past week, i felt depressed" (1 = rarely or none of the time, 2 = some or little of the time, 3 = occasionally or a moderate amount of time, 4 = most or all of the time, 5 = prefer not to answer). the item was recoded into a j o u r n a l p r e -p r o o f dichotomous depression indicator where "rarely or none of the time" was coded as 0 and "some or little of the time" to "most or all of the time" were coded as 1. "prefer not to answer" responses were coded as missing. poor parental sleep was measured by a single item indicator from the center for epidemiologic studies depression scale (cesd-r; radloff, 1977) : "during the past week, my sleep was restless" (1 = rarely or none of the time, 2 = some or little of the time, 3 = occasionally or a moderate amount of time, 4 = most or all of the time, 5 = prefer not to answer). the item was recoded into a dichotomous depression indicator where "rarely or none of the time" was coded as 0 and "some or little of the time" to "most or all of the time" were coded as 1. "prefer not to answer" responses were coded as missing. protective factors. parental support, perceived control over the covid-19 pandemic, and acceptance were assessed as possible protective factors. parental support was measured using the parental support subscale from the parent-child relationship inventory (pcri; gerard, 1994) . the parental support subscale consists of nine items measuring the level of emotional and social support a parent receives. parents indicate the extent to which they agree or disagree with each item (1 = strongly agree, 2 = agree, 3 = disagree, 4 = strongly disagree). example items include: "when it comes to raising my child, i feel alone most of the time", "i sometimes feel overburdened by my responsibilities as a parent", and "i have someone to help me do chores around the house." three items are reverse coded, items are summed, and higher scores indicate positive parenting characteristics (i.e., higher parental support). in this sample, the pcri parental support subscale demonstrated acceptable reliability (α = .71). perceived control over the covid-19 pandemic was measured by the present control subscale of the perceived control over stressful events scale (frazier et al., 2011) . the present j o u r n a l p r e -p r o o f control subscale consists of eight items measuring the extent to which individuals believe they have control over a current event; we asked parents to respond to each item with regard to the covid-19 pandemic and how they have felt in the past two weeks (1 = strongly disagree, 2 = disagree somewhat, 3 = agree somewhat, 4 = strongly agree). example items include: "there isn't much i can do to help myself feel better about the event", "how i deal with this event now is under my control", and "when i am upset about the event, i can find a way to feel better." four items are reverse coded, items are summed, and higher scores indicate better perceived control over the stressful event. in this sample, the present control subscale demonstrated acceptable reliability (α = .79). regulation questionnaire-short form (cerq/sf; garnefski et al., 2001) . the acceptance subscale consists of two items measuring an individual's thoughts and cognitive strategies after having experienced a negative event (1 = never, 2 = sometimes, 3 = regularly, 4 = often, 5 = always). items include: "i think that i have to accept that this has happened" and "i think i have to accept the situation." items are summed and higher scores indicate better coping (i.e., higher acceptance). in this sample, the cerq/sf acceptance subscale demonstrated acceptable reliability (α = .78). primary outcomes. perceived stress and child abuse potential were assessed as the primary outcomes of the study. perceived stress was measured by the perceived stress scale (pss-10; cohen et al., 1983) . the pss-10 consists of 10 items measuring individuals' thoughts and feelings regarding potentially stressful situations that occurred in the past month (0 = never, 1 = almost never, 2 = sometimes, 3 = fairly often, 4 = very often). example items include: "in the last month, how often have you been upset because of something that happened unexpectedly?", j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 12 "in the last month, how often have you felt nervous and stressed?", and "in the last month, how often have you found that you could not cope with all the things that you had to do?" items are summed and higher scores indicate greater perceived stress. in this sample, the pss-10 demonstrated good reliability (α = .83). child abuse potential was measured using a revised version of the brief child abuse potential inventory (cap inventory; milner, 1986; ondersma et al., 2005) . the original cap inventory (milner, 1986) consists of 160 total items, which are shown to distinguish parents who may be abusive from those who may not (milner & wimberely, 1980) . the cap inventory can be divided into factor scales describing psychological difficulties and interactional problems (e.g., distress, rigidity, family conflict, loneliness). to reduce participant burden, for this study, 10 items were used from a brief version of the cap inventory (ondersma et al., 2005) , specifically from the loneliness, rigidity, and family conflict factor scales in which participants were asked whether they agree or disagree with several statements. example items include: "my family fights a lot", "children should never disobey", and "a child needs very strict rules." items are summed and higher scores indicate greater child abuse potential. in this sample, the revised cap inventory demonstrated good reliability (α = .80). descriptive statistics (means, standard deviations, and percentages) were used to describe the sample demographic characteristics and study variables. because emerging research has shown that families of diverse racial and ethnic identities may be disproportionately impacted by the covid-19 pandemic, chi-square and one-way anova analyses were conducted to examine whether there were racial and ethnic differences in covid-19 risk factors, mental health risk factors, and protective factors. next, relationships among key variables of interest were j o u r n a l p r e -p r o o f running head: stress, parenting, and covid-19 13 examined using pearson's bivariate correlations. finally, hierarchical multiple regression analyses were conducted to test correlates of: (1) parental perceived stress and (2) child abuse potential. for each of the two regression models, correlates were entered sequentially in four steps: first, demographic characteristics; second, covid-19 risk factors; third, mental health risk factors; and fourth, protective factors. this allowed for an assessment of the unique relationships between covid-19 and mental health risk factors and study outcomes as well as whether protective factors mitigated perceived stress and child abuse potential over and above these risks. prior to conducting hierarchical multiple regression analyses, we examined variables for missingness using little's test of missing completely at random (mcar; little, 1988) and found that values were not missing at random (p = .39); therefore, missing data were handled with listwise deletion. to examine responses to the single open-ended question regarding how participants' lives have been affected by covid-19 and recommendations to isolate at home, qualitative template analysis was used to explore stressors as well as potential positive experiences. common themes were then identified within these categories, which allowed for a hierarchical method of coding in which broad themes were used to encompass more specific codes and/or patterns (padgett, 2008) . the themes identified were then summed across participant responses to calculate their frequencies and percentages. two of the authors coded a subset of the questions with high rates (85%) of interrater reliability. detailed sample characteristics are provided in table 1 . participants included parents, ages 18 to 55, of a child under the age of 18 years. the majority of participants were mothers and racially/ ethnically identified as non-latinx white, followed by latinx, other or mixed race/ ethnicity, or black/ african american. participants were mostly married or had a partner living in the home and had some college or higher education. participants indicated that they received some form of financial assistance, with most participants receiving medicaid coverage, followed by food stamps, women, infants, and children (wic), free or reduced cost childcare, unemployment, cash assistance/ temporary assistance for needy families (tanf), housing assistance, alimony/ child support, or other financial assistance. regarding stressors from covid-19, participants reported experiencing approximately 3.5 stressors, on average, which most often included stressors due to changes in parent mood or general stress, followed by changes in parent's relationship/ interactions with child(ren) and with partner, parent physical health, and child(ren)'s academics/ learning and physical health. over half of the sample knew someone who tested positive for covid-19 and slightly over one-quarter of the sample knew someone who died from covid-19. most participants also indicated that they experienced high levels of anxiety symptoms, depressive symptoms, and poor sleep in the past one to two weeks. participants reported that there were on average approximately two adults and two children in their households. [insert table 1 about here] regarding racial and ethnic differences in covid-19 risk factors, mental health risk factors, and protective factors, there were no significant differences across racial and ethnic groups for knowing someone who tested positive, x 2 (3) = 4.03, p = .26; or died from covid-19, 3.60, sd = 1.41) was also significantly higher than the mean score of covid-19 related stressors for black/ african american parents (m = 2.14, sd = .90; 95% ci, .28 -2.62). results of the bivariate correlations among key study variables can be found in table 2 . the first hierarchical multiple regression model regressed parental perceived stress on demographic characteristics, covid-19 risk factors, mental health risk factors, and protective factors. results for each block are reported separately in text, while results for the hierarchal multiple regression model retained from the fourth block can be found in table 3 .05) were significantly and negatively associated with perceived stress, indicating that these factors may mitigate and protect against increased perceived stress. [insert table 3 .14, p < .001). specifically, parental support (b = -.23, p < .001) and perceived control over the covid-19 pandemic (b = -.11, p < .05) were significantly and negatively associated with abuse potential, indicating that these factors may mitigate and protect against increased risk of child abuse potential. [insert table 4 about here] participants were asked to describe how their life has been affected by covid-19 and recommendations to isolate at home. several themes were identified from the single open-ended question inquiring about their experiences. table 5 provides the frequency and percentages of themes that emerged from participant responses. participants reported that they experienced increased stressors in the areas of: (1) loss of employment or income/ inability to provide for family, (2) uncertainty about the future, (3) inability to see family or friends or socialize with others, (4) relationship difficulties, (5) general stress and feeling tired, (6) poor mental health, (7) poor physical health, (8) meltdowns and/or boredom from children, (9) difficulties managing children's academics, (10) fear of contracting the virus or hearing about people dying from the virus, (11) lack of supports, and (12) difficulties managing work from home. loss of employment or income and an inability to provide for their family was the most salient stressor. for example, one parent stated, "it's been difficult trying to pay my bills with being unemployed at the moment." another parent said, "[our] financial health has been very poor with both of us out of work and not qualified for unemployment. this leads to greater stress." some parents reported negative impacts on their own health and well-being in order to care for their children. specifically, one parent expressed that "to make sure my baby is taken care of during this time…i sometimes go without food." the inability to see family or friends or socialize with others was also a common stressor among participants. one parent stated, "i have found myself to be stressed and on edge constantly. i miss interacting with people outside my home and office." parents reported that their mental health has also been impacted by the stay-at-home restrictions. one parent reported, "depression has affected us in our household." other parents reported experiencing multiple stressors that intersect across themes. for example, a parent expressed "experiencing fear and anxiety about contracting the virus." this parent also stated, "i was laid off my job, so i feel worried financially about the future." one parent described that the inability to go outside increases stress and "more meltdowns" from their children. another parent reported: it's been mentally and physically stressful dealing with quarantine. small house. my husband has been recovering from… [an] injury and now has to be a teacher, housekeeper, and try to heal himself at the same time. it's hard for me to go to work every day as an essential worker and be exposed to potential infection. finances are a mess and we are relying on food stamps and food banks/ free dinners. despite participants reporting an increase in stressors, some parents reported no change or positive change as a result of recommendations to self-isolate and stay at home. positive or neutral themes emerged as follows: (1) (earls et al., 2008; lau et al., 2008) . disease control and prevention, 2020; hooper et al., 2020) , and several studies indicate that stress processes in families may differ both within and across racial and ethnic groups (gomel et al., 1998; goosby et al., 2012) . particularly for latinx families, familismo is commonly recognized as a core latinx value. familismo involves strong identification with and attachment to a nuclear extended family as well as an obligation to provide both material and emotional support to one's family (calzada et al., 2012) . the absence of family support due to the stay-athome restrictions and physical distancing could explain latinx parents' higher report of covid-19 related stressors compared to other racial and ethnic groups. however, the cultural context regarding physical distancing and social support were not systematically assessed in this study and should be considered in future research examining the impact of covid-19 on domains of parenting, health, and wellness. although information concerning covid-19 is continuing to evolve, results were somewhat unexpected with regard to participants' reports of knowing someone who tested positive or died from covid-19; over half of the sample reported knowing someone who tested positive for the virus, with approximately one-quarter of the sample knowing someone who died. consistent with prior research, however, it was anticipated that families would report experiencing financial strain during the pandemic and this is another major source of stress among families. indeed, lower-income individuals tend to live in urban settings, in more crowded conditions both by neighborhood and household composition, and are more likely to be employed in public-facing occupations (e.g., services and transportation), which may be a barrier to physical distancing (webb-hooper et al., 2020) . furthermore, the economic crisis in 2008 also affected the labor market and individual's health and wellness (mucci et al., 2016) , thereby highlighting the toll that health and economic crises may have on general perceptions of stress j o u r n a l p r e -p r o o f and mental health. qualitative findings provide further insight into participants' experiences about how their life has been affected by covid-19 and the stay-at-home restrictions, specifically with regard to their financial, physical, and mental health as well as their children's well-being and ability to assist with their children's academics. an accumulation of stressors due to covid-19 is a key risk factor implicated in higher parent-perceived stress whereas anxiety and depression are associated with both higher parentperceived stress and child abuse potential. these results suggest that the greater number of stressors experienced from covid-19 may act as a salient determinant of general parental stress, but negative perceptions of stress could be particularly higher among parents who also report feelings of anxiety and depression. that cumulative stressors from covid-19 did not significantly relate to increased risk of child abuse potential in this sample is favorable. although cumulative stress, in general, is strongly associated with child maltreatment, our findings indicate that stressors specific to covid-19 may not intensify maltreatment risk, though future research is needed to test the long-term implications of the covid-19 pandemic on myriad child and family outcomes. further research would also benefit from the use of standardized measures to better assess the impacts of covid-19 on risk of child maltreatment and to replicate these results. given that some parents expressed positive benefits as a result of the stay-at-home restrictions, such that recommendations to isolate at home also corresponded with more time spent with their children, it is possible that these positive changes had an impact on parenting. in contrast, certain sociodemographic and mental health risk factors were linked to risk of abuse potential. specifically, families receiving financial assistance and parents with higher symptoms of anxiety and/or depression were at increased risk. these findings corroborate extant research j o u r n a l p r e -p r o o f demonstrating robust relationships between low income, mood disorders, and maltreatment risk (drake & johnson-reid, 2014; liu & merritt, 2018; lorant et al., 2003) . in spite of the positive associations among covid-19 and mental health risk factors, parental perceived stress, and child abuse potential, protective factors also mitigated adverse outcomes. specifically, parents' present perceived control over the covid-19 situation decreased their perceptions of stress and risk of child abuse potential. a large body of research examining perceived control shows that parents with higher perceived control over life events are more likely to be able to use available resources to manage stressors (duchovic et al., 2009) . despite limited existing research of perceived control during the covid-19 pandemic, research shows that with regard to health-related outcomes, in general, levels of perceived control predict changes in health over time (infurna et al., 2011) . moreover, in the parenting context, parents with lower present control have higher levels of parenting stress (harrison & sofronoff, 2002) . because families are likely to experience a lack of support from external sources, such as from schools or childcare settings during the covid-19 pandemic, it is important that the support they receive within the home impacts their perceived stress and parenting. indeed, findings from the current study suggest that the level of emotional and social support a parent receives is significantly associated with lower perceptions of stress and risk of child abuse potential. these findings are promising because, in light of the difficulties associated with the global pandemic, families who may be coming together to provide support may also be alleviating stress and its consequences. given that the majority of participants were mothers, support for mothers, in particular, should be considered as research indicates they take on more of the household and familial duties regardless of socioeconomic status (bianchi et al., 2012) . while perceptions of control over the covid-19 situation and parental support were protective for parents in this sample, other cognitive strategies, such as their level of acceptance after having experienced a negative event was not. prior research shows that interventions that promote acceptance, such as mindfulness, may mitigate general stress (e.g., baer et al., 2012) . however, given that the majority of parents reported anxious and/or depressive symptomology and that individuals with clinical-level concerns are more likely to have lower levels of acceptance (roemer et al., 2009) , it is possible that it may be difficult to manage the uncertainty and unintended consequences associated with covid-19. longitudinal research and randomized controlled trials are needed to determine the directionality and causality of these associations. this study has several limitations. the sample was relatively small and only families involved with service agencies and educational settings in the rocky mountain region of the united states were invited to participate. the sample was also largely comprised of mothers who racially and ethnically identified as non-latinx white. although results here may be similar to those found in racially and ethnically diverse families living in different geographic regions, findings cannot be broadly generalized. in fact, minoritized families are more likely to experience greater social inequities than their white counterparts, which may in turn exacerbate risk of poor outcomes during global health crises. despite efforts to reduce participant burden by administering a brief survey, a limitation of this study was that the survey included single item indicators to assess mental health risk. in addition, a revised version of the child abuse potential inventory was used. although this may serve as a proxy for child maltreatment potential, further research should use both objective and subjective measures with known psychometric properties to reliably and validly assess mental health and maltreatment risk. also, the survey was only administered in english, limiting the j o u r n a l p r e -p r o o f opportunity to capture the experiences of non-english speaking parents. the sample also included parents with a child under the age of 18 years; thus, this study included a large age range of children, which may influence the impact of covid-19 related stressors as well as the importance that protective factors may have in determining stress and parenting outcomes. finally, this study was cross-sectional. therefore, causal inferences cannot be made. longitudinal research is needed to evaluate the effects of the covid-19 pandemic and prolonged stay-at-home restrictions among diverse families across the world. in addition, more research is needed to better understand how children's age may influence parent stressors and child abuse potential during the covid-19 pandemic. despite these limitations, these findings elucidate the important associations of covid-19 related stressors and mental health risk with parental perceived stress and child abuse potential and the role that protective factors may play in mitigating these adverse associations. as such, this study has several implications for prevention and intervention programming to reduce parental stress and child abuse potential during the covid-19 pandemic. for example, given that higher perceived control was associated with lower perceived stress, mindfulnessinformed or cognitive-behavioral strategies that are known to promote flexible responding in situations may improve outcomes for parents (brown et al., 2018; pagnini et al., 2016) . moreover, in addition to the emotional and social support a parent receives within their family, providers and educators serving families could connect families with other community resources to broaden their support networks. also, providing culturally responsive whole-family programs and services that benefit children and parents as well as their extended family may offer additional support, particularly for latinx families and families impacted by greater financial strain (prime et al., 2020) . this may include supporting transportation costs, implementing telehealth services, or ensuring a culturally safe practice environment. finally, policy solutions, including expanding community partnerships and streamlining partnerships across service sectors that aim to reduce disparities and promote more equitable outcomes for marginalized children and families are also needed (campbell, 2020) . in conclusion, this study examined the risk and protective impacts of the covid-19 pandemic in relation to parental perceived stress and child abuse potential among families in the western united states. agencies that serve families during the pandemic will benefit knowing about stressors parents experience due to covid-19 and what factors may increase or reduce stress and risk of child maltreatment. as most research has been conducted outside the context of a global health crisis with significant health, social, and financial implications, the current research provides preliminary insight into factors affecting families during the covid-19 pandemic. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f note. n = 157; b = unstandardized coefficient, (se) = standard error,  = beta (standardized coefficient), ci = 95% confidence interval; total r 2 = r square for the final retained model from block 4; reference category indicated in (). *p < .05, **p < .01, ***p < .001. note. n = 156; b = unstandardized coefficient, (se) = standard error,  = beta (standardized coefficient), ci = 95% confidence interval; total r 2 = r square for the final retained model from block 4; reference category indicated in (). *p < .05, ***p < .001. table 5 prevalence of qualitative themes from participants' experiences regarding theme n (%) loss of employment or income /inability to provide for family 52 (34.7) uncertainty about the future 9 (6.0) inability to see family or friends or socialize with others 25 (16.7) medicaid and covid-19: at the center of both health and economic crises weekly change in mindfulness and perceived stress in a mindfulness-based stress reduction program family functioning, perceived control, and anxiety: a mediation model racial and ethnic disparities in access to and use of paid family and medical leave: evidence from four nationally representative datasets the role of negative parental attributions in the associations between daily stressors, maltreatment history, and harsh and abusive discipline housework: who did, does or will do it, and how much does it matter? race, racism, and health: disparities, head: stress, parenting, and covid-19 mechanisms, and interventions the psychological impact of quarantine and how to reduce it: rapid review of the evidence a pilot randomized trial of a mindfulness-informed intervention for child welfare-involved families familismo in mexican and dominican families from low-income, urban communities an increasing risk of family violence during the covid-19 pandemic: strengthening community collaborations to save lives covid-19 in racial and ethnic minority groups child abuse & neglect: risk and protective factors mediating effects of parental stress on harsh head: stress, parenting parenting and parent-child relationship during coronavirus (covid-19) pandemic in singapore toward an ecological-transactional model of community violence and child maltreatment: consequences for children's development developmental perspectives on the etiology, intergenerational transmission, and sequelae of child maltreatment. new directions for child development a global measure of perceived stress parenting stress and child adjustment: some old hypotheses and new questions parenting stress engaging in rather than disengaging from stress: effective coping and perceived control mindfulness and coping with stress: do levels of perceived stress matter? poverty and child maltreatment promoting child and adolescent mental health in the context of the hiv/aids pandemic with a focus on sub-saharan africa beyond reading, writing, and arithmetic: the role of teachers and schools in reporting child maltreatment perceived past, present, and future control and adjustment to stressful life events perceived control and adjustment to trauma: a comparison across events longitudinal association of county-level economic indicators and child maltreatment incidents negative life events, cognitive emotion regulation, and emotional problems parent-child relationships inventory (pcri): manual the effects of economic hardship on family relationships among african american, latino, and euro-american families ethnic differences in family stress processes among african-americans and black carribeans the dark side of inclusion: undesired acceptance increases aggression stress exposure and depression in disadvantaged women: the protective effects of optimism and perceived control adhd and parental psychological distress: role of demographics, child behavioral characteristics, and parental cognitions loneliness matters: a theoretical and empirical review of consequences and mechanisms head: stress, parenting, and covid-19 generalized anxiety disorder, depressive symptoms and sleep quality during covid-19 outbreak in china: a web-based cross-sectional survey relations between parenting stress, parenting style, and child executive functioning for children with adhd or autism racial and ethnic residential segregation and household structure: a research note examining dynamic links between perceived control and health: longitudinal evidence for differential effects in midlife and old age the sars (severe acute respiratory syndrome) pandemic in hong kong: effects on the subjective wellbeing of elderly and younger people cognitive and coping processes in emotion a cumulative risk model of child physical maltreatment head: stress, parenting, and covid-19 33 potential: findings from a community-based study the association between perceived provider discrimination, health care utilization, and health status in racial and ethnic minorities protective factors among families with children at risk of maltreatment: follow up to early school years a test of missing completely at random for multivariate data with missing values familial financial stress and child internalizing behaviors: the roles of caregivers' maltreating behaviors and social services socioeconomic inequalities in depression: a meta-analysis the psychological impact of covid-19 pandemic in italy: a lesson for mental health prevention in the first severely hit european city maternal variations in stress reactivity: implications for harsh parenting practices with very young children predictors of parenting stress for abusive and nonabusive mothers the child abuse potential inventory: manual prediction and explanation of child abuse the correlation between stress and economic crisis: a systematic review the brief child abuse potential inventory: development and validation qualitative methods in social work research perceived control and mindfulness: implications for clinical practice child maltreatment as a function of cumulative family risk: findings from the intensive family preservation program barriers to care among racial/ethnic groups under managed care mental health before and during the covid-19 pandemic: a longitudinal probability sample survey of the uk population the lancet psychiatry. advance online publication racial residential segregation, socioeconomic disparities, and the white-black survival gap risk and resilience in family well-being during the covid-19 pandemic the ces-d scale: a self-report depression scale for research in the general population understanding compliance and psychological impact of the sars quarantine experience mindfulness and emotion regulation difficulties in generalized anxiety disorder: preliminary evidence for independent and overlapping contributions parenting stress and anger expression as predictors of child abuse potential parenting stress among child welfare involved families: differences by child placement the triple p-positive parenting program: a systematic review and meta-analysis of a multi-level system of parenting support the etiology of cognitive competence: a systems perspective infants at risk: assessment of cognitive functioning fourth national incidence study of child abuse and neglect department of health and human services, administration for children and families a brief measure for assessing head: stress, parenting, and covid-19 37 generalized anxiety disorder: the gad-7 department of health & human services, administration for children and families, administration on children, youth, and families using social and behavioural science to support covid-19 pandemic response covid-19 and racial/ethnic disparities the role of parental stress in physically abusive families the effect of material hardship on child protective service involvement range parent age relationships inventory -parent perceived control over stressful events -present control n = 183; percentages that do not equal 100% indicate missing data; categories of financial assistance and stressors from covid-19 poor physical health (e.g., lack of physical activity n = 150; qualitative themes from participants reporting on their experiences from covid-19 are not mutually exclusive all authors declare that there are no conflicts of interest. the authors are indebted to our community partners for their time and extremely grateful to the families who shared their experiences with us. running head: stress, parenting, and covid-19 key: cord-342047-pm3i54mb authors: du preez, andrea; law, thomas; onorato, diletta; lim, yau m.; eiben, paola; musaelyan, ksenia; egeland, martin; hye, abdul; zunszain, patricia a.; thuret, sandrine; pariante, carmine m.; fernandes, cathy title: the type of stress matters: repeated injection and permanent social isolation stress in male mice have a differential effect on anxietyand depressive-like behaviours, and associated biological alterations date: 2020-09-21 journal: transl psychiatry doi: 10.1038/s41398-020-01000-3 sha: doc_id: 342047 cord_uid: pm3i54mb chronic stress can alter the immune system, adult hippocampal neurogenesis and induce anxietyand depressive-like behaviour in rodents. however, previous studies have not discriminated between the effect(s) of different types of stress on these behavioural and biological outcomes. we investigated the effect(s) of repeated injection vs. permanent social isolation on behaviour, stress responsivity, immune system functioning and hippocampal neurogenesis, in young adult male mice, and found that the type of stress exposure does indeed matter. exposure to 6 weeks of repeated injection resulted in an anxiety-like phenotype, decreased systemic inflammation (i.e., reduced plasma levels of tnfα and il4), increased corticosterone reactivity, increased microglial activation and decreased neuronal differentiation in the dentate gyrus (dg). in contrast, exposure to 6 weeks of permanent social isolation resulted in a depressive-like phenotype, increased plasma levels of tnfα, decreased plasma levels of il10 and vegf, decreased corticosterone reactivity, decreased microglial cell density and increased cell density for radial glia, s100β-positive cells and mature neuroblasts—all in the dg. interestingly, combining the two distinct stress paradigms did not have an additive effect on behavioural and biological outcomes, but resulted in yet a different phenotype, characterized by increased anxiety-like behaviour, decreased plasma levels of il1β, il4 and vegf, and decreased hippocampal neuronal differentiation, without altered neuroinflammation or corticosterone reactivity. these findings demonstrate that different forms of chronic stress can differentially alter both behavioural and biological outcomes in young adult male mice, and that combining multiple stressors may not necessarily cause more severe pathological outcomes. animal research has been paramount in investigating the association between stress and major depressive disorder (mdd) 1 . exposure to individual or multiple stressors in rodents can alter the immune system 2,3 and the hypothalamic-pituitary-adrenal (hpa) axis 4,5 , decrease hippocampal neurogenesis 6, 7 and induce anxiety-and depressive-like behaviours 8, 9 . these areas of research have predominately utilized either (i) unpredictable chronic mild stress (ucms) models, which incorporate a variety of both physical and psychosocial stressors 10 , or (ii) a social-stress-based model (e.g., social isolation or social defeat stress), which employ predominately psychosocial stressors 11, 12 . however, one main limitation to this area of research is that there is no discrimination between the types of stress used, as most studies predominately incorporate either one or a combination of both these stress types. thus, the distinction between the effect of different types of stress on animal behaviour and physiology has been largely unexplored. this idea is not entirely novel in a clinical setting, with research showing that psychological, sexual and physical maltreatment can differentially affect mental health [13] [14] [15] [16] . for example, hodgdon et al. 15 recently demonstrated that psychological, sexual and physical abuse lead to distinct behavioural profiles and this research warrants being investigated in a preclinical setting, which, to our knowledge, is yet to be done. thus, is one type of stress exposure more potent than another and, if so, does this matter? this has already been partially addressed by a few preclinical studies showing how only ucms and/or predator stress, but not restraint stress, can induce depressive-like behaviour 17, 18 . it also has been shown that exposure to psychosocial stress leads to a much wider range of depressive-like behaviours and more pronounced increases in pro-inflammatory cytokine profiles than exposure to predominately physical stress and/or ucms 4, 17 . although these lines of research have shed some light on the impact of different types of stress, only one of these studies directly compared different stress paradigms in the same study and, moreover, was limited in the biological domains assessed, having focused specifically on the neuroendocrine system 4 . therefore, a more comprehensive evaluation is needed, one that focuses on multiple behaviours and biological outcomes resembling the complexity of mdd. therefore, with this in mind, we aimed to investigate the effect of two well-established stressors, social isolation 1, 11 and repeated injections [19] [20] [21] , both for 9 weeks in young adult male animals. although depression can develop at any age between early childhood and older adulthood, both national and cross-national epidemiological studies report that the first onset of depression most frequently occurs in the 20s to early 30s [22] [23] [24] , and thus the animal equivalent of a young adult was purposely selected for study. we measured anxiety-and/or depressive-like behaviour and corticosterone responsivity, systemic inflammation, neuroinflammation and hippocampal neurogenesis-biological outcomes all associated with mdd 25 . we wanted to establish whether we can discriminate between the effects of repeated injection and permanent social isolation with respect to the behavioural and biological outcomes, and whether combining these stressors alters the severity of the associated behavioural and/or biological outcomes. experiments were conducted with male balb/canncrl mice (n = 50), aged 6-7 weeks, weighing 22-26 g, obtained from charles river laboratories (margate, kent, uk). animals were housed under standard conditions (19-22°c, humidity 55%, 12 : 12 h light : dark cycle with lights on at 07:00, food and water ad libitum) and had a 1-week period, prior to stress exposure, to acclimatize to the biological services unit and experimenter. during habituation, all animals were housed in sibling pairs. all housing and experimental procedures were carried out in compliance with the local ethical review panel of king's college london and the uk home office animals scientific procedures act 1986. for the rationale behind the chosen strain, sex and age of the mice used, see supplementary material. mice were exposed to one of three stress treatments for a 9-week duration. each treatment comprised one or two distinct stressors that was either in the form of repeated injection, which has been previously shown to differentially alter stress responses in balb/c mice 20 and affective outcomes in outbred rats with high and low emotional reactivity 21 , or permanent social isolation, which has consistently been associated with depressivelike phenotypes 8, 9, 11 . in brief, three groups of mice were exposed either to repeated injection only (group 2), permanent social isolation only (group 3) or a combination of both stressors (hereafter referred to as combined stress; group 4), whereas one group remained stress free (group 1). group sample sizes were based on numbers typically used in animal research using chronic stress models 1, 10 . animals, either singly or as a sibling pair, were randomly assigned by a chance procedure to their respective groups after the initial habituation period. for all forms of assessment, the experimenter was blinded to group status and all animals were handled regularly. figure 1 defines in more detail the experimental groups and depicts the experimental timeline and housing conditions. intraperitoneal injections were carried out according to recommended guidelines and as previously described 26, 27 . briefly, animals were scruffed and injected with saline volumes of 10 ml/kg body weight administered between 10:00 and 12:00 daily. injection naive animals were handled but not injected. for details on the injection procedure, see supplementary material. body weight and food intake was measured weekly during the first 6 weeks of stress exposure (fig. 1) . for details on these measures, see supplementary material. anxiety-like behaviour was assessed using the open-field test (oft) 28 and novelty suppressed feeding test (nsft) 29 , whereas depressive-like behaviour was measured using the sucrose preference test (spt) 30 and porsolt swim test (pst) 31 -all as previously described. behavioural testing was carried out after 6 weeks of stress exposure (fig. 1) . for details on the general conditions on all behavioural testing and the methods for all behavioural assays, see supplementary material. blood sampling, processing and storage whole blood, from a tail cut, was collected between 09:00 and 12:30, both 24 h before and 30 min after acute stress exposure, i.e., the pst, at the end of behavioural testing. samples were centrifuged at 3000 r.p.m. for 10 min at 4°c and plasma was removed and stored at −80°c. for details on blood sampling, see supplementary material. corticosterone levels were measured in duplicates from plasma samples collected at both blood collection points using commercially available enzyme-linked immunosorbent assay (elisa) kits (enzo life sciences, switzerland), according to the manufacturer's instructions. absorbance values were converted into concentrations using cubic spline four parameter logistics. plasma cytokine levels were determined using the multiplex screening assay based on magnetic luminex® xmap® technology as previously described 32 . using a custom-made mouse premixed multi-analyte magnetic luminex screening assay (r&d systems, minneapolis, usa), levels of interleukin (il)-1β, il2, il4, il6, il10, il12, c-reactive protein, vascular endothelial growth factor (vegf), insulin-like growth factor and tumour necrosis factor (tnf)-α, in blood plasma collected at both blood collection points, were measured according to the manufacturer's instructions. median fig. 1 schematic representation of the experimental procedure. mice were exposed to one of four experimental conditions after an initial 1week habituation period: (i) stress-free control group (socially-housed in pairs and injection naive-group 1); (ii) repeated injection stress (sociallyhoused in pairs and repeatedly injected-group 2); (iii) social isolation stress (permanent social isolation and injection naive-group 3); and (iv) combined stress (permanent isolation and repeatedly injected-group 4). behavioural testing began after 6 weeks of treatment and was carried out under stringent environmental control, using standardized test procedures, between 09:00 and 15:00, unless otherwise stated. blood was collected 24 h before and 30 min after acute stress exposure, i.e., the porsolt swim test, in the final week of behavioural testing. animals were culled 24 h later and brain tissue extracted. a housing for sibling pair-housed animals (groups 1 and 2): large techniplast cages (45 × 32 × 20 cm) were set up with sawdust, a red house, white house, nesting material and a plastic enrichment tube. larger cages with more nesting material, shelter and enrichment were deemed necessary to reduce aggression between sibling pair-housed animals. b housing for singly-housed animals (groups 3 and 4): small tecniplast cages (32 × 16 × 14 cm) were set up with sawdust, a white house and nesting material. note: a larger sample size was used for control animals (n = 20) given the risk of group housing such an aggressive strain (deacon, 2006 ; charles rivers laboratories international, inc., 2012; www.criver.com). for details on the elisa and luminex protocols, inter-and intra-assay variability, and kit sensitivity. brain tissue collection and sectioning animals were transcardially perfused and brains quickly extracted as previously described 33 . all brain tissue was coronally sectioned at a thickness of 40 μm using a leitz freezing microtome (microm hm430, carl zeiss ltd, cambridge, uk) as previously described 34, 35 . for details on brain tissue collection, sectioning and storage, see supplementary material. proliferative cells, immature neurons, microglia, astrocytes and mature astrocytes were visualized using ki67, doublecortin (dcx), ionized calcium-binding adapter molecule-1 (iba1), glial fibrillary acidic protein (gfap) and s100β, respectively, using free-floating immunohistochemistry as described previously 36 . for protocol details, antibodies used and representative images, see supplementary material and supplementary fig. 1 . the cell density of immunopositive ki67, dcx, iba1, gfap and s100β cells in the prefrontal cortex (pfc) and/ or the dentate gyrus (dg) of the hippocampus were estimated by stereological analysis with stereoinvestigator software (mbf bioscience, williston, vt) using the optical fractionator module as previously described 37 . for details on the stereological methods, see supplementary material. whole-slide digital images were provided by the ucl iqpath slide scanning service, using a leica scn400f scanner. representative images of the hippocampus and the pfc were captured with aperio imagescope software v12.2.2 (leica biosystems, uk) at ×2 magnification. to quantify and compare relative levels of immunoreactivity for iba1-and gfap-positive cell staining in the dg and pfc, thresholding analyses using imagej software v1.51 38 were performed as described previously 39, 40 . see supplementary material for details of the thresholding methodology. using imagej software v1.51 38 sholl and skeleton analyses were performed as previously described [39] [40] [41] . for all analyses, 18 iba1 cells were randomly selected across three hippocampal sections per mouse. three mice per experimental group were used for morphological quantification, equating to a total of 54 iba1 cells per group. for details of the morphometric methodology, see supplementary material. given that gfap is also a marker for neural stem cells 42 , to determine whether changes observed in gfap immunoreactivity were related specifically to astrocytes, sections were fluorescently double-labelled to determine the extent of co-localization of gfap with sox2-a neural stem cell specific marker 43 . immunofluorescent double labelling was carried out as previously described 44 . images of double-labelled sections were obtained using a leica sp5 confocal microscope. specifically, an objective of ×63 (oil immersion, na 1.42) was used for each image captured and wavelength laser lines of 405 nm (diode laser), 488 nm (argon laser) and 594 nm (hene laser) were used. all images were acquired as confocal stacks of ten images separated by a 0.64 μm z-axis step size. each image was taken at a resolution of 1024 × 1024 pixels, with the picture dwell time set to 3.36, giving a rate of 0.185 zplanes per second, and each frame was averaged four times to reduce signal noise. additional settings of gain, offset and pinhole size were optimized prior to imaging and were held constant for all images at 600 v, 800 v and 1.0 airy unit, respectively. for each confocal stack, the percentage of doublelabelled cells for gfap and sox2 in the dg of the hippocampus was determined using methods as outlined previously 45 . briefly, 100 gfap-positive cells per mouse were counted and the percentage of radial glial cells (gfap+/sox2+) and astrocytes (gfap+/sox2−) were calculated. for each mouse, cells were counted from a total of 12 acquired z-stacks across 6 hippocampal sections. dcx-positive cell morphology in the dg was visually classified as previously described 46 . in brief, dcx-positive cells were classified according to four neuroblast subtypes based on their level of maturation, and the cell density for each neuroblast cell type was determined using stereological analyses as aforementioned. for details on the classification process, see supplementary material and supplementary fig. 3 . statistical comparisons were conducted in ibm spss statistics v.23 (ibm ltd, portsmouth, uk) and consisted of independent samples t-tests, one-way or two-way analyses of variance (anova), repeated-measures twoway anova, three-way factorial anova, repeatedmeasures generalized linear mixed modelling, mann-whitney, or kruskal-wallis, followed by bonferroni or dunn's post hoc analyses where appropriate. all data were assessed for normality using probabilityprobability plots and the kolmogorov-smirnov test, and for homogeneity of variance using the levene's test. for data that did not conform to normality and/or homoscedasticity non-parametric statistical tests were applied. all tests carried out were two-sided and the alpha criterion used was p < 0.05. data are represented as the mean and sem or the median and interquartile range. all animals were included in all analyses. for a more detailed description of the analytical approach, see supplementary material. we first assessed the behavioural effects associated with chronic stress exposure and found that all stress-exposed mice, irrespective of the type of stress, exhibited significantly increased anxiety-like behaviour in the oft (fig. 2a) relative to control animals, as shown by significantly less time in the centre of the oft arena (−59% in repeatedly injected animals, −92% in the socially isolated mice and −74% in animals exposed to combined stress spent (fig. 2a) ). moreover, all groups of stress-exposed mice displayed an increased latency to feed in the nsft relative to control animals ( fig. 2b ) but due to the significant increases in anxiety also observed in these mice in the oft, the nsft was heavily confounded by anxiety, as the task is run in a novel arena. therefore, it was not possible to use this test to specifically assess anhedonia. interestingly, only mice exposed to permanent social isolation exhibited signs of significant depressive-like behaviour when compared with controls (fig. 2c, d) , as shown by increased anhedonic-like behaviour (exposed animals consuming 16% less sucrose in the spt; fig. 2c ), and behavioural despair (exposed animals spending 38% more time immobile in the pst; fig. 2d) . notably, behaviour in the oft and nsft was not confounded by locomotor activity (supplementary fig. 4a, b) , and the observed differences in the spt were not attributed to either total liquid consumption (supplementary fig. 4d ) or food consumption ( supplementary fig. 4e) . moreover, the observed differences in the behavioural assays were not confounded by olfactory ability (supplementary fig. 4c ) or by social dominance (supplementary table 1) . for further detail on all other behavioural readouts, see supplementary fig. 5 and for a full summary of all the behavioural changes associated with the three chronic stress exposures, see supplementary table 2 . chronic stress does not alter baseline corticosterone levels after stress exposure, but the type of stress differentially alters corticosterone responsivity we next sought to uncover some of the biological alterations associated with these behavioural perturbations in stressed-exposed mice, focusing first on peripheral changes related to hpa axis functioning. we found no significant differences in baseline corticosterone levels across experimental groups after chronic stress exposure (i.e., 24 h before acute stress), and all mice showed a 256% average increase in plasma corticosterone in response to the pst (fig. 3a) . interestingly, corticosterone levels 30 min after the acute stress did significantly differ between experimental groups (fig. 3a) . on average, levels increased by threefold after the pst; however, mice exposed to repeated injection had a 41% larger increase in corticosterone levels compared with controls, whereas socially isolated mice had a 22% smaller increase in corticosterone levels compared with controls, and mice exposed to combined stress had a stress response similar to that of controls (fig. 3a) . for a detailed summary of all the peripheral corticosterone data for each of the three chronic stress exposures, see supplementary table 3 . repeated injection decreases tnf-α and il4; social isolation increases tnf-α, but decreases il1β, il2, il4, il10 and vegf; and injection and social isolation combined decreases il1β, il4 and vegf we next examined the impact of chronic stress exposure on peripheral inflammatory profiles, both at baseline (i.e., 24 h before the pst) and after the acute stress. interestingly, we found that there was very little pre vs. post effect of the acute stress on any of the cytokines ( fig. 3b-g) . specifically, only socially isolated mice exhibited significant differences in cytokine plasma levels post-pst relative to baseline, with a decrease in il2 and an increase in vegf levels in response to acute stress. in terms of between-group comparisons vs. control mice, mice exposed to repeated injection had significantly lower baseline tnf-α and post-pst il4 levels (fig. 3b, e) . conversely, socially isolated mice had significantly higher baseline and post-pst tnf-α levels, lower baseline and post-pst il10 levels, together with lower levels of baseline il1β and vegf, and post-pst il2 and il4 ( fig. 3b-g ). mice exposed to combined stress had consistently lower il4 and vegf at baseline and post-pst, and lower il1β levels post-pst (fig. 3c, e, g) . intriguingly, no significant differences in il6 or il12 were found between (or within) experimental groups, preand/or post-pst ( supplementary fig.6 a, b) . for a detailed summary of all the inflammatory data for each of the three chronic stress exposures see supplementary table 3 . it is also noteworthy that none of the reported inflammatory changes were artifacts of social hierarchy (supplementary table 4 ). given that glial cells play an important role in immune system functioning 47 , we next examined both microglial and astrocyte biology in both the hippocampus and pfctwo particularly stress-sensitive regions 48 . repeated injection increases iba1-positive cell density and promotes a dystrophic cell morphology in the ventral dg, whereas social isolation decreases iba1-positive cell density and induces a ramified cell morphology in the dorsal dg looking at microglial biology first, we found that exposure to repeated injections and social isolation reduced iba1 immunoreactivity in the dg of the hippocampus, with a similar pattern, but not reaching statistical significance, for the combined stress (fig. 4a, b) . these effects in the dg had specific regional selectivity, with decreases that were significant in the ventral dg for the repeatedly injected mice (−19%) and in the dorsal dg for the socially isolated mice (−25%) (fig. 4a, b) . interestingly, none of the conditions affected iba1-positive cell immunoreactivity in the pfc (supplementary fig. 7a, b) . to further understand the relevance of these changes in iba1 immunoreactivity, we next assessed cell density and morphology in these specific regions of the dg. interestingly, despite both repeated injections and social isolation showing a decrease in iba1 immunoreactivity, repeatedly injected mice had a 22% increase in iba1 cell density in the ventral dg (fig. 4c) , while socially isolated mice had a 24% decrease in iba1 cell density in the dorsal dg (fig. 4d) . moreover, although injected mice had an increased iba1 cell density in the ventral dg, the morphological characteristics of these cells were altered, such that they assumed a more dystrophic morphology, potentially explaining the decreased iba1 immunoreactivity data for this group (fig. 4e , f and supplementary table 5 ). in direct contrast, iba1 cells of socially isolated animals assumed a more ramified morphology, with no difference in the amount of space each cell occupies observed ( fig. 4g and supplementary table 5 ). it is also noteworthy that cell soma size and average process length was not different between experimental groups (supplementary table 5 ). social isolation increases gfap-positive cell immunoreactivity and s100β-positive cell density in the dg in addition to investigating the impact of chronic stress on microglial biology, we also examined astrocyte biology in both the dg and pfc. interestingly, only social isolation stress increased gfap immunoreactivity in the dg relative to all other groups (47%)-a change not dg region specific (fig. 5a, b) -and similar to iba1 data, there were no differences in gfap immunoreactivity in the pfc between experimental groups ( supplementary fig. 7c, d) . as gfap is also expressed in neural progenitor cells, immunofluorescent double labelling was subsequently used to further characterize these cells in socially isolated and control animals. interestingly, our data revealed that all mice irrespective of exposure had a radial glia to astrocyte cell ratio of 9 : 1 ( supplementary fig. 8) . given that only~10% of gfap-positive cells in the dg were astrocytes (i.e., controls: 12%; isolated: 11%), stereological cell density estimates were next obtained to determine whether the observed differences in gfap immunoreactivity were related to one or both gfappositive cell types. specifically, we found that social isolation significantly increased the number of radial glial cells only in both the dorsal (25%) and ventral dg (26%) (fig. 5c, d) . as only a small proportion of gfap-positive cells were a1 astrocytes in the context of our work, s100β-positive cell density-typically a marker of a2 astrocytes-was next examined. similar to gfap, we found that social isolation significantly increased s100β-positive cell density in both the dorsal (52%) and ventral dg (65%) (fig. 5e, f) . finally, given the associations between hippocampal neurogenesis, depression, stress and inflammation 49 , we investigated the impact of the three chronic stress paradigms on both proliferation and differentiation in the dg. we found that chronic stress exposure did not alter hippocampal volume (supplementary fig. 9a ) or ki67 cell density in the dg (supplementary fig. 9b, c) for any of the exposure groups. however, mice exposed to repeated injection, or the combined stress, did show a decrease in overall dcx-positive cell density (−48%) in the dorsal dg relative to controls (fig. 6a, b) . surprisingly, socially isolated mice had an overall dcx-positive cell density similar to controls. to gain a better understanding of whether these reductions in dcx cell density were related to all dcx cell types, or whether they were specific to a particular stage of dcx maturation, we classified all dcx-positive cells into one of four morphology types. compared with control mice, we found a decrease in all neuroblast cell types (−66%) and in early post mitotic cells (−55%) in the dorsal dg for repeatedly injected mice and mice exposed to combined stress, respectively. interestingly, we also found an increase in post mitotic stage neuroblasts (71%), in the ventral dg, in socially isolated animals. no differences in intermediate stage neuroblast cell density were found for any of the exposure groups (fig. 6c) . none of the reported brain-related changes were artifacts of social hierarchy (supplementary tables 6 and 7) , and for a full summary of all these changes, see supplementary table 8 . for the first time, we show how exposure to different types of chronic stressors elicits distinct behavioural and biological phenotypes in both the periphery and the brain. overall, our results clearly demonstrate that the type of chronic stress exposure can indeed matter. our most interesting finding is that distinct types of chronic stress differentially alter hippocampal neuroinflammatory and (see figure on previous page) fig. 3 effect(s) of repeated injection, social isolation and combined stress on corticosterone responsivity and systemic inflammation. a mean (±sem) plasma corticosterone levels before and after acute stress exposure. b mean (±sem) plasma tnf-α levels before and after acute stress exposure. c mean (±sem) plasma il1β levels before and after acute stress exposure. d mean (±sem) plasma il2 levels before and after acute stress exposure. e mean (±sem) plasma il4 levels before and after acute stress exposure. f mean (±sem) plasma il10 levels before and after acute stress exposure. g mean (±sem) plasma vegf levels before and after acute stress exposure. *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). analyses: repeated-measures two-way anova with bonferroni post hoc comparison. neurogenic profiles, which may be the basis by which the different behavioural phenotypes ultimately manifest. based on our data, we believe that the neurogenic profiles observed are a functional consequence of the neuroinflammatory changes associated with each stress exposure, given that microglia and astrocytes play an important role b representative photomicrographs of the ventral (i) and dorsal (ii) dentate gyrus stained for iba1 for repeatedly injected and socially isolated mice, respectively, all relative to controls. images captured at ×2 and ×20 magnification, scale bar = 50 μm. c mean (±sem) iba1-positive cell density in the ventral dentate gyrus for repeatedly injected mice (t [28] = 2.7, p = 0.02). d mean (±sem) iba1-positive cell density in the dorsal dentate gyrus for socially isolated mice (t [28] = 4.2, p = 0.001). e representative photomicrographs and associated sholl masks of iba1 cells in the ventral dentate gyrus of repeatedly injected and control animals. f representative photomicrographs and associated skeletons of iba1 cells in the ventral dentate gyrus of repeatedly injected and control animals. g representative photomicrographs and associated sholl masks of iba1 cells in the dorsal dentate gyrus of socially isolated and control animals. images e-g were taken at ×40 magnification, scale bar = 40 μm. in sholl masks, red denotes a greater degree of branching complexity, whereas blue denotes the lowest degree of branching complexity. *p < 0.05; **p < 0.01; ***p < 0.001 (adjusted p-values). analyses: two-way anova with bonferroni post hoc comparison or independent samples t-test. in maintaining synaptic integrity 50 . moreover, given that glucocorticoids and peripheral inflammation are the two most well-known pathways through which stress can affect microglia/astrocyte structure and functioning 51-53 , we believe that altered hpa axis activity and cytokines levels, also found in our models, are the key systems involved in altering microglia/astrocyte/neurogenesis and ultimately behaviour. our data suggest that repeated injections promotes hpa axis hyperactivity (possibly related to the anxious phenotype), as shown by increased corticosterone reactivity to stress, together with lower tnfα and il4 levels, indicating a hypercortisolemia-associated inhibition of the peripheral immune system 54 . moreover, hpa axis hyperactivity can lead to increased microglial activation 53 , which is also seen in repeatedly injected mice, as indicated by the increased iba1 cell density, reflecting microglia rapidly proliferating once activated 55 , and iba1 cell morphology resembling reactive microglia 51 , given that these cells occupy less overall space, possess a shorter maximum process length, and have fewer processes and reduced branching ramification. interestingly, previous research demonstrates that activated microglia assume a phagocytotic role and contribute to the apoptosis of newborn neurons 56 , and consistent with this we observe a concomitant decrease in neuronal differentiation in these same animals. given that there is no change in ki67, which is predominately expressed during the earlier critical neurogenic period, and there is a decrease in more mature neuroblasts (but not in the intermediate stage), we can conclude that the majority of apoptosis is likely occurring during the later 1-3 week window, a critical period for newborn cell survival 52 . however, it is notable that we also see a significant reduction in proliferative stage neuroblasts, which supports that some cell death also occurs in the earlier critical period, although as apoptosis was not quantified in our study, this requires validation. contrary to repeated injection, permanent social isolation promotes hpa axis hypoactivity as shown by decreased corticosterone reactivity to stress, together with higher tnfα (i.e., an exact mirror image of the repeated injections), together with decreased il10, il1β, vegf and il4. importantly, il10 is an important mediator for suppressing the immune response and directly inhibits the proliferation and production of tnfα 57 . furthermore, increased tnfα and decreased il10 are both strongly associated with depressive-like behaviour and clinical depression 58-60 -a behavioural phenotype found only in our socially isolated animals. as with repeated injection, we believe that these different changes in hpa axis/immune system activity promote the microglial/astrocyte-associated abnormalities observed in socially isolated mice 61 . for example, the reduced levels of corticosterone and the increased levels of pro-inflammatory cytokines promote microglial overactivation 44, 62 , which can lead to increased microglial apoptosis 63 and hyper-ramification 62 , which we observe in our socially isolated animals, as indicated by a decrease in iba1 cell density and an increase in the internal complexity of these cells. moreover, we find an activation of neuroprotective a2 astrocytes, as shown by the increased gfap-and s100β-positive cell density, the latter of which is specifically associated with a2 astrocytes 64 , findings that have already been described in association with stress [65] [66] [67] , including in our own in vitro work mimicking 'stress in a dish' with low concentration of glucocorticoids 68 . as microglia and astrocytes are vital for synaptogenesis and/or synaptic integrity/maintenance 56, [69] [70] [71] , and that their structure is intrinsically linked to their function 72 , we speculate that the changes in neurogenesis following social isolation are functionally linked to the observed neuroinflammatory changes. indeed, in the socially stressed animals, we see a specific increase in mature neuroblasts, suggesting an overall impairment in synaptic pruning and/or apoptosis inhibition. given that we observe a reduced number of iba1 cells with an altered morphology, and an increase in a2 astrocyte activation, we believe that these glial cells are not able to adequately perform their function of synaptic pruning and/or overcompensate their neuroprotective-associated functions 66 . further evidence to support these functional abnormalities comes from our finding that vegf is reduced in these animals, since vegf regulates synaptic pruning and synapse formation 73 , as well as neurogenesis and microglia 74 , and that astrocytes can directly secrete vegf 75, 76 . interestingly, we not only find a wider regional specificity of chronic stress exposure for both neuroinflammatory and neurogenic profiles between the pfc and the hippocampus, but also regional specificity within the dg. although, the finding that regional differences exist is not novel 48, 77 , finding no impact of chronic stress on the pfc is contrary to previous research 48, 62, 78, 79 . however, the type and duration of stress exposure can have important implications on microglia and/or astrocyte biology 36 , and we cannot out rule that changes in the pfc may have occurred earlier than measured in this study. although the basis for the regional specificity within the dg is unclear, one potential explanation could relate to the microglial response, which might occur at a different rate, or endure for different periods of time, in response to each of the stress exposures. moreover, given that our differentially stressed animals respond so distinctively to acute stress, a differentially altered microglial response represents a parsimonious explanation and especially when glucocorticoids are well-known modulators of microglial function 80 . it is also notable that the ventral hippocampus (relative to the dorsal) responds entirely differently to glucocorticoids, such that it has a reduced firing frequency accommodation and more depolarization-associated spikes 81 . this differential response may allow for a longer window of acquisition when activated 82 and given the prominent role of the ventral hippocampus in inhibiting the hpa axis, raises the possibility that the differential actions of glucocorticoids on synaptic function may be relevant to stress regulation. in our work, this could potentially contribute to the regional specificity observed for both neuroinflammatory and neurogenic profiles in stress-exposed animals. regarding a broader interpretation of the functional significance of the neurogenesis-related findings, it is wellknown that the dorsal dg plays a role in memory and cognition, while the ventral dg controls stress responsivity and emotional processing 83 . therefore, it is not entirely surprising that dcx in the ventral region of the dg is specifically altered in the context of permanent social isolation, which promotes a robust depressive-like phenotype, and a decrease in stress responsivity. moreover, these preferential effects on the ventral dg have previously been reported [84] [85] [86] [87] [88] . although our data on social isolation aligns with the functional relevance of the ventral dg, it is unknown whether this applies to repeated injection and its associated changes in the dorsal dg when cognitive behaviour was not measured. however, it is noteworthy that anxietylike behaviour and cognitive impairment are closely associated in the context of chronic stress [89] [90] [91] and future research should therefore measure cognitive functioning to extend upon our findings. unlike repeated injection and/or social isolation, we find that combined exposure to these stressors surprisingly does not induce depressive-like behaviour, alter microglial/ astrocyte biology, or alter hpa axis activity, contrary to previous research 36, 48, 92, 93 . perhaps the most powerful conclusion of our study is that there appears to be no synergistic, potentiating effect in combining such different stressors, and in fact the two specific phenotypes of hypercortisolemia/reduced inflammation (repeated injection) and hypocortisolemia/increased inflammation (social isolation) seem to cancel each other out, leading to no differences in these biological systems. however, we do observe a specific decrease in overall neuronal differentiation in these stressed animals, for which altered cell death and/or cell proliferation will still ultimately account for these changes. moreover, these animals also exhibit specific decreases in il4, il1β and vegf, and given that vegf is an important pro-neurogenic growth factor 94, 95 , and that cytokines can independently modulate neurogenesis 96, 97 , these changes in immune system functioning could account for the observed decrease in neurogenesis. pertinently, a lack of il4 specifically has previously been shown to promote anxiety-not depressive-like behaviour 98 -a behavioural outcome also observed in our combined stressexposed mice. therefore, the observed decrease in il4, together with reduced neurogenesis, could ultimately contribute to the aberrant behavioural profile of these animals. although we clearly demonstrate that differential types of stress can indeed promote unique phenotypes, we believe that nociception could potentially explain these differential outcomes, especially when repeated injection initiates an acute pain response, whereas social isolation does not. the immune system, hpa axis and nociception are all intrinsically linked 99, 100 , and therefore this could account for some of the observed biological and behavioural phenotypes associated with the two types of stress. for example, we specifically see reduced tnfα in repeatedly injected animals, and tnfα is an important inflammatory mediator of pain 101 , the inhibition of which has been shown to alter pain perception 102 . thus, the observed biological changes associated with our repeated injection paradigm could be a reflection of the pain response and/or a change in nociception sensitivity. pertinently, chronic pain has been consistently associated with anxiety 103-105 -a behavioural outcome observed for our repeatedly injected animals. furthermore, preclinical studies using electric foot shock paradigms, which also elicit pain, likewise report an increase in anxiety-like behaviour 106, 107 , and clinical studies using repeated pain stimulation show that, despite pain habituation, exposed individuals report increased anxiety 108 . interestingly, previous research shows how repeated injection differentially alters affective outcomes in rats with high and low emotionality, with low responders showing no change in depressive-like states 21 . thus, repeated injection in the context of our work could differentially alter the emotional reactivity of our inbred mice, such that our repeatedly injected animals become low emotional responders. this could also account for why injection stress seems to override the effects of social isolation within the combined paradigm and why no apparent additive effect was observed for these animals. regarding the independent impact of permanent social isolation, it is unsurprising that this particular stressor, which is more ethological in nature, promotes aberrant behavioural and biological changes across multiple domains, especially in a gregarious species. mice are social due to the various advantages that an organized social structure provides in terms of mating selection, resource allocation, and social status 109 . therefore, by socially isolating animals that are biologically and evolutionarily suited to social living, it is understandable how the removal of social structure could impair physiology, and/ or physiological responses that then ultimately manifest into aberrant behaviours. indeed, preclinical research consistently shows how chronically isolated rodents have increased anxiety-and depressive-like behaviour 8, 11, 110 , together with altered hpa axis activity 4, 11, 110 , and specific increases in the pro-vs. anti-inflammatory balance, i.e., increased tnfα/il6 and decreased il2 2,111 -outcomes all observed in our socially isolated animals. moreover, in humans, who are also a social species, the lack of social support is an important risk factor for affective disorders and social isolation/loneliness has been consistently associated with depression [112] [113] [114] [115] . interestingly, a recent study even demonstrates how social integration can be protective against inflammation in young black women 116 . although our work was designed to ensure maximum validity, it is unknown how generalisable our findings are relative to wider animal research when the species/strain, age and sex of the animal can impact both the behavioural and neurobiological phenotypes associated with stress exposure [117] [118] [119] [120] . due to financial and time constraints for such a huge programme of research, we only used young adult male animals for our research, which represents a limitation particularly when the prevalence of depression is significantly higher in women, in adolescence and old age [121] [122] [123] , and when research using female rodents is underrepresented 124, 125 . however, given that males are less susceptible to clinical depression, one could speculate that in the context of our work, female balb/c mice could be equally, if not more, sensitive to the impact of the different types of stress. future work should prioritise investigating the impact of different stressors in both male and female animals and in different strains/species, and it is our ambition to extend our work to female animals in the near future. moreover, it remains unknown whether the observed biological changes are direct consequences of chronic stress exposure, indirect changes from other biological system alterations, or simply adaptive compensation. by characterizing biological phenotypes at the end of treatment, based on a single snapshot, we cannot determine the temporal sequence of these changes, and more importantly, may have missed earlier biological alterations. future work should aim to collect behavioural readouts, blood samples, and tissue from parallel groups to determine the temporal sequence of these reported outcomes and more fully explore the mechanistic links. despite these limitations, this is the first time that a study has assessed all of these biological processes together and done an extensive evaluation of both microglial biology and neurogenesis. moreover, we have also taken into account the importance of the hippocampal dorsalventral axis 126 . in summary, the outcomes of our study provide some novel insight into the changes that occur in behaviour, in peripheral stress and inflammatory systems, and within the brain, following exposure to different types of chronic stress, and how the type of stress can make a difference in terms of these changes. we demonstrate that the type of stress exposure could differentially alter depressive symptomology and/or its biological basis, thus informing the molecular underpinning of the clinical studies showing that different types of abuse and maltreatment can differentially affect mental health. moreover, our work gives clinically-relevant insights into the effects of social isolation, a condition that can increase morbidity and mortality 127, 128 and can specifically lead to mdd [112] [113] [114] [115] , consistently with our behavioural data, while the relationship between repeated injections and anxiety may be relevant to the impact of recurrent medical treatments on mental health. finally, given the global impact of the coronavirus outbreak, for which social isolation has increased significantly as a consequence, understanding the psychological and biological effects of social isolation has become fundamentally important in further understanding, and then subsequently alleviating, the impact that social isolation may have worldwide. the recent progress in animal models of depression social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or n-acetyl cysteine menthone confers antidepressant-like effects in an 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synthesis anxiety, depression, loneliness and social network in the elderly: longitudinal associations from the irish longitudinal study on ageing (tilda) social integration and quality of social relationships as protective factors for inflammation in a mationally representative sample of black women strain differences in sucrose preference and in the consequences of unpredictable chronic mild stress sex differences and phase of light cycle modify chronic stress effects on anxiety and depressivelike behavior age-related changes in behavior in c57bl/6j mice from young adulthood to middle age stability of inbred mouse strain differences in behavior and brain size between laboratories and across decades why is depression more prevalent in women? depression and c-reactive protein in us adults: data from the third national health and nutrition examination survey age differences in major depression: results from the national comorbidity survey replication (ncs-r) female mice liberated for inclusion in neuroscience and biomedical research sex bias in neuroscience and biomedical research differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus loneliness and social isolation as risk factors for mortality: a meta-analytic review. perspect disentangling loneliness: differential effects of subjective loneliness, network quality, network size, and living alone on physical, mental, and cognitive health this study was funded by janssen pharmaceutica as part of a large programme of research on depression and inflammation awarded to c.m.p., s.t., c.f. and p. a.z. c.m.p. has received additional research funding from the medical research council (uk) and the wellcome trust for research on depression and inflammation as part of two large consortia that also include johnson & johnson, gsk and the lundbeck foundation, and from the company eleusis benefit corporation, which is interested in research on depression and inflammation. the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies this paper at (https://doi.org/ 10.1038/s41398-020-01000-3).received: 20 july 2020 revised: 12 august 2020 accepted: 3 september 2020 key: cord-334890-7lpe8wa6 authors: fitzgerald, eamon; hor, kahyee; drake, amanda j. title: maternal influences on fetal brain development: the role of nutrition, infection and stress, and the potential for intergenerational consequences date: 2020-09-10 journal: early hum dev doi: 10.1016/j.earlhumdev.2020.105190 sha: doc_id: 334890 cord_uid: 7lpe8wa6 an optimal early life environment is crucial for ensuring ideal neurodevelopmental outcomes. brain development consists of a finely tuned series of spatially and temporally constrained events, which may be affected by exposure to a sub-optimal intra-uterine environment. evidence suggests brain development may be particularly vulnerable to factors such as maternal nutrition, infection and stress during pregnancy. in this review, we discuss how maternal factors such as these can affect brain development and outcome in offspring, and we also identify evidence which suggests that the outcome can, in many cases, be stratified by socio-economic status (ses), with individuals in lower brackets typically having a worse outcome. we consider the relevant epidemiological evidence and draw parallels to mechanisms suggested by preclinical work where appropriate. we also discuss possible transgenerational effects of these maternal factors and the potential mechanisms involved. we conclude that modifiable factors such as maternal nutrition, infection and stress are important contributors to atypical brain development and that ses also likely has a key role. which may associate with deficiencies in specific nutrients, has also been associated with effects on the response to stress in young adulthood 15 . studies in animal models aimed at delineating the long-term consequences of in utero undernutrition have demonstrated adverse effects on neurodevelopment. for example in mice, exposure to a low protein diet during gestation affects neural progenitor populations at several points during embryogenesis in both the cortex and the ganglionic eminence, and in adulthood these mice display deficiencies in short term memory 16 . in 2017 62% of women in england were overweight and of these, 30% were obese 17these figures include women of childbearing age. maternal obesity during pregnancy is associated with increased adiposity and cardiometabolic risk factors in adult offspring 18 . maternal obesity is also associated with impaired neurodevelopment and executive functioning 19, 20 and with adverse neuropsychiatric outcomes in children 20 , including attention deficit hyperactivity disorder (adhd) 21 and autism spectrum disorder (asd) 22, 23 . studies in mouse models of maternal obesity suggest that there are adverse effects on brain development and behaviour 24, 25 and in non-human primates, exposure to a high fat diet during pregnancy is associated with an increase in offspring anxiety-like and repetitive behaviours 26 . some of these effects may be mediated by epigenetic alterations, for example in mice, exposure to excess dietary ω-6 poly-unsaturated fatty acids (used to model a western high fat diet) has been associated with altered dna methylation and chromatin architecture in the offspring cortex 27 . population data from england and wales in 2014 indicated that 5% of pregnant women had diabetes 28 , of which 87.5% had gestational diabetes (gdm). gdm is a condition which arises during pregnancy, typically resolves postnatally and is closely associated with bmi 28,29 . diabetes during pregnancy is associated with a range of fetal effects across various organ systems, including an increased incidence of neural tube defects 30, 31 . in mice, hyperglycaemia during pregnancy is associated with disrupted neurogenesis in the embryo 32 and with apoptosis in epithelial cells within the neural tube 33 , and additionally affects social behaviours in adulthood 34 . some of the effects of maternal obesity on the offspring may be mediated by the associated increase in inflammation. higher levels of maternal inflammation during pregnancy are associated with an increased risk of neurodevelopmental delay during childhood and mediate the effect of prenatal environmental adversity on child neurodevelopmental delay 35 . exposure to maternal obesity during the antenatal period may result in the fetus being more susceptible to other insults such as infection or inflammation; for example, in mice, high fat diet-induced diabetes during pregnancy can potentiate the transcriptional response to a subsequent inflammatory stimulus in the fetal brain 36 . this is in line with the multi-hit j o u r n a l p r e -p r o o f journal pre-proof hypothesis which is commonly cited for schizophrenia but may have relevance to other neurological disorders 37 . pregnant women are more susceptible to infection and display an increased inflammatory response to some pathogens, but the mechanisms behind this are largely unknown 38, 39 . maternal viral infections during pregnancy may increase the risk of psychiatric disease in the offspring, with the time of infection being particularly important. for instance, a large danish study found a significant association between maternal hospitalisation for influenza during the first trimester and asd in her offspring 40 40 and in a large american study, there was a marked increase in psychiatric disorders in the adult offspring of mothers who experienced a bacterial infection during pregnancy 48 . this was dependent on the severity of infection and a higher incidence was seen in males 48 . stereotyped, repetitive behaviors during childhood 52, 53 . in rodents, maternal immune activation with poly i:c results in altered social interaction, communication and repetitive behaviours in adult offspring 54 . one potential mechanism which might mediate this is through maternal cytokine release; in mouse models, maternal interleukin (il-)6 has been shown to mediate the effects of poly i:c on fetal neurodevelopment 55 . a separate study indicated the maternal production of il-17a in response to poly i:c was involved in the pathogenesis of offspring neurodevelopmental disorders 56 . this leads to a hypothesised model in which maternal il-6 is stimulated by a viral infection, which in turn activates th17 cells to produce il17a, which crosses the placenta to affect offspring neurodevelopment. models of bacterial infection during pregnancy in rodents have also given important insights into atypical neurodevelopment induced by infection. prenatal lps in rodents results in an increase in repetitive behaviours 57 , reduced social interaction 58 , anxiety like behaviour 59 and spatial memory defects 60 in adult offspring. in rats, prenatal lps is associated with an altered lipid profile in 8 week old offspring 61 and alterations in dopaminergic signalling 62 , which has been hypothesised to be central in the aetiology of schizophrenia 63 . stress can be broadly described as an imbalance between the environment and an individual's perception of their resources to cope with that environment. maternal stress can occur as a result of a diverse range of environmental perturbations and crucially, the experience of stress is highly individually specific. therefore there are many domains of stress which can be associated with pregnancy 64 . while stress during pregnancy affects women worldwide, pregnancy-related stress in lower and middle income countries appears to be associated with a greater risk of adverse outcome in offspring 65 . offspring exposed to high levels of prenatal maternal stress or anxiety are at a higher risk of developing depression 66 , asd 67 , schizophrenia 68 and adhd 69 as well as various emotional and behavioural problems 70 ; with the timing of stress and the sex of the fetus playing important roles in the outcome of these studies. there is also evidence to suggest that maternal antenatal depression and socioeconomic status can interact with a polygenic score for major depressive disorder to modulate risk 71 . moreover, good maternal mental health during pregnancy is positively associated with cognitive abilities in the offspring at 2 years of age 72 . there are many factors that need to be accounted for in these studies. for instance some traditional metrics to assess stress may not be validated for use in pregnancy 64 . further, studies typically focus on periods of increased stress and neglect, but fail to measure positive or uplifting experiences which may counterbalance these 73 . the questionnaires used to measure stress during pregnancy may also be sensitive to underlying personality traits, perhaps underscored by the substantial j o u r n a l p r e -p r o o f heritable component of stress as measured by these methods 74 . moreover, stress experienced during pregnancy tends to continue during postnatal life, making assigning the importance of pre-and postnatal stress in mediating outcomes difficult 75, 76 . to address this, some studies have focussed on the effects of short-lasting large-scale environmental perturbations during pregnancy. for example, stress associated with experiencing an earthquake during the first trimester has been associated with a shorter length of gestation 77 . there is also evidence for effects on neurodevelopmental outcome, as offspring born to mothers who were pregnant during the 1998 ice storm crisis in quebec had lower cognitive and language abilities at 5.5 years of age when compared to control infants from pregnancies outwith this period 78 . there are a number of rodent models of maternal stress during pregnancy including exposure to a lactating female, forced swimming, noise and restraint stress 79 and the first rodent experiments describing altered behaviour in the offspring of stressed mothers was published more than 60 years ago 80 . in this study the adult offspring showed increased activity in the open field (a preclinical proxy of anxiety like behaviour), a finding which has been replicated in many subsequent studies (see weinstock 2017 for a full review 79 ). prenatal stress in rodent models has also been shown to affect other cognitive domains in offspring, such as spatial memory 81, 82 and social behaviour 83 . to account for the effects of pre-and postnatal stress, offspring cross-fostering studies have been performed. cross-fostering the offspring of stressed mothers onto a control non-stressed mother at birth results in no differences in anxiety or social related behaviours in stress-exposed offspring and controls, suggesting that the postnatal environment is important for some effects 84, 85 , but differences in learning memory, novel object recognition, long-term potentiation and risk of depression remain 86-88 . many of the effects of maternal stress on the fetus may be attributable to fetal glucocorticoid overexposure 89 . the enzyme 11β-hydroxysteroid dehydrogenase type 2 is present within the placenta and is responsible for converting active glucocorticoids in the maternal circulation (cortisol in humans, corticosterone in rodents) into an inactive form (cortisone or 11-dehydrocorticosterone). however, the enzyme has finite capacity and some active glucocorticoid may cross to the fetus, particularly in the presence of the increased maternal circulating glucocorticoid concentrations which may occur in stressful situations 90 . the administration of synthetic glucocorticoids, which bypass this enzyme, is commonplace in the management of women at risk of preterm birth and significantly improves morbidity and mortality in preterm babies 91 . however, there have been concerns about the deleterious effects of exposure to excess glucocorticoids (endogenous or exogenous) on the developing brain, with clinical studies reporting long-term effects on the function of the hypothalamic-pituitary-adrenal axis and behaviour in exposed children [92] [93] [94] [95] [96] . this is supported by data from a number of animal studies (reviewed by mcgowan and j o u r n a l p r e -p r o o f journal pre-proof matthews 95 ). although evidence from both human and animal studies suggests that excess fetal exposure to glucocorticoids can have adverse consequences on the brain, the effects of pregnancy related stress cannot be entirely attributed to glucocorticoid exposure 97 . maternal stress in rodents is also associated with an accumulation of catecholamines in the fetal circulation 98 , an increase in kynurenic acid in the placenta and fetal brain 99 , changes in oxytocin signalling 83 , and an altered maternal microbiome and associated in utero cytokine levels 100 . finally, maternal stress may interact with other factors e.g. infection to modulate offspring neurodevelopmental outcome. for example concurrent infection and stress during the second trimester result in a higher adolescent depression score in offspring then either alone 101 . a growing number of studies suggest that 'programmed' effects might not be limited to the first generation offspring i.e. those directly exposed in utero, but may be transmitted to a number of factors such as poor nutrition, stress and infection during pregnancy have all been associated with adverse effects on fetal neurodevelopment. one important consideration is that the long-term risks associated with exposure to an adverse intra-uterine environment are not evenly distributed across society. low socio-economic status (ses) is associated with an array of adverse pregnancy outcomes even in countries with established antenatal care programs 112,113 which may in part be due to a reduced awareness of service provision 114 . women of lower ses have an increased likelihood of exposure to risk factors during pregnancy 115 and ses is independently associated with future cognition 115, 116 . in higher 117 as well as lower-and middle-income countries 118 lower ses is associated with measures of unhealthy behaviour for example poor nutrition, smoking and low levels of physical activity, including amongst women of reproductive age 119 . increased levels of infections have been seen in pregnant women with a lower ses. for instance, an increased presence of the infectious bacterium h. pylori 120 (which has been linked with hyperemesis gravidarum 121 ) and an increase in urinary tract infections 122 . this is important for public health policy since many of the behaviours associated with lower ses are potentially amenable to change. for example, income supplementation, independent of a changing family dynamic or characteristic can reduce the prevalence of clinically relevant externalising behaviours in a low income population 123 . increasing the provision of 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increased risk of hyperemesis gravidarum: a meta-analysis urinary tract infections during pregnancy key: cord-333224-grjbz5u7 authors: effati-daryani, fatemeh; zarei, somayeh; mohammadi, azam; hemmati, elnaz; ghasemi yngyknd, sakineh; mirghafourvand, mojgan title: depression, stress, anxiety and their predictors in iranian pregnant women during the outbreak of covid-19 date: 2020-09-22 journal: bmc psychol doi: 10.1186/s40359-020-00464-8 sha: doc_id: 333224 cord_uid: grjbz5u7 background: pregnancy as a sensitive period of a woman’s life can be affected by various psychological factors that can have adverse effects on the woman, her fetus and future baby. since covid-19 is a new phenomenon with limited information available, it may have adverse psychological effects on pregnant women. therefore, this study was conducted to determine the status of depression, stress, anxiety and their predictors in iranian pregnant women during the outbreak of covid-19. methods: this descriptive-analytical cross-sectional study was performed on 205 pregnant women covered by tabriz health centers in iran. the sampling method used was cluster sampling. the data collection tool was the socio-demographic characteristics questionnaire and the dass-21 (depression, anxiety and stress scale-21), which were completed online by pregnant women. the general linear model was used to determine the predictive factors of depression, anxiety and stress. results: the mean (sd) score of depression, stress, and anxiety were 3.91 (3.9), 6.22 (4.25), and 3.79 (3.39), respectively; the score range of 0 to 21. depression, stress, and anxiety symptoms were observed in 32.7, 32.7, and 43.9% of the participants, respectively, with varying degrees from mild to very severe. based on the adjusted general linear model, variables of education level, spouse’s job and marital life satisfaction were the predictors of depressive symptoms. variables of spouse’s education level, spouse’s support, marital life satisfaction and the number of pregnancies were the predictive factors of anxiety symptoms and the variables of spouse’s education level, household income sufficiency, spouse’s support and marital life satisfaction were predictors of stress symptoms. conclusions: considering the role of marital life satisfaction, high level of spouse’s education and income in reducing symptoms of stress, anxiety, and depression in pregnant women in critical situations such as the prevalence of covid-19, it seems that using strategies to promote marital life satisfaction and socio-economic status can play an effective role in controlling anxiety and reducing stress and depression in pregnant women. the sars-cov-2 is a coronavirus belonging to the group of beta-coronaviruses. covid-19 is the third most well-known animal coronavirus disease after sars and mers, both of which belong to the group of betacoronaviruses [1] . the exact route of transmission of the disease is not yet known, but it is thought that covid-19 can be transmitted through droplets, close contact, aerosols, and possibly through feces-mouth, and patients in the incubation period can transfer the virus to others [2, 3] . covid-19 is rapidly advancing in the world and its mortality rate is increasing day by day. during the high prevalence of pandemics, different groups of the population, including pregnant women, as a vulnerable group, are exposed to high levels of psychological damage [4] . the unknown nature of the virus, as well as the lack of adequate information about its transmission, reproduction, risk factors, mortality, and disease-causing effects on pregnancy and the fetus [5] can pose risks not only to people's physical health but also to their mental health. it can lead to psychological effects, including stress, anxiety and depression [6] . although psychological change is one of the major changes in mothers during pregnancy, psychological care should not be neglected as these changes can lead to damage [7] ; because stress, anxiety, and depression are likely to have adverse effects on the mother and fetus. complications of stress during pregnancy include preterm labor, low birth weight, and delayed neuropsychiatric development in children born to these mothers [8] . depression during pregnancy can also have adverse effects on the fetus, the most important of which are preterm labor and low birth weight [9] . anxiety during pregnancy also increases the risk of preterm labor [10] , low birth weight [11] , preeclampsia and cesarean delivery [12] . in various studies, depression and anxiety have been reported to be about 10% during pregnancy [13] , which is considerable depending on the conditions and living environment [14] , especially in the second and third trimesters of pregnancy [15] ; it was considerable in women with a history of depression, too [16] . depressed women suffer from poor physical health and poor quality of life [17] . unfortunately, reviewing the available studies, it is found that a research investigating the pandemic effect of covid-19 disease during pregnancy and its psychological disorders was not available in iran. however, a canadian study found that pregnant women had higher levels of stress, anxiety and depression compared to the time before the covid-19 outbreak [18] . another study in turkey by durankus et al. (2020) showed that the covid-19 pandemic could cause psychological effects in pregnant women. they showed that the levels of anxiety and depression symptoms in pregnant women increased during the covid-19 pandemic [19] . in most cases, depression, anxiety, and stress are not detected or treated during pregnancy [7] . however, it is possible to improve the health of mothers and infants by identifying those women who have symptoms of anxiety and depression, and their risk factors during pregnancy [13] . consequently, considering the effects of mental disorders on pregnancy and infancy, it is possible to improve the psychological state of pregnant women, prevent complications by recognizing the psychological status of pregnant women, provide information and emotional support and other psychological interventions, and help to improve the mental state of mothers, and prevent unwanted complications. therefore, this study was conducted to determine the status of depression, stress, anxiety and their predictive factors in pregnant women during the outbreak of covid-19 in iran. this cross-sectional study was conducted after obtaining a license from the ethics committee of the vice chancellor for research and technology of tabriz university of medical sciences (code: ir.tbzme-d.rec.1398.1303) on 205 pregnant women who had a file in the health centers of tabriz-iran in 2019. tabriz is one of the metropolises of iran in which about 1897 people between march to april (the last day of sampling in this study) were identified with this disease during the covid-19 pandemic [20]. the criteria for entering the study included having a file in the health centers of tabriz, the desire to participate in the study, having a mobile phone and a healthy pregnancy. the exclusion criteria were a history of mental illness, medical problems during pregnancy, and high-risk pregnancies. sampling method was a two-step cluster sampling. twenty-two health centers (one-fourth of all health centers) selected randomly using the www.random.org website. the researcher extracted the list of pregnant women covered by the centers along with their phone numbers through the integrated health system (sib) and randomly selected the women based on the sample size determined for each center. then, the researcher made phone calls to the selected women and, while briefly explaining the objectives of the research and the confidentiality of the information, examined them in terms of eligibility criteria and, if eligible, they would participate in the study. since it was not possible to do the research in the traditional way and complete the paper questionnaire, they were asked to go online to answer the socio-demographic characteristics questionnaire, and dass-21 depression anxiety and stress questionnaire, the links of which was sent to them via whatsapp app. each participant completed the socio-demographic and obstetrics characteristics questionnaire and the dass-21 (depression, anxiety and stress scale-21) by the link to the questionnaires. the socio-demographic characteristics questionnaire included questions on age, level of education, job, spouse's age, level of education and job, sufficiency of monthly income for living expenses (this variable was measured by using a subjective item categorized in three levels including completely sufficient, fairy sufficient and insufficient), spouse's support level (this variable was measured by using a subjective item categorized in four levels including extremely high, high, moderate and poor), marital life satisfaction (this variable was measured by using a subjective item categorized in four levels including extremely high, high, moderate and poor), and obstetrics questions included the number of pregnancies, gestational age, and sex of the fetus based on ultrasound. the dass-21 is a shortened version of dass-42 and includes 21 questions and 3 subscales of stress, depression, and anxiety (7 questions for each subscale). the score for each question is a score from not at all (0) to very high (3). the score is calculated for each scale separately and the overall score is not calculated. the minimum score for each subscale is zero and the maximum is 21, and a higher score indicates a worse situation [21] . the dass21 questionnaire is commonly used in the pregnant population, especially in iran, due to the limited number of questions and simple sentences with simultaneous assessment of stress, anxiety and depression [22] . this instrument is in the public domain, and therefore, it can be freely used in research or practice [23] . this questionnaire can also be used in non-clinical populations [22, 23] . in iran, the validity of dass has been confirmed using forward-backward translation, factor analysis and criterion validity. the correlation between the beck depression inventory (bdi) and the depression subscale was 0.7, in the range of the zung self-rating anxiety scale (sas), the anxiety subscale was 0.67 and in the range of the perceived stress scale (pss), and the stress subscale was 0.49. in addition, its reliability was reported to be 0.73 for the anxiety subscale, 0.81 for the depression subscale and 0.81 for the stress subscale [24] . its reliability in pregnant women in tabriz (the setting of this study) for the variables of depression, anxiety and stress has been calculated as 0.80, 0.72 and 0.80, respectively [25] . in this study, the internal reliability using cronbach's alpha was 0.80 for the anxiety subscale, 0.78 for the depression subscale and 0.77 for the stress subscale. sample size was estimated as 97 individuals with considering d (precision) = 0.1 about the mean score of stress (m = 7.21), standard deviation = 3.63 [26] , and α = 0.05. as it was a cluster sampling, final sample size was calculated as 194 individuals with respect to the design effect of 2.0. at the end, 205 pregnant women were studied. data were analyzed using spss (version 24.0, spss inc., chicago, il). descriptive statistics including frequency, percentage, mean and standard deviation were used to describe the socio-demographic and obstetrics characteristics of the participants, depression, anxiety and stress. normal distribution of data was examined using skewness and kurtosis. the variables of depression, anxiety and stress didnot have a normal distribution. therefore, logarithmic transformation based on 10 (log 10) was used for these variables with abnormal distribution. then, the normality of log-transformed variables was checked again and all of them showed a normal distribution. general linear model test (adjusted, unadjusted) was used to determine the relationship between sociodemographic characteristics and depression, anxiety and stress. between the end of march 2020 and the beginning of april 2020, 205 people (with 40% participation) were examined. none of the participants had been affected by covid-19. the mean (standard deviation) of the participants and their spouses age were 29.3 (5.5) and 34.2 (5.6) years, respectively. about half of women (55.6%) had a university degree (44.4%) and the rest had a secondary high school and diploma degrees, and about 85% were housewives. about half of the spouses of the participants (48.3%) had a university degree and the rest (51.7%) had a high school degree, and diploma, and about half of them (45.4%) had freelance jobs. about half of women (43.9%) lived in private houses and 41.5% lived in rented houses, and about two-thirds of women (62.9%) reported relatively sufficient family income. according to ultrasound, 42% of fetuses were male. more than two-thirds of participants (70.3%) were in the second half of their pregnancy, and more than half (57.1%) experienced their first pregnancy. two-thirds reported high and very high levels of marital support (69.8) and marital life satisfaction (75.1) ( table 1) . the mean (standard deviation) score of depression, stress and anxiety were 3.91 (3.9), 6.22 (4.25) , and 3.79 (3.39), respectively, from the score range of 0 to 21. based on the scores obtained, they were divided into normal, mild, moderate, severe and very severe subgroups; 67.3% of women had normal status and 32.7% had symptoms of depression. in addition, 67.3% of people were normal in relation to stress and 32.7% of them had symptoms of stress. in the anxiety test, 56.1% of participants were normal and 43.9% of them had symptoms of anxiety ( table 2 ). the results of the present study showed no statistically significant relationship between pregnancy trimester and depression, stress and anxiety (p > 0.05). according to the unadjusted general linear model, there was a significant relationship between depression scores and spouse's level of education, spouse's job, spouse's support, and marital life satisfaction (p < 0.05). based on the adjusted general linear model, and by adjusting other variables, variables of spouse's level of education, spouse's job, and marital life satisfaction have been significantly associated with depression scores (p < 0.05) and they were able to predict 24.7% of the variance of depression score in pregnant women during the prevalence of covid-19. in addition, according to the unadjusted general linear model, there was a significant relationship between the anxiety score and the spouse's level of education, spouse's support, marital life satisfaction and the number of pregnancies (p < 0.05). based on the adjusted general linear model, and by adjusting other variables, the four variables of spouse's educational level, spouse's support, marital life satisfaction and number of pregnancies were significantly associated with the anxiety score (p < 0.05) and were able to predict 19.0% of anxiety score variance in pregnant women during the prevalence of covid-19. based on the unadjusted general linear model, there was a significant relationship between the stress score and age, education level, spouse's age, spouse's educational level, spouse's job, household income sufficiency, spouse's support, and marital life satisfaction (p < 0.05). based on the adjusted general linear model, and by adjusting other variables, the variables of spouse's level of education, sufficiency of household income, spouse's support and marital life satisfaction have a significant relationship with stress score (p < 0.05) and it was possible to predict 21.6% of stress score variance in pregnant women during the prevalence of covid-19 (table 3 ). the results of the study showed that 32.7, 32.7, and 43.9% of the participants had depression, stress and anxiety symptoms, respectively, with varying degrees from mild to very severe. based on the adjusted general linear model, variables of education level, spouse's job and marital life satisfaction were the predictors of depressive symptoms. variables of spouse's education level, spouse's support, marital life satisfaction and the number of pregnancies were the predictive factors of anxiety symptoms and the variables of spouse's education level, household income sufficiency, spouse's support and marital life satisfaction were the predictors of stress symptoms. in the present study, 67.3% of women had normal depression and stress and 32.7% had varying degrees of depression and stress. in terms of anxiety, 56.1% of people were normal, and 43.9% suffered from varying degrees of anxiety during the pandemic of sars-cov-2 disease. in line with the present study, in effati et al. [26] study on pregnant women and in a similar setting to the present study (tabriz-iran) (2018), more than half of women were normal in terms of depression, stress and anxiety and about 36, 32, and 27% of women experienced varying degrees of depression, stress, and anxiety symptoms, respectively. comparing the two groups, women's stress and depression symptoms levels were expected to be more severe during the coronavirus outbreak, while the severity of these problems was almost the same as when the coronavirus did not exist in the community. in this regard, it can be said that pregnant women, due to the importance of their fetus and its emotional attachment, may take care of themselves and follow the health advices of sars-cov-2 seriously. therefore, they should have more peace of mind and confidence, followed by less stress, anxiety and depression. during the covid-19 pandemic, the results of berthelot et al., [18] study showed that pregnant women had higher levels of stress, anxiety, and depression compared to the pregnant women who were examined before the pandemic, which is inconsistent with the results of our study. a possible reason for this discrepancy may be the cultural and social differences between our setting and their study. another study by durankus et al., (2020) [19] found that more than one-third of pregnant women had symptoms of depression and anxiety during the covid 19 pandemic, which is almost in line with the findings of our study. in a case-control study by lee et al., during the outbreak of sars, the results of anxiety in women who were pregnant during the outbreak of sars were only slightly higher than in women who were pregnant before the outbreak of sars and the rate of depression did not differ significantly between the two groups [4] . perhaps the reason for not increasing or slightly increasing of the severity of anxiety, stress and depression symptoms during the outbreak of diseases such as sars and covid-19 is that the disease is new or not taken seriously by people in the first spread. due to the newness of covid-19 disease and the lack of a study in the field, it was not possible to interpret the results of the present study in pregnant women with similar conditions in other studies. in the present study, there was a significant relationship between spouse's level of education with depression, anxiety and stress symptoms. women whose husbands had a non-university education were less likely to report depression, anxiety, and stress compared to those with a university degree. in a study by salmalian et al., [27] there was a significant association between spousal education and depression before and after childbirth, so that as the level of education was lower, depression was higher. in a study on the general population [28] , the level of education had a reverse statistical relationship with the three variables of depression, anxiety and stress. as the level of education increased, depression, stress and anxiety were reduced. the results of both studies are inconsistent with the results of the present study. education can open people's eyes and make them understand the situation, and increase their reaction to the events, especially in critical situations such as the prevalence of covid-19. while people with nonuniversity education may not have an idea of the bad condition and be less sensitive to the crisis of the outbreak of the disease, or may even be unaware of the dimensions of the crisis and the depth of the tragedy. while people with university education are expected to have more accurate follow-up of the deterioration of the situation from various sources such as scientific journals, cyberspace, media, etc. in addition, this increases the severity of depression, stress and anxiety symptoms and this causes a high level of depression, stress and anxiety in them and those around them. in our study, there was a significant relationship between spouse's job and symptoms of depression, so that women whose husbands were shopkeepers had more symptoms of depression than those whose husbands were employees. salmalian et al. [27] reported a significant relationship between spouse's job and pre and postpartum depression. depression was more common in women whose husbands had lower-paying jobs, which is consistent with our results. in the present study, there was a significant relationship between marital life satisfaction with depression, anxiety and stress scores during covid-19 prevalence. depression, stress and anxiety scores were lower in women who were satisfied and very satisfied with their lives compared to those who were moderately satisfied. in their study, bakhshi et al. [29] showed that with increasing severity of depression among men and women, their marital life satisfaction decreased. odinka et al. [30] in their study of low-risk women in the postpartum period also found a significant association between the severity of depression and anxiety and marital life satisfaction. the results of both studies were consistent with the present study. in our study, anxiety and stress scores were significantly higher in women with high levels of support from their spouses than in those with moderate levels of support. however, one study reported high anxiety and fear of childbirth in women who had poor support from their husbands or dissatisfaction with their husbands' support [31] . in addition, the results of a study showed that in 86% of pregnant women, the support of the husband during pregnancy has reduced their stress symptoms, and more than 90% of them have reported a sense of emotional security following the support of the husband [32] . the results of both studies are inconsistent with the results of our study. one of the possible causes of this mismatch could be that due to the depth of emotional relationships, high dependence and attachment to the spouse, the fear of losing him, his falling ill with covid-19 disease is greater among those supported by spouses, and this can increase their stress and anxiety. it is also possible that the stress and anxiety caused by the covid-19 pandemic in the mother will be so great that simply the support of the spouse cannot play an effective role in reducing it. in our study, anxiety scores were significantly lower in women who experienced their first and second pregnancies than in those in the third and more pregnancies. dunkel schetter et al. [33] showed a high level of pregnancy anxiety in women during their first delivery. in their study of pregnant women, rezaee et al. [34] did not report a difference in the number of parities between anxious and non-anxious women. perhaps the reason for the lower anxiety in low parity in the covid-19 pandemic in this study is the high relationship of mothers with low parity with health centers, which helps to obtain sufficient and accurate information and reduce their anxiety. according to available sources, this study is the first to investigate the depression, stress and anxiety of iranian pregnant women and their predictive factors during the prevalence of covid-19, and the random sampling of participants is another strength of the study. one of the limitations of this study is the crosssectional nature of it, the relationships shown between socio-demographic variables with symptoms of stress, depression and anxiety cannot accurately reflect the causal relationship. another limitation was that those who could have a mobile phone with internet connection could participate in this study. although 100% of the women studied had a cell phone, only 60% had a phone with this feature. therefore, as a limitation, this study may not be the representative of pregnant women in iran in general. in addition, the low level of participation was another limitation, as about half of pregnant women completed the questionnaire online. perhaps the reason for this is the recent online method of collecting data in iran, where all previous projects with pregnant women have been done in person. in the present study, marital life satisfaction and a high level of spousal education and income were associated with reduced symptoms of stress and anxiety in pregnant women. according to the results of the present study, low levels of stress, anxiety and depression in pregnant women during covid-19 prevalence can be a sign of successful training in controlling negative emotions during crisis by health centers and mass media. on the other hand, given the effective role of marital life satisfaction in reducing stress and 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from southeastern nigeria defining childbirth fear and anxiety levels in pregnant women women's perception of husbands' support during pregnancy, labour and delivery demographic, medical, and psychosocial predictors of pregnancy anxiety predictors of mental health during pregnancy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank authorities of tabriz university of medical sciences for the scientific and ethical approval and financial support of this research. we also sincerely thank all women who participated in this study.authors' contributions mm, am and fed: study concept and design, acquisition of the data, analysis and interpretation of the data, and drafting of the manuscript; fed, am, sgh and eh acquisition of the data; mm and am: critical revision of the manuscript for important intellectual content; fed, sz and am analysis and interpretation of the data, drafting of the manuscript, and statistical analysis; mm, study supervision. the author(s) read and approved the final manuscript. the authors received financial support from the tabriz university of medical sciences. the funder had no role in the study design, data collection and analysis or manuscript production. the data that support the findings of this study are not publicly available due to ongoing analyses but are available from the corresponding author, m. mirghafourvand, upon reasonable request.ethics approval and consent to participate this study was approved by the ethics committee of tabriz university of medical sciences (code: ir.tbzmed.rec.1398.1303). the informed written consent was obtained from all participants. all authors have read through the final version of the manuscript and given consent for publication in bmc psychology. all authors of this manuscript have no financial or non-financial conflicts of interest to declare. key: cord-309892-z7rb7adi authors: traylor, claire s.; johnson, jasmine; kimmel, mary c.; manuck, tracy a. title: effects of psychological stress on adverse pregnancy outcomes and non-pharmacologic approaches for reduction: an expert review date: 2020-09-24 journal: am j obstet gynecol mfm doi: 10.1016/j.ajogmf.2020.100229 sha: doc_id: 309892 cord_uid: z7rb7adi both acute and chronic stress can cause allostatic overload, or long-term imbalance in mediators of homeostasis, that results in disruptions in the maternal-placental-fetal endocrine and immune system responses. during pregnancy, disruptions in homeostasis may increase the likelihood of preterm birth and pre-eclampsia. expectant mothers traditionally have high rates of anxiety and depressive disorders and many are susceptible to a variety of stressors during pregnancy. these common life stressors include financial concerns and relationship challenges and may be exacerbated by the biologic, social and psychologic changes occurring during pregnancy. in addition, external stressors such as major weather events (e.g., hurricanes, tornados, floods) and other global phenomena (e.g., the covid-19 pandemic) may contribute to significant stress during pregnancy. this review investigates recent literature published about the use of non-pharmacologic modalities for stress relief in pregnancy and examines the interplay between psychiatric diagnoses and stressors, with the purpose of evaluating the feasibility of implementing non-pharmacologic interventions as sole therapies or in conjunction with psychotherapy or psychiatric medication therapy. further, the effectiveness of each non-pharamacologic therapy in reducing symptoms of maternal stress is reviewed. mindfulness meditation and biofeedback have shown effectiveness in improving one’s mental health such as depressive symptoms and anxiety. exercise, including yoga, may improve both depressive symptoms and birth outcomes. expressive writing has successfully been applied post-partum and in response to pregnancy challenges. though some of these non-pharmacologic interventions can be convenient and low cost, there is a trend towards inconsistent implementation of these modalities. future investigations should focus on methods to increase ease of uptake , ensure each option is available at home, and a standardized way to evaluate whether combinations of different interventions may provide added benefit. both acute and chronic stress can cause allostatic overload, or long-term imbalance in 23 mediators of homeostasis, that results in disruptions in the maternal-placental-fetal endocrine 24 and immune system responses. during pregnancy, disruptions in homeostasis may increase the 25 likelihood of preterm birth and pre-eclampsia. expectant mothers traditionally have high rates of 26 anxiety and depressive disorders and many are susceptible to a variety of stressors during 27 pregnancy. these common life stressors include financial concerns and relationship challenges 28 and may be exacerbated by the biologic, social and psychologic changes occurring during 29 pregnancy. in addition, external stressors such as major weather events (e.g., hurricanes, 30 tornados, floods) and other global phenomena (e.g., the covid-19 pandemic) may contribute to 31 significant stress during pregnancy. 32 this review investigates recent literature published about the use of non-pharmacologic 33 modalities for stress relief in pregnancy and examines the interplay between psychiatric 34 diagnoses and stressors, with the purpose of evaluating the feasibility of implementing non-35 pharmacologic interventions as sole therapies or in conjunction with psychotherapy or 36 psychiatric medication therapy. further, the effectiveness of each non-pharamacologic therapy 37 in reducing symptoms of maternal stress is reviewed. mindfulness meditation and biofeedback 38 have shown effectiveness in improving one's mental health such as depressive symptoms and 39 anxiety. exercise, including yoga, may improve both depressive symptoms and birth outcomes. 40 expressive writing has successfully been applied post-partum and in response to pregnancy 41 challenges. though some of these non-pharmacologic interventions can be convenient and low 42 cost, there is a trend towards inconsistent implementation of these modalities. future 43 investigations should focus on methods to increase ease of uptake , ensure each option is 44 stress is a frequently used, ambiguous term. 1 the 'allostatic model' has been developed to 48 help clarify these ambiguities and explain the effects of acute versus chronic stress responses. 2 49 initial conceptions of stress response were centered around homeostasis, a concept referring to 50 self-regulating processes that maintain the stability of an individual's essential systems. cohen 51 et al. provide a working definition of stress as "when environmental demands tax or exceed the 52 adaptive capacity of an organism, resulting in psychological and biological changes that may 53 place persons at risk for disease." 3 these environmental demands may be internal, relating to 54 an individual's disposition, or external, relating to an individual's life circumstances. acute stress 55 is an intense, but relatively short-lived response to stressors whereas chronic stress is the result 56 of unresolved stressors that are experienced for a longer period of time. 57 repeated or chronic stress contributes to the cumulative allostatic load-the "wear and tear 58 on the body," or a sum of the lifetime stress exposure. 4,5 allostatic load increases over time and 59 represents physiologic consequences of heightened neural or neuroendocrine responses. 6 60 hypothalamic-pituitary-adrenal (hpa) axis hormones -including cortisol, catecholamines such 61 as epinephrine, and cytokines -are all primary mediators impacting allostasis. 2 when these 62 primary mediators go beyond the limits of homeostatic mechanisms and become unbalanced, 63 the body is only able to sustain this state without negative effects for a limited time. 2 a 64 prolonged imbalance of these primary homeostatic mediators results in allostatic overload. 65 additionally, chronic stressors or repeated acute stressors may result in changes related to 66 glucocorticoid genes, by alterations of the epigenome and/or transcriptome, and hasten 67 disease. however, though the initial stressful insult cannot always be prevented, epigenetic and 68 transcriptomic effects are dynamic, and potentially reversible through treatment. 7-12 69 stress is common among pregnant women. in 2009-2010, data from the us-wide centers 71 for disease control pregnancy risk assessment monitoring system found that nearly 75% of 72 j o u r n a l p r e -p r o o f postpartum mothers reported at least one major stressful event in the year leading up to delivery 73 of their baby. 13 the most commonly cited stressors experienced during pregnancy included 74 moving to a new address, arguing with a partner more than usual, serious illness and 75 hospitalization of a family member, and inability to pay bills. in addition, external stressors such 76 as extreme weather events (e.g., hurricanes, tornados, floods) and other global adverse events 77 (e.g., the covid-19 pandemic) may contribute to significant acute and chronic stress during 78 pregnancy. reports of perceived stress varied widely by race and ethnicity, with non-hispanic 79 american indian/alaska native women reporting highest levels of stress and non-hispanic 80 asian women reporting the lowest. 13 81 chronic stress may be associated with adverse pregnancy outcomes via a positive feedback 83 loop. maternal-placental-fetal neuroendocrine interaction and immune responses are stress-84 sensitive, and thus may affect birth outcomes. maternal stress is associated with cortisol 85 release. 14,15 high cortisol levels reduce lymphocyte sensitivity to glucocorticoids by binding to 86 glucocorticoid receptors; subsequently, as steroid resistance is developed, there is an increased 87 release of pro-inflammatory cytokines. 15 furthermore, maternal stress influences circulating 88 levels of inflammatory markers by increasing pro-inflammatory cytokines interleukin-1β, 89 interleukin-6, and tumor necrosis factor α, and decreasing anti-inflammatory cytokine 90 interleukin-10. 14 these inflammatory markers dampen the immune system response, increasing 91 the susceptibility to adverse pregnancy outcomes, such as ptb. 14 women who develop adverse 92 pregnancy complications requiring early delivery or resulting in other maternal or neonatal 93 morbidities may then experience additional stress, furthering the loop. 94 previous studies have found that minority and low-income pregnant women may have 95 higher baseline levels of cortisol compared to women of other situations, supporting the 96 association between chronic stress, increased allostatic load, and higher rates of adverse 97 pregnancy outcomes among these high risk obstetric populations. 16 one stressor of particular 98 j o u r n a l p r e -p r o o f concern to pregnant women of color in the us is racism. racism (and/or discrimination) is 99 typically defined as differential treatment based on ones' skin color or racial identity. racism 100 affects over 68% non-hispanic black women in the u.s. it is hypothesized that racism may be a 101 significant contributor to the disparities in adverse birth outcomes (e.g., ptb and preeclampsia) 102 among non-hispanic black women compared to non-hispanic white women. 15 the chronic 103 stress associated with racism contributes to an increased allostatic load and a more rapid 104 decline in health during an individual's lifetime-it is a major contributing factor to the 105 'weathering hypothesis.' weathering is the premature aging of the body due to endurance of 106 adverse events -this can be both physically and psychological. this hypothesis is supported by 107 studies suggesting that age-related increases in preterm birth are higher among non-hispanic 108 black women compared to non-hispanic white women 17 and that racial and ethnic disparities in 109 ptb and other adverse pregnancy outcomes persist among women of high socioeconomic 110 status. 18 111 acute stress, chronic stress, and allostatic overload have all been associated with a variety 113 of adverse pregnancy outcomes, including spontaneous ptb, preeclampsia, neonatal morbidity, 114 and low birth weight (table 1) . 14, 15, 19 in turn, ptb (regardless of indication) is associated with a 115 higher risk of short-term neonatal morbidities (including neurologic, pulmonary, cardiovascular, 116 gastrointestinal, immune and metabolic complications) and long-term complications among 117 survivors (e.g., cerebral palsy, neurodevelopmental delay, visions problems, and hearing 118 loss). 20-26 as the total allostatic load increases, the likelihood of adverse pregnancy outcomes 119 may also increase ( figure 1) . 120 natural disasters and adverse national and international events (e.g., pandemics) provide a 121 unique opportunity to study the effects of a 'universally stressful' exposure on pregnancy 122 outcomes. while it can be difficult to navigate ethical considerations of human research 123 involving an imposed stress variable, populations and individuals intrinsically experience varying 124 j o u r n a l p r e -p r o o f levels of impact, and thus stress exposure, in the event of a population-wide stressor. these 125 stressful experiences -including destructive weather events (e.g., hurricanes, earthquakes) and 126 more chronic population-wide stressors (e.g., local political unrest, war, pandemics) may also 127 activate both the acute and chronic stress response feedback loops in pregnant individuals. 128 depression and anxiety during pregnancy is very common. one in five women will have an 130 anxiety disorder in pregnancy, 27 and 10-14% of women in the general obstetric population meet 131 criteria for major depression during pregnancy. 28 however, anxiety and depressive symptoms 132 are as high as 25-50% in pregnancy, when symptoms are present but insufficient to meet full 133 diagnostic criteria for a specific anxiety or depressive disorder. 29-31 in a study of pregnant low-134 income black women engaging in home-visiting programs in an urban environment, over 20% 135 met full criteria for major depressive disorder in pregnancy, 31 further supporting that non-136 hispanic black women who face higher stress also have higher rates of depression. 137 women with a previous diagnosis of depression or anxiety prior to pregnancy, past 138 pregnancy or delivery complications including pregnancy loss and stillbirth, history of adverse 139 life events (e.g., abuse), and particularly those with multiple traumatic events have a higher 140 allostatic load and higher rates of antenatal depression and anxiety. 32 similar to biologic findings 141 seen in those with chronic stress, women with major depressive disorder during pregnancy 142 have increased pro-inflammatory cytokines and a blunted cortisol awakening response. 33 143 further, prenatal anxiety is associated with increased cortisol levels and pro-inflammatory 144 cytokines. 34 women with both severe depression and severe anxiety during the third trimester 145 had higher levels of il-6, il-2, il-9 and il-17a. 35 146 147 though many pregnant women are exposed to both acute and chronic stressors, not all 149 women who are exposed have adverse pregnancy outcomes. this may explain, in part, why 150 j o u r n a l p r e -p r o o f pregnancy outcomes remain variable in the setting of more widespread adverse events. the 151 sum of an individual's prior social experiences (both positive and negative), and their reaction to 152 these experiences influence whether exposure to new acute or chronic stressors disrupts 153 homeostasis and results in disease or adverse outcomes. hogue and colleagues positioned 154 racial discrimination and spontaneous ptb within a stress and coping framework whereby 155 effective coping may reduce the negative impact of the stress of discrimination, but ineffective 156 coping may allow the stress to cause a disruption in homeostasis and contribute to adverse birth 157 outcomes. 36 others report that though women have elevated risk of spontaneous ptb when 158 reporting lifetime racism (or 1.5, 95% ci 0.9-2.8) and racism in the past year (or 2.5, 95%1.2-159 5.2), this risk can be abrogated by "active coping." 37 finally, though one study of 3,021 women 160 in canada found that stress was a significant risk factor for ptb (or 1.73, 95% ci 1.07-2.81), 161 the risk of prematurity was highest among those with low levels of social support or optimism. 38 162 based on these data, the successful reduction on the biological effects of stress during 163 pregnancy has the potential for profound impacts on maternal health and pregnancy outcomes 164 for certain populations. though pharmacologic therapy including selective serotonin reuptake 165 inhibitors (ssris) and benzodiazepines have a role in treating depression or anxiety in 166 pregnancy, pharmacologic treatments for the different alterations in stress responses that have 167 built up over time are more elusive, and the effect on pregnancy outcomes remains under-168 investigated. of concern, benzodiazepine use in combination with ssri therapy has been 169 associated with worse adverse behavioral effects in the infants. 39 furthermore, though rates of 170 depression are high, only 8.6% receive adequate treatment in pregnancy, and data regarding 171 remission rates of depression with medication during pregnancy are limited. 40 finally, women may be taking appropriate doses of pharmacotherapy but need or desire 186 an additional adjunct therapy. psychotherapy also has a role in the treatment of stress during 187 pregnancy through cognitive behavioral therapy and other approaches, but is excluded from this 188 review because access to psychotherapy may be limited due to provider availability or financial 189 concerns. therefore, the objective here is to review the available use of low-cost, logistically 190 feasible, non-pharmacologic therapies to reduce stress during pregnancy (figure 2 ), and to 191 discuss future avenues for research and clinical care in this area. 192 section 2: interventions to reduce stress during pregnancy 193 meditation is a mental exercise that improves attention and emotional self-regulation. 195 some types of meditation include mindfulness, breathing, mantra recitation, and visualizations. 46 196 grounded in buddhist origins, mindfulness is described as attention to and awareness of 197 present perceptions. 47 it is a form of experiential processing in which instances are observed 198 from a wider perspective that recognizes influential judgements and associations. 45 in contrast, 199 during conceptual processing, an individual evaluates situations within the context of self-200 concern. in recent literature, mindfulness has been of prominent interest as a potential 201 j o u r n a l p r e -p r o o f therapeutic tool because it is low cost and only requires a relatively short investment of time 202 each day. an integrative review of mindfulness in the workplace reported improvements in 203 attention, cognitive capacity, emotional reactivity, self-regulation, and stress response. 48 as a 204 targeted intervention for stress, mindfulness has shown effectiveness in reducing negative 205 outcomes such as anxiety, depression, and chronic pain. 46 206 in pregnancy, mindfulness has potential as a therapy to reduce stress and improve birth 207 in a randomized controlled trial of non-pregnant students with chronic, pain, and anxiety, 228 mindful breathing facilitated by a 12-minute smartphone-based task is proposed to decrease 229 hrv. 52 similarly, hrv biofeedback is a non-invasive technique that utilizes metronomic 230 breathing while monitoring one's parasympathetic activity to improve hrv measures. 43 hrv 231 biofeedback has been shown to improve control in response to negative situations, 43 and has 232 been associated with reductions in self-reported perceived stress and anxiety. 53 two studies of 233 perinatal women, one including women with threatened preterm labor and the other including 234 women in the early postpartum period utilized hrv biofeedback versus control groups. [54] [55] [56] in 235 the study of antenatal women with threatened preterm labor, 48 women were randomized to 236 hrv biofeedback vs. standard care at an average 29 weeks' gestation; those randomized to 237 hrv biofeedback had a decrease in their perception of chronic stress during the study period, 238 and the rate of ptb was lower than in the control group (13% vs. 33%; p value=not significant, 239 exact value not provided in manuscript). 55 in the early postpartum study, use of hrv 240 biofeedback was associated with significant improvements in hrv measures and in scores on 241 the edinburgh postnatal depression scale compared to those who did not use hrv feedback. 54 242 in anxiety symptoms and improvement in psychological well-being. 57 taken together, these 244 data suggest that biofeedback -particularly when linked to hrv monitoring -may be a method 245 of determining who is responding physiologically to mindfulness and deep breathing practices. 246 studies have already shown efficacy in reducing perceived stress and anxiety during 247 pregnancy, including among pregnant women with threatened preterm labor. 248 according to the national center for health statistics, over 35 million american adults 250 actively practiced yoga in 2017, and this number is still growing. 58 yoga is the most commonly 251 used complementary health approach in the u.s. and consists of three aspects: physical 252 postures (asanas), breathing techniques (pranayama), and meditation (dhyana). 58 originating 253 from india as a spiritual practice, yoga has grown and evolved into many different styles such as 254 hatha, iyengar, bikram, and integrated approaches. 59 while hatha yoga is the most popular 255 form, these different styles do not significantly differ in the probability of reaching positive 256 conclusions in recent research (p=0.191). 59 257 yoga is a popular non-pharmacologic intervention available to pregnant women that may 258 improve both birth outcomes and mental health. prenatal yoga classes are commonly available 259 across the united states. in addition, yoga instruction specifically tailored for pregnant women is 260 available for 'free' online through smartphone apps and on publicly available websites. in a 261 study investigating the effects of prenatal yoga on birth outcomes, 84 women with depressive 262 symptoms were randomized to yoga, massage therapy, or standard prenatal care from 20 to 32 263 weeks' gestation. a greater improvement in depression scores, decreased anxiety scores, 264 decreased anger scores, decreased back and leg pain scores, and increased relationship 265 scores were seen for those in both the yoga group and the massage therapy group, but not the 266 control group. in addition, those in the yoga and massage group delivered later (mean 38.6 and 267 38.4 weeks', respectively) than those in the control group (mean 36.7 weeks' gestation). 60 a 268 taiwanese study evaluating the effects of prenatal yoga on stress and immune function 269 intervention. however, it is it important to note that the trials evaluated in both analyses were 289 typically preliminary with small sample sizes. 290 as defined by the american college of sports medicine, exercise is "a type of physical 292 activity consisting of planned, structured, and repetitive bodily movement" that is produced by 293 skeletal muscle contraction and leads to increased energy expenditure. 65 exercise is known to 294 help prevent and treat metabolic or cardiovascular diseases; it has also been shown to reduce 295 depressive symptoms. 65 the american college of obstetrics and gynecology recommends 296 women with uncomplicated pregnancies should complete moderate-intensity physical activity for 297 at least 20-30 minutes on most or all days of the week. 66 historically, intense exercise has been 298 assumed to have negative consequences for mother and child. 66 in fact, women tend to reduce 299 their physical activity during pregnancy and less than half of pregnant women meet exercise 300 recommendations. 61 while there may be a theoretic concern regarding vigorous exercise in 301 some situations (e.g., women with premature cervical dilation or threatened preterm labor), 302 there is minimal evidence to suggest harm, and one observational study of women with short 303 cervix suggested a higher risk of preterm birth among women with activity restrictions compared 304 j o u r n a l p r e -p r o o f to those without such restrictions. 67 an analysis of exercise and pregnancy loss referencing 6 305 cohort studies and reviews including over 120,000 women found that regular exercise for up to 306 7 hours a week, including low-and high-intensity activity, is not associated with increased rates 307 of miscarriage. 68 308 the benefits of exercise during pregnancy span both physical and mental capacities. a 309 systematic review and meta-analysis of low-impact physical activity in pregnancy evaluated 30 310 randomized-controlled trials and 51 cohort studies for maternal-child health outcomes. 69 regular 311 exercise was associated with lower weight gain during pregnancy, a lower likelihood of 312 gestational diabetes mellitus, and a lower risk of preterm delivery. less is understood about the 313 relationship between stress and exercise in pregnancy. however, there is a direct association 314 between low exercise frequency and higher reports of stress-related symptoms. 70,71 one 315 randomized controlled trial of 167 women explored depression reduction and exercise during 316 pregnancy. 72 women randomized to the exercise group completed three 60-min sessions of 317 supervised physical activity per week throughout pregnancy. compared to the control group, 318 women in the exercise intervention group scored significantly lower on the center for 319 epidemiologic studies depression scale (7.76 ± 6.30 vs. 11.34 ± 9.74, p = .005) at the end of 320 the study. 72 321 expressive writing involves a personal and often emotional reflection of thoughts or memories; it 323 focuses on detailing one's feelings while writing with the purpose of potentially easing emotional 324 trauma. it was developed as a type of therapy by james w. pennebaker in the late 1980s after 325 his research found that writing for 15 minutes a day for at least 3 consecutive days about 326 previous distressing experiences was associated with significantly fewer visits to a physician in 327 the following months. 73 subsequently, various medical disciplines have started using expressive 328 writing; it is an attractive therapeutic option as it is accessible, customizable, does not require 329 significant time commitment, and is low cost. additionally, it may be more convenient compared 330 with traditional psychotherapy given the lack of mental health professionals in many areas, 331 though it may also be effective if the pregnant woman is able to share some of her writing with a 332 health care professional. although a quantitative survey of pregnant women found that women 333 preferred video telehealth therapy compared to computer-assisted therapy and self-guided 334 online therapy, 74 psychotherapy access is limited for many individuals due to lack of providers 335 and other barriers including financial and scheduling logistics. pregnant women rated 336 computer-based support as acceptable, 74 meditation and mindfulness, biofeedback, yoga, exercise, and expressive writing have 361 not been explored as possible stress therapy options until recent years. therefore, research 362 conducted on these interventions is largely preliminary and prospective. support for these 363 interventions, particularly expressive writing, could benefit from more randomized, controlled 364 trials with larger sample sizes. likely there may be characteristics that make some women more 365 responsive to on over another (e.g., anxious depression versus non-anxious depression). in 366 addition, combining some of these might also lead to greater results, e.g., a mindfulness 367 practice that also includes expressive writing. 368 non-pharmacologic interventions -including, but not limited to those discussed here -369 have the potential to be affordable and widely available. additionally, with the rising popularity of 370 online classes and support groups, all of the above interventions can be completed at-home. 371 this holds great potential for times when leaving one's home is not possible-a period where 372 situational stress can even be elevated. for example, the methods for stress reduction outlined 373 here provide a safe and potentially effective way for women to reduce stress during pregnancy 374 during a pandemic. non-pharmacologic interventions also provide patients with a choice of 375 preference; patient autonomy will likely improve participation and engagement. expressive 376 writing, mindful meditation, biofeedback, yoga, and exercise provide a broad range of options in 377 terms of metacognition and physicality. finally, these interventions are novel strategies that may 378 be readily available to women of all social and racial / ethnic backgrounds, and the benefits of 379 such could help to close the health disparities gap with respect to maternal and fetal health 380 outcomes. these may also augment pharmacologic treatment of depression and anxiety just as 381 therapy and pharmacotherapy combined can be most effective for those with severe depression 382 or anxiety. 81,82 383 thus, non-pharmacologic interventions for stress reduction provide a viable option 384 available to women during pregnancy. nevertheless, improvements in the implementation of 385 these options should be explored. providers must also take into consideration the unique 386 situation and variable social determinants of health that may make some stress reducing 387 modalities difficult to routinely practice -such as one's work schedule, caregiver needs, unstable 388 housing and/or internet access, etc. in one study, the viability of using expressive writing was 389 studied through agreement of participation and completion of full intervention. around 8,000 390 eligible women were contacted, approximately 1,400 replied to the study invitation, and 854 391 agreed to participate. 83 however, by the 6-month follow-up, only 290 women remained. 68 392 additionally, less than 30% of the women in both writing groups fulfilled the intervention 393 conditions completely. 83 most of the women who did not choose to participate marked "too busy" 394 as the reason for their decline. these results reveal that although expressive writing is 395 theoretically more convenient than other interventions, many patients struggled to complete the 396 intervention conditions to the full extent. horsch's study supports this idea as although 94 of the 397 105 eligible mothers contacted agreed to participate, only 54 completed the study through to the 398 3-month follow-up. 84 further, it is unknown whether combinations of these approaches -e.g., 399 expressive writing and yoga or exercise -might improve pregnancy outcomes. finally, 400 incentivizing the use effective non-pharmacologic therapies for providers could also help with 401 update of the modalities discussed in this review. in the future, ways to incentivize completion 402 and emphasize the potential importance of writing therapy and meditation should be researched 403 for all stakeholders. 404 expressive writing, meditation, mindfulness, biofeedback, yoga, and exercise are effective 406 therapies for emotional and physical health. while these methods have not always been 407 considered for medical treatment of individuals, new research is continually revealing the 408 potential of these interventions to improve health outcomes. for example, other non-409 pharmacologic approaches to reduce stress in pregnancy -including music therapy and a 410 smartphone app specifically designed to reduce stress in pregnancy -have also been 411 evaluated, but more data are needed to determine the effectiveness of these additional 412 modalities. 85, 86 the application of these accessible and widely available interventions to reduce 413 stress during pregnancy. in turn, by limiting the negative implications associated with stress in 414 pregnancy, these non-pharmacologic options could help improve birth outcomes. research 415 should continue to expand on the strengths of each intervention as effective, affordable, 416 adaptable to make them a more convenient option for stress alleviation in addition to research 417 on how these non-pharmacologic methods could further enhance the effects of pharmacologic 418 therapies to treat psychiatric conditions. 419 the stress reaction: a historical perspective measuring stress: a guide for health and social scientists stress, adaptation, and disease. allostasis and allostatic load weathering" and age patterns of 439 allostatic load scores among blacks and whites in the 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and meta-analysis a stress coping app for 648 hospitalized pregnant women at risk for preterm birth key: cord-288101-pij16jaa authors: li, jun-yu; yong, yan-hong; gong, dong-liang; shi, lin; wang, xiao-min; gooneratne, ravi; yadnyavalkya, patil; ju, xiang-hong title: proteomic analysis of the response of porcine adrenal gland to heat stress date: 2019-02-28 journal: research in veterinary science doi: 10.1016/j.rvsc.2018.11.004 sha: doc_id: 288101 cord_uid: pij16jaa abstract heat stress (hs) and its associated pathologies are major challenges facing the pig industry in southern china, and are responsible for large economic losses. however, the molecular mechanisms governing the abnormal secretion of hs-responsive hormones, such as glucocorticoids, are not fully understood. the goal of this study was to investigate differentially expressed proteins (deps) in the adrenal glands of pigs, and to elucidate changes in the immune neuroendocrine system in pigs following hs. through a functional proteomics approach, we identified 1202 peptides, corresponding to 415 proteins. of these, we found 226 deps between heat-stressed and control porcine adrenal gland tissue; 99 of these were up-regulated and 127 were down-regulated in response to hs. these deps included proteins involved in substrate transport, cytoskeletal changes, and stress responses. ingenuity pathway analysis was used to identify the subcellular characterization, functional pathway involvement, regulatory networks, and upstream regulators of the identified proteins. functional network and pathway analyses may provide insights into the complexity and dynamics of hs-host interactions, and may accelerate our understanding of the mechanisms of hs. rising global temperatures have been accompanied by increased interest in researching the detrimental effects of heat stress (hs) on the swine industry. pigs enter a state of hs when the ambient temperature exceeds their thermal neutral zone (16-22°c for adult pigs) (coffey et al., 1995) . due to their high production of metabolic heat, accelerated fat deposition, and lack of sweat glands, pigs there are more sensitive to hs than many other mammals (d'allaire et al., 1996) . heat stress in pigs not only decreases food intake and body weight gain, but also has immunosuppressive effects, all of which may result in large economic losses for the swine industry (cruzen et al., 2015; pearce et al., 2013) . for instance, hs is estimated to cost the us swine industry losses of over $300 million each year (st-pierre et al., 2003) . understanding the stress-associated mechanisms involved in immune system function and the increased susceptibility of livestock to heat-related illness is more important now than ever, as the majority of emerging animal diseases are zoonotic and can potentially threaten public health. when exposed to a high-temperature environment, the central nervous system of mammals, including livestock, engages in physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (hpa) axis and the sympatho-adrenal axis. the predominant hormone regulating the synthesis and secretion of adrenal glucocorticoids is adrencocorticotropic hormone (acth) (minton, 1994) . in pigs, cattle, and sheep, both corticotropin-releasing hormone and vasopressin regulate the secretion of acth, suggesting that these two proteins interact to enhance acth secretion. acth acts on the adrenal gland with the circulation, it to induces the expression and secretion of glucocorticoids, which suppress the production of cytokines and other pro-inflammatory mediators, including tnf-α, interferon-γ, il-1β, il-11, il-12, il-8, and prostaglandins (wilckens and rijk, 1997) . glucocorticoids also facilitate the release of anti-inflammatory mediators, such as transforming growth factor-α, il-10, and il-4, and have apoptotic effects and strong anti-proliferative properties in immune cells (visser and nagelkerken, 2002) . ultimately, cytokines activate the release of glucocorticoids, which in turn suppress cytokine synthesis fin a negative feedback loop (barrat et al., 2002; haddad et al., 2002) . thus, it is of interest to understand how higher core temperatures alter adrenal function. isobaric tag for relative and absolute quantification (itraq) is a powerful quantitative proteomics technique. in recent years, several proteomic studies have explored protein expression in a number of porcine cells and tissues, including pulmonary alveolar macrophages (lu et al., 2013) , mesenchymal stem cells (huang et al., 2015) , liver (liu et al., 2016) , heart (cabrera et al., 2012) , and intestine (colladoromero et al., 2015) . nevertheless, no large-scale proteomic analysis to date has examined the molecular complexes or pathways involved in the pathogenesis of the hpa axis. the purpose of the present study was to investigate protein expression in the adrenal gland of pigs in response to hs, and to elucidate potential changes in that occur in the endocrine system under hs. pigs were maintained and studied in accordance with the national institutes of health (nih) guidelines for the care and use of laboratory animals, and all protocols were approved by the guangdong ocean university animal care and use committee, china. six castrated bama miniature pigs (sus scrofadomestica) 3 months, weighing 30-40 kg were obtained from the bama miniature pigs breeding farm in the guangxi zhuang autonomous region of china. the six pigs were randomly divided into a heat stress group (3 barrows, ha) and a control group (3 barrows, ca). control pigs were housed with an ambient temperature of 28 ± 3°c, and the relative humidity was kept at approximately 90%. pigs assigned to the heat stress treatment were kept at 35 ± 1°c(maintained using an artificial climate chamber) in a manmade climate room, with a relative humidity of approximately 90%. all pigs were given access to water ad libitum. diet (see diet composition table) was formulated according to the recommended nutrient allowances for this breed of pig and the feeding was done twice a day, in the morning as well as in the evening. pigs were euthanized by a head-only electric stun tong apparatus on the 7th day under heat stress, followed by manual exsanguination. immediately after slaughter, adrenal tissue was removed and weighed. subsequently, tissues were washed with pbs to remove any blood and contaminants on the tissue surface. adrenal tissue was placed into sterile tubes and snap frozen in liquid nitrogen three pigs were used in the control group and three pigs were used in the heat-stressed group. the adrenal tissues of the 3 pigs in each of the groups were pooled together to form one pooled sample and utilized as one sample for further analyses. once in the laboratory, frozen specimens were stored at −80°c until biochemical and molecular analyses were performed. frozen samples of adrenal tissue from all pigs in the two groups were crushed in a mortar containing liquid nitrogen. the powder (approximately 100 mg per sample) was transferred to a sterile tube containing 1 ml lysis buffer (lb; containing 7 m urea, 2 m thiourea, 4% chaps, 40 mm tris-hcl, ph 8.5, 10 mm dithiothreitol, dtt). tissue homogenate was further disrupted using an ultrasonic cell disruptor (vcx130, usa) at 20% power output for 10 min, cycling between 2 s on and 4 s off. afterwards, the lysate was centrifuged at 25,000 ×g for 30 min at 4°c, and the supernatant was collected for protein quantification. protein concentration was measured using the pierce bca protein assay kit (thermo scientific, usa). protein digestion was performed as per the fasp procedure described by wisniewski, zougman et al. (wiśniewski et al., 2009) ; and the resulting peptide mixture was labeled using the itraq reagent-4plex multiplex kit (ab sciex, framingham, usa), according to the manufacturer's instructions. after 2 h of incubation at room temperature, labeled samples were mixed at equal ratios. subsequently, labeled peptides were combined and fractionated by strong cation exchange (scx) chromatography (han et al., 2015) and desalted on c18 cartridges (66872-u; sigma, st. louis, mo, usa). the dried peptide mixture was reconstituted and acidified with 2 ml buffer a (10 mm kh 2 po 4 in 25% of acn, ph 3.0) and loaded onto a column (4.6 × 250 mm). peptides were eluted at a flow rate of 1 ml/ min with a gradient of 0%-5% buffer b (2 m kcl, 10 mm kh 2 po 4 in 25% of acn, ph 2.7) for 5 min, 5-10% buffer b for 10-15 min, 10%-30% buffer b for 25-35 min, and 30%-50% buffer b for 35-50 min. the elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. the tryptic peptides were extracted, and the peptide mixtures were concentrated by speed vac centrifuge to dryness, and were again dissolved with 2% acetonitrile (acn) in 0.1% formic acid before lc-ms/ms analysis. the fractions from 2.3 were subjected to liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis. initially, samples were loaded onto pre-columns (180 μm × 20 mm; 5 μm-c18; waters, usa). peptide mixtures were separated on analytical columns (100 μm × 100 mm; 1.7 μm-c18; waters, usa) at a flow rate of 300 nl/ min over 60 min. thermo easy-nlc is a binary buffer system used for high performance liquid chromatography (hplc), consisting of 0.1% formic acid (buffer a) and 80% acetonitrile (acn) in 0.1% formic acid (buffer b). the related liquid phase gradient was as follows: 0-40 min with 5% to 35% buffer b; 40-45 min with 35%-80% buffer b; and 45-50 min with 80% buffer b. peptides eluted by hplc were directly injected into a q-exactive mass spectrometer (thermo fisher scientific). data were acquired in the positive ion mode with a selected mass range of 300-1800 mass/charge (m/z). q-exactive survey scans were obtained at 70,000 (m/z 200) and 17,500 (m/z 200), with the resolution for higher-energy collisional dissociation (hcd) spectra and maximum ion injection times fixed at 20 ms and 60 ms, respectively. dynamic exclusion (40.0 s duration) was used. ms/ms data were collected using the top 10 most abundant precursor ions. the normalized collision energy was 30 ev, and the underfill ratio defined as 0.1%. the instrument was operated with peptide recognition mode enabled. protein identification was performed using the mascot 2.3.02 search engine (matrix science, london, uk). according to the relative abundance of different itraq tags, the peptides derived from different groups were quantified by the scaffold software, and represent the ratio of one group to another. the relative quantification of the protein is calculated by using the relative quantification of the peptide, which is expressed as the average ratio. to determine the deps in the adrenal gland between the hs and the control groups, the average ratio of identified proteins was calculated by proteinpilot based on the weighted average log ratios of the peptides. the deps were further analyzed for significant down-or upregulation, which was not determined by the size of the ratio but was calculated by software perseus. a cutoff level of significance of 5% (or p < 0.05) was chosen as a criterion. gi numbers of all significantly regulated proteins and some unaltered proteins were imported into the ingenuity pathway analysis software (ipa, www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as gene ontology, uniport and trembl. the canonical pathways and proteins interaction network of the deps were analyzed using the ipa. statistical analysis was performed using spss statistics 22.0. differences analysis in physiology indices between the ha group and the ca group were performed using a t-test, and p < 0.05 was taken to indicate statistical signifcance. we measured the body temperature, heart rate and respiratory rate of pigs at days 1, 3 and 6. it was found that body temperature and heart rate were increased significantly in heat-stressed pigs at days 1 and 6. (p < 0.05), however, the difference between body temperature and heart rate was not significant at 3 days. compared to controls, the respiratory rate, too increased substantially in pigs under heat stress at days 1, 3 and 6 (p < 0.01) (see table 1 ). the sample size used for this was 3 pigs each in control and hs groups respectively. we used the itraq method to identify proteins differentially expressed in the adrenal gland between the heat stress and control groups. we identified 226 differentially expressed proteins (deps), of which 99 were up-regulated and 127 were down-regulated in the ha group vs. the ca group. table 2 provides information about 18 key deps, 9 upregulated and 9 downregulated (see table 2 ). all the deps have been provided in the supplementary table 1. all protein and peptide identifications were obtained by database searching and stringent data filtering. the lc-ms/ms analysis produced 21,879 spectra, corresponding to 1202 unique peptides; 415 the effect of heat stress on the body temperature, heart rate and respiratory rate at days 1, 3, and 6. we found that heat stress increased the body temperature (bt), heart rate (hr) and respiratory rate (rr) of pigs significantly. the sample size used for this was 3 pigs each in control and hs groups respectively. the asterisk '*' and double-asterisk '**' denote a significant difference between stressed pigs and control pigs on the same day (p < 0.05 and 0.01, respectively). the units of the above indices are centigrade (°c), beats per minute and breaths per minute, respectively. proteins were identified at a false discovery rate (fdr) of ≤0.01 (fig. 1a) . the molecular weights of the identified deps ranged from 0 to 20 kd (n = 39), 20-40kd (n = 57), 40-60kd (n = 48), 60-80kd (n = 38), 80-100kd (n = 14), or > 100 kd (n = 30) (fig. 1b) . in addition, the identified deps had high peptide coverage; 85% of deps had > 10% sequence coverage, and 52% of deps had > 20% sequence coverage (fig. 1c) . 79.65% of the identified deps were represented by three or more peptides (fig. 1d) . to gain functional insights into the cellular proteome, gene ontology annotation was used to determine the subcellular localization of the 226 identified deps. the 226 deps identified from adrenal gland tissue altered by heat stress localized to various subcellular regions: high density lipoprotein particles (1.9%), extracellular area (15.0%), endoplasmic reticulum (7.9%), protein-lipid complexes (2.3%), cytoplasmic lipoprotein particles (2.3%), triglyceride-rich lipoprotein particles (1.9%), serosa (2.8%), platelet alpha particles (2.8%), endoplasmic reticulum inherent (1.9%), extracellular matrix (4.2%), chromatin (2.3%), pigment granules (5.6%), extracellular space (2.3%), serosa integrity (2.3%), neuronal processes (10.7%), secretory granules (5.1%), cytoplasmic vesicles (15.4%), and cytoskeleton (13.3%). (fig. 2) . gene identifications of the identified deps (supplementary table 1 ) were converted to human geninfo identifier (gi) numbers, since the pig genome database has poor annotations compared to the human genome, and because many proteins were unassigned or uncharacterized. to better understand these 226 deps, we used ingenuity pathways analysis (ipa) tool to examine canonical pathways; the top 20 enriched pathways are shown (fig. 3) , including pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. the deps identified in adrenal gland tissue during heat stress by itraq were clustered according to different functions. four functional groups were found: diseases and disorders, molecular and cellular functions, physiological system development, and toxicity functions were significantly enriched (p ≤ 0.05; fig. 4 ). the 226 deps from adrenal gland under heat stress, which correspond to 24 diseases and disorders (fig. 4a) , included proteins that are related to neurological disease, psychological disease, metabolic disease, skeletal and muscular disorders, hereditary disorders, hematological disease, immunological disease, inflammatory disease, inflammatory response, respiratory disease, dermatological disease and conditions, connective tissue disorders, infectious disease, cardiovascular disease, cancer, and endocrine system disorders. these 226 deps were assigned to 25 molecular and cellular function groups (fig. 4b) , including cell death and survival, molecular transport, cellular growth and proliferation, cellular assembly and organization, cellular function and maintenance, cellular movement, lipid metabolism, small molecule biochemistry, free radical scavenging, protein degradation, protein synthesis, and cell morphology. these deps were also enriched in 18 physiological system developmental functions groups (fig. 4c) , including tissue development, nervous system development and function, organ morphology, organismal development, embryonic development, hematological system development and function, immune cell trafficking, and tissue morphology. finally, these deps were enriched in 9 toxicity function groups (fig. 4d) , including renal necrosis/cell death, liver hyperplasia/ hyperproliferation, kidney failure, cardiac inflammation, and heart failure. proteins that changed significantly in the adrenal gland under heat stress were mapped to 13 functional networks (fig. 5) . the main networks of interest correspond to (1) cell assembly and tissue, cellular function and maintenance (fig. 5a); (2) cell migration, intercellular signal interactions (fig. 5b) small molecule biochemistry (fig. 5d ). proteins that are present in these pathways and identified as up-regulated deps in our analysis are depicted in shades of red; those that were identified as down-regulated deps are shown in green. proteins in the network but not identified as deps in our study are depicted in white. we also predicted the upstream regulators of adrenal deps by ipa analysis and found that cytokines, kinases, chemical agents, chemical kinase activators, mature micrornas, and growth factor were activators of these deps, as an example, chemokine(c-c motif)ligand 5, curcumin and brain derived neurotrophic factor while cytokines, mature microrna, auxins, transcription regulators, and chemicals were inhibitors of these deps, such as, il-6, ccaat enhancer-binding protein β and dexamethasone etc. these predicted upstream regulators of deps responsive to heat stress may have an important role in regulating hormone secretion and signal transduction in pigs. . the previous research of our laboratory found that: the analysis of plasma cortisol levels in bama miniature pigs revealed that the levels increased with the duration of heat stress. although there was no significant difference in the cortisol levels of control and heat-stressed pigs on the first day. however, at subsequent time points, cortisol levels were significantly higher in heat-stressed pigs compared with those in the control animals on the 7th day (ju et al., 2014) . in the present study, we used a method combining proteomics and bioinformatics to identify proteins that are differentially expressed in the adrenal gland of pigs under heat stress, and to elucidate molecular pathways and cellular functions that might mediate the heat stress response through these deps. we identified a total of 226 deps in the pig adrenal gland under heat stress conditions. ipa analysis software was used to analyze the cell localization, molecular function, signal pathway, regulatory network, and upstream regulators of these deps, which laid the foundation to elucidate mechanism of heat stress and stress-induced immunosuppression. the function of tubulin is mainly to interact with microtubule-associated proteins and related proteins that activate microtubule structures and maintain microtubule polymerization and depolymerization, modulate cell morphology, and are involved in cell division, cell movement, and transport of intracellular substances. cytoskeletal changes are associated with trans-cellular membrane trafficking. together, β-tubulin and α-tubulin (table 2 ) participate in the formation of microtubules, the integrity of which is essential for the segregation of chromosomes during cell division, the maintenance of cell shape, and the intracellular trafficking of macromolecules and organelles. changes in β-tubulin and vimentin levels have been detected in sars-cov (jiang et al., 2005) and infectious bursa disease virus (ibdv) (zheng et al., 2008a) . β-tubulin was one of the deps identified in this study, and its expression was approximately 2.2-fold up-regulated in adrenal tissue under heat stress, suggesting that differentially expressed cytoskeletal proteins could promote stress response in the adrenal gland. further large-scale studies are required to understand the roles and interrelation of β-tubulin and vimentin in porcine heat stress response. the heat shock protein (hsp) response is a highly conserved cellular response to external stress in all species. hsps take part in antigen presentation, intracellular trafficking, and apoptosis, and acting as molecular chaperones by assisting nascent polypeptides in assuming their proper conformations (khar et al., 2001) . several hsps were identified as down-regulated deps in this study, including hsp27 and hsp60. several members of the hsp family are expressed on the surfaces of cells; these can stimulate immune effector cells directly or can play crucial roles in antigen cross-priming. in this situation, hsps act as shuttle molecules for exogenous antigens and can directly stimulate t cells by prompting apc cytokine secretion (kotsiopriftis et al., 2005) . hsps appear to play a part of the innate immune response since the emergence of phagocytes in early multicellular organisms, and were commandeered for adaptive immune responses with the appearance of immune specificity (srivastava, 2002) . hsp70 is an endogenous ligand for the toll-like receptors (tlrs) that bind to microorganism-and tumor-specific antigens, then combine with major histocompatibility complexes (mhc) i and ii, which activate tumor-specific pathogens and t cells (han et al., 2009 ). mortaz found that high temperature induced mouse bone marrow mast cells to release hsp70, and the secretion of hsp70 in turn activated the toll-like receptor 4(tlr4) pathway (mortaz et al., 2006) . the expression of hsp70 is enhanced under stress conditions, and is generally considered to act through its role as a molecular chaperone, but recent reports indicate that hsp70 also modulates inflammatory responses by inhibiting activation of the inflammatory transcription factor, nuclear factor-kappa b (zheng et al., 2008b) . in addition, hsp70 may directly interfere with cell death pathways, such as those involved in apoptosis and necrosis (yenari et al., 2005) . in this study, hsp70 was up regulated(1.52 fold reduction) in porcine adrenal gland tissue under heat stress, further indicating the role of hsp70 in adrenal gland injury and highlighting its relevance to inflammatory responses. hsp60 has direct modulatory effects on inflammatory cells, and can activate monocytes and granulocytes to produce inflammatory cytokines, tumor necrosis factor-α (tnf-α), il-12 and il-6 (wells and malkovsky, 2000) . hsp60 can also specifically bind tlrs, especially tlr4, which is involved in human and rat atherosclerotic lesions (grundtman et al., 2011) . hsp27 belongs to a family of small heat shock proteins, can affect protein assembly, and may also participate in protein degradation. this conclusion follows directly from data suggesting that heat stress reduces the expression of heat shock proteins, handicapping their ability to induce a protective immune response when immunocytes are confronted with foreign entities. antigen presentation by hsps activates the innate and adaptive immune systems to initiate an acute response to stress factors, and suppression of hsps obstructs this response. these properties allow hsps to be used in immunotherapy in novel ways, which could lead to a greater understanding of how heat stress modulate the immune response, and why heat stress induces immunosuppression in pigs afflicted by post weaning multi-system wasting syndrome (pmws). histone h2a was significantly down-regulated in porcine adrenal gland under heat stress. ipa analysis shows that histone h2a plays a fig. 5 . ingenuity pathway analysis of proteins significantly altered in heat stressed pigs. red, up-regulated proteins; green, down-regulated proteins significantly; white, proteins known to be in the network but were not identified in our study. the colour depth shows the magnitude of the change in protein expression level. the shapes are suggestive of the molecular class(i.e., protein family). lines connecting the molecules indicate the relationship between molecules. dashed lines demonstrate indirect interactions, and solid lines demonstrate direct interactions. the arrow styles demonstrate specific molecular relationships and the directionality of the interaction. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) role in cancer, infectious diseases, reproductive system diseases, liver diseases, and immune diseases. however, in a bovine proteome study, histone h2a was considered a new natural immune molecule, and was down-regulated in neutrophils of immunosuppressed dairy cows. the immunological function of neutrophils releases a series of dna, histones, and antimicrobial peptides, forming a microbial kill 'trap' (lippolis and reinhardt, 2008; john and lippolis, 2005; kimura et al., 2006) . histone h2a belongs to a group of conserved eukaryotic cationic proteins that are involved in antibacterial activity, and function in dna folding. further studies are needed to determine if down-regulation of h2a expression in the adrenal gland under heat stress conditions is related to immunosuppression. annexin (anxa) is a calcium-dependent phospholipid-binding protein widely found in eukaryotes. phosphatidylserine (ps) is transferred from the inside of cell membrane to the outside after cell apoptosis. in the presence of calcium ions, anxa5 has a high affinity for ps, and can specifically bind to ps exposed on cell membrane. thus, anxa5 specifically recognizes apoptotic cells, and acts as a novel molecular probe to detect apoptosis. anxa5 is one of the most widely distributed and abundant members of the anxa family, and is involved in the anti-coagulation activity, calcium channel activity, protein kinase inhibitory activity, and many other important physiological processes. anxa5 is also closely related to inflammatory responses and cell stress (gauer et al., 2013; lokman et al., 2011) . anxa1, 2, and 11, were down-regulated deps identified in our study. these proteins participate in cell function and maintenance, intercellular communication and interaction, cell survival and death, cell migration, neurological diseases, and other processes. whether these down-regulated proteins are involved in the regulation of heat stress-induced apoptosis is still unclear (gu et al., 2014) , and must be explored in further studies. cui performed similar studies on heat stressed pig livers and found that chronic heat stress caused a reduction in the weight of the liver. they also found 45 proteins in the liver that were differentially abundant between heatstressed pigs and control pigs. proteins found were responsible for heat shock and oxidative stress response, cellular apoptosis, metabolism, signal transduction, cytoskeleton and immune system responses. their research found that heat caused an innate immune response whereas the voluntary diet restriction resulted in stress and cellular apoptosis (cui et al., 2016) . in summary, in this study we identified deps in the porcine adrenal gland under heat stress were characterized, and we constructed a comprehensive network of protein regulation in the porcine adrenal gland in response to heat stress. although many significantly differentially regulated proteins and pathways are closely related to the symptoms or pathological responses of heat stress, further functional investigations ought to be carried out to facilitate our understanding of the pathogenic mechanisms and molecular responses of pigs to heat stress. further work may identify novel therapeutic targets or lead to the development of new methods of preventing heat stress. in conclusion, we identified 226 deps, of which 99 were up-regulated and 127 were down-regulated, from pig adrenal gland tissue under heat stress. among these 226 deps, tubulin and heat shock protein (hsp) such as hsp70, hsp60, hsp27, and histone h2a might be involved in the regulation of heat stress. ingenuity pathways analysis (ipa) tool examined canonical pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. our expression profiles provide new insights into the key proteins involved in heat stress in the pig. supplementary data to this article can be found online at https:// doi.org/10.1016/j.rvsc.2018.11.004. in vitro generation of interleukin 10-producing regulatory cd4+ t cells is induced by immunosuppressive drugs and inhibited by t helper type 1 (th1)-and th2-inducing cytokines altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning feeding growing-finishing pigs to maximize lean growth rate quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to salmonella typhimurium effects of long term heat stress in utero or during finishing on pork carcass composition chronic heat stress induces immune response, oxidative stress response, and apoptosis of finishing pig liver: a proteomic approach sow mortality associated with high ambient temperatures membrane modulates affinity for calcium ion to create an apparent cooperative binding response by annexin a5 heat shock protein 60 and immune inflammatory responses in atherosclerosis heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic-pituitary-adrenal revolving axis soluble b and t lymphocyte attenuator possesses antitumor effects and facilitates heat shock protein 70 vaccine-triggered antitumor immunity against a murine tc-1 cervical cancer model in vivo itraqbased quantitative analysis of hippocampal postsynaptic density-associated proteins in a rat chronic mild stress model of depression proteomic analysis of porcine mesenchymal stem cells derived from bone marrow and umbilical cord: implication of the proteins involved in the higher migration capability of bone marrow mesenchymal stem cells quantitative analysis of severe acute respiratory syndrome 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cancer progression twodimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (itraq) labeling approac function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals acetylsalicylic acid-induced release of hsp70 from mast cells results in cell activation through tlr pathway the effects of heat stress and plane of nutrition on metabolism in growing pigs interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses economic losses from heat stress by us livestock industries 1 expression of the il-10 receptor on human monocytes is moderately suppressed by glucocorticoids heat shock proteins, tumor immunogenicity and antigen presentation: an integrated view glucocorticoids and immune function: unknown dimensions and new frontiers universal sample preparation method for proteome analysis antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection proteomics analysis of host cells infected with infectious bursal disease virus anti-inflammatory effects of the 70 kda heat shock protein in experimental stroke not applicable. the authors of this study have no conflicts of interest. jl conceived the study designed the experiments, prepared the samples for mass spectrometry, analyzed the mass spectrometry data, conducted the experimental work created the figures and wrote the manuscript. yy participated in the enrichment analysis, created the pathway figure, aided in revising the manuscript. dg, ls and pt analyzed the mass spectrometry data, participated in the enrichment analysis, participated in writing and revision of the manuscript. rg obtained and analyzed flow cytometry data and aided revising the manuscript. xwang was responsible for animal care xj provided the financial support, and designed the experiments and revised the manuscript. all authors have confirmed the final version of the manuscript. key: cord-340128-qxkopvot authors: schreibauer, elena christina; hippler, melina; burgess, stephanie; rieger, monika a.; rind, esther title: work-related psychosocial stress in small and medium-sized enterprises: an integrative review date: 2020-10-13 journal: int j environ res public health doi: 10.3390/ijerph17207446 sha: doc_id: 340128 cord_uid: qxkopvot background: work-related psychosocial stress can cause mental and physical illnesses resulting in high costs for the individual, the economy and society. small and medium-sized enterprises (smes) employ the majority of the world’s workforce and often have fewer financial and human resources compared to larger businesses. the aim of this review is to summarize current knowledge on work-related stress in smes according to well-established guidelines categorizing psychosocial factors at work. methods: a systematic database search was carried out in pubmed, psycinfo, psyndex and business source premiere from march to june 2019, updated in january 2020. data of included studies were analyzed and mapped into five themes: “work content and task”, “organization of work”, “social relations”, “working environment” and “new forms of work”. results: after full-text screening, 45 out of 116 studies were included for data extraction. studies were very heterogeneous and of varying quality, mostly applying a cross-sectional study design. psychosocial factors in smes have been researched with a focus on the work patterns “work organization” and “work content and task”. conclusions: this review underlines the need for more and better quality research of psychosocial factors in smes, particularly in relation to ongoing and new challenges in the workplace, including stressors related to the process of digitalization or the development of safe working conditions during the emerge of new infectious diseases. according to the international labour organization (ilo), 2.8 million workers die from work-related diseases each year and there are about 374 million non-fatal work-related injuries annually [1] . this results in human and economic costs of about 4% of the annual global gross domestic product [1] . additionally, work-related psychosocial risks, comprising issues such as work-related stress, have been identified as significant risks in the field of occupational health and safety over the last two decades [2, 3] . however, as of yet only a few countries have drafted specific regulations on psychosocial risks (e.g., the 2014 belgian royal decree on the prevention of psychosocial risks at work or the colombian resolution 2646 on risk assessment and management of psychosocial hazards from 2008) or have implemented existing policies in their national occupational safety and health legislation [4, 5] . furthermore, there is little evidence whether and how these recommendations and policies can be implemented in a working environment with limited human and financial in order to support enterprises with the development of instruments and interventions to reduce psychosocial risks, knowledge on relevant work-related psychosocial factors is essential. between 2014-2017, the german federal institute for occupational safety and health (baua) conducted several literature reviews to describe the scientific evidence regarding mental health in the working world [35] . based on well-established theoretical models including the job-demand-control-model [36] , the effort-reward-imbalance-model (eri) [37] or the organizational justice (oj) model [38] , a framework was developed to categorize the results into four subject areas: "working task", "leadership and organization", "working time" and "technical factors". the reviews did, however, not specifically focus on the situation of employers and employees working in smes. furthermore, the joint german occupational safety and health strategy (gda) published recommendations for implementing psychosocial risk assessments listing five work patterns (i.e., work characteristics) that have been identified as primary stress factors in the workplace [39] : • work content and task (e.g., job autonomy, completeness of tasks, emotional demands); • organization of work (e.g., working time and processes); • social relations (e.g., aspects of hierarchy, leadership and managerial abilities); • working environment (e.g., physical factors, working equipment); • new forms of work (e.g., spatial and temporal mobility). to organize the results of this review, we used this classification because it comprises all of the psychosocial factors published by the baua [35] and integrates factors published by other well-established international frameworks (eu-osha (the european agency for safety and health at work) [12] , who (world health organization) [40] and ilo [5] ). furthermore, the gda-classification includes a systematic table and detailed examples of work-related psychosocial demands which provided a useful framework for a clear assignment of the studies identified in this review. to the best of our knowledge, this review is the first to summarize and categorize the current evidence on work-related psychosocial demands with a specific focus on small and medium sized enterprises to identify gaps in current knowledge and provide a systematic overview of which psychosocial factors, outcomes and economic sectors have been considered to date. the subsequent research questions guided our review: (1) what is the current state of knowledge on psychosocial demands in smes? (2) which outcomes and economic sectors have been examined? we conducted an integrative review according to the five-stage method described by whittemore et al. [41] . to select relevant studies, we used the peo criteria (population, exposure, outcome) which are applied frequently in evidence-based health research [42, 43] . for our study, we defined the criteria as follows: p = workforce in smes (with smes defined according to the eu definition: number of employees < 250 [44] ) and e = psychosocial demands as defined by the gda [39] . we did not predefine any outcomes for the literature search because we did not want to limit our review to certain health or health-related outcomes. the literature search was carried out between march and june 2019 and updated in january 2020 with individually adapted search strings in medical (pubmed), psychological (psycinfo, psyndex) and economic (business source premiere) databases. additionally, we performed a hand search of the reference lists of the studies included. the search terms followed this general scheme: terms related to "workforce in smes" and terms related to "psychosocial demands". we also considered relevant medical subject headings (mesh)-terms and search terms previously published [45] . terms including "family business(es)" were excluded because this resulted in a considerable number of articles not relevant in our context. for example, many articles dealt with the financing of micro-enterprises or the family income situation in developing countries. the complete search strategy for pubmed and the applied search string is presented in supplementary table s1 . we included all types of peer-reviewed quantitative and qualitative studies as well as literature reviews relevant to the sme setting considering at least one of the psychosocial demands listed by the gda [39] . articles that did not report employment figures, but whose surveyed enterprises were designated as smes, were included if their sme definition complied with the european definition [44] . if multiple publications were based on the same dataset, all papers meeting the inclusion criteria were selected. we limited our search to articles published in german or english from january 2000 onwards, the beginning of the fourth industrial revolution promoting digital processes in the working environment [46, 47] introducing new stressors to the workforce such as digitized performance monitoring or information overload [47, 48] . furthermore, we excluded study protocols as well as publications which tested and/or validated questionnaires on psychological demands or work-related stress. we also excluded reviews because we considered the original research and did not consider any kind of "grey literature". after the removal of duplicates, all studies were transferred to rayyan [49] , a free web application for systematic review screening. two raters (e.c.s. and s.b.) screened titles and abstracts independently, according to the predefined inclusion and exclusion criteria. articles that could not be judged by title and abstract were included in the full text screening, also independently executed by two raters (e.c.s. and m.h.). lack of agreement was solved in consensus discussions with a third reviewer (e.r.). a tabular scheme for data extraction (author, year, country, year of data collection, topic, study design, data collection methods, type of enterprise, sample size, industrial classification of the business, investigated psychosocial demands, outcomes, and data collection instruments) was compiled and used for data extraction. we used the agency for healthcare research and quality ahrq study design algorithms [50] to classify the study design if it was not reported. the allocation of the studies to economic sectors was carried out according to the international standard industrial classification of all economic activities (isic) [51] . we amended the classification of the baua-project [35] to categorize the outcome variables of the studies into these subcategories: general (work-related) stress outcomes, health, well-being, factors affecting cardiovascular health, mental health, musculoskeletal system, social relations, and business-related outcomes. different tools were used for critical appraisal: the special unit for review evidence (sure) checklist for cross-sectional studies [52] the sure-checklist of randomized controlled trials and other experimental studies [53] , the sure-checklist for qualitative studies [54] , and the joanna briggs insitute (jbi)-critical appraisal tool [55] for text and opinion articles to assess the quality of narrative reviews. we identified 3658 studies through electronic data base searching (see prisma-flowchart [56] figure 1 ). after the removal of duplicates, 3460 studies remained for title and abstract screening, including eleven articles detected by hand search. for the full-text-screening, 116 articles were eligible, of which 45 were included in the full text analysis (figure 1 ). the first article suitable for this review was published in 2004. since then, the annual number of publications was relatively low (<5/year) with a peak in 2018, when nine articles were published. due to our main database search in the first half of 2019 (hand search update march 2020), it can be assumed that studies published in 2019 were not completely indexed at that time. the first half of 2019 (hand search update march 2020), it can be assumed that studies published in 2019 were not completely indexed at that time. the samples of the studies were very heterogeneous. the number of participants varied between seven and 23,000. five studies included only sme-managers and enterprise owners [57] [58] [59] [60] [61] . some studies used data from nationwide surveys that provided a large sample size and included a range of economic sectors and branches [29, 30, [62] [63] [64] [65] [66] [67] . other studies included only participants of a single enterprise [68] [69] [70] [71] . a complete summary of the study characteristics can be found in supplementary table s2 . most of the studies included were carried out in europe (n = 24) and in asian countries (n = 13). other studies were conducted in australia [57, 72] , the united states [73] and new zealand [74] . four studies from japan [62, [64] [65] [66] seemed to use data from the same survey but examined different outcomes. no studies from africa or south america met our inclusion criteria, and four studies were transnational [61, [75] [76] [77] . the majority of studies were cross-sectional (n = 37), analyzing a variety of psychosocial factors in the sme-setting, mainly using data from nationwide surveys or specifically designed questionnaires for particular companies or settings. we also identified qualitative studies (n = 2), narrative reviews (n = 3), and intervention studies (n = 3). one qualitative study [69] looked at the samples of the studies were very heterogeneous. the number of participants varied between seven and 23,000. five studies included only sme-managers and enterprise owners [57] [58] [59] [60] [61] . some studies used data from nationwide surveys that provided a large sample size and included a range of economic sectors and branches [29, 30, [62] [63] [64] [65] [66] [67] . other studies included only participants of a single enterprise [68] [69] [70] [71] . a complete summary of the study characteristics can be found in supplementary table s2 . most of the studies included were carried out in europe (n = 24) and in asian countries (n = 13). other studies were conducted in australia [57, 72] , the united states [73] and new zealand [74] . four studies from japan [62, [64] [65] [66] seemed to use data from the same survey but examined different outcomes. no studies from africa or south america met our inclusion criteria, and four studies were transnational [61, [75] [76] [77] . the majority of studies were cross-sectional (n = 37), analyzing a variety of psychosocial factors in the sme-setting, mainly using data from nationwide surveys or specifically designed questionnaires for particular companies or settings. we also identified qualitative studies (n = 2), narrative reviews (n = 3), and intervention studies (n = 3). one qualitative study [69] looked at psychosocial resources instead of focusing on psychosocial risks like most other studies. however, as a single-case study, there are issues with the transferability of the results. the other qualitative study [78] aimed to identify challenges faced by novice community pharmacists at transition to independent practitioners. the three narrative reviews [79] [80] [81] focused on changing work characteristics in smes during the first years of the 21th century. cooper [80] analyzed the changing world of work and its impact on employees and their families in that period. he focused on new forms of work, affecting most of all employees in smes, as well as self-employed and workers outsourced in virtual organizations. allan et al. [79] investigated challenges of online learning in smes. yuhshy [81] discussed work-and family-related stress as significant problems of smes. we identified only three intervention studies [71, 73, 82] : casteel et al. [73] evaluated an intervention to reduce violent crimes in smes. magnavita [71] reported an intervention to reduce psychosocial risks at the workplace in smes. he described a cost-effective participatory model and emphasized the usefulness of regular health examinations as they can be used to identify problems in the workplace climate and work organization. in a controlled intervention study, torp [82] reported results of a 2-year training program for managers of small and medium-sized enterprises. he examined the program's impact on the companies' health and safety management systems and on the mental and physical health of employees. applying the sure-criteria [53] , the internal validity of these studies was rated as medium quality, because of some methodological limitations and limited representativeness. furthermore, we identified four studies investigating the differences of job stress in smes compared to large firms [30, 31, 63, 67] . whereas tsai et al. [31] found higher levels of job stress and "higher favorable attitudes toward managers" in smes, lai et al. [63] obtained no firm size effect on overall job stress after adjusting for covariates including individual and organizational characteristics (information on job tenure, contractual status, gender, age, marital status, number of children, caring responsibility, long-term illness, academic qualification, weekly pay, work condition changes, organizational support) in an european (u.k.) sample. yeh et al. [67] reported higher job demands, higher job insecurity, lower job autonomy and lower career prospect in smes, compared to those of large private enterprises and the public sector. encrenaz et al. [30] investigated the influence of the size of enterprises on mental health and the mediating role of perceived working conditions in this relationship by measuring the outcome "anxious/depressive episodes" in employees. between micro-enterprises compared to the others, there were differences in perceived working conditions: taking working conditions into account, the risk of "depressive/anxious episodes" was larger in micro-enterprises. as summarized in table 1 , most studies investigated several work characteristics with a focus on factors related to 'work content and task' (n = 33) and 'organization of work' (n = 31), followed by factors related to 'social relations' (n = 30), 'new forms of work' (n = 8) and the 'working environment' (n = 7). only one study considered all five work characteristics to measure work quality in smes [29] . a relatively high number of studies assessing the work characteristics 'work content and task' included the factors 'freedom of action' (n = 15), 'responsibility' (n = 10) and 'emotional demands' (n = 9). multiple studies also investigated factors related to 'organization of work', mostly looking at 'work time' (n = 16), 'work process' (n = 20), and 'communication/cooperation' (n = 13). however, the correlations of these factors with the outcomes were not always reported. many studies also investigated the work characteristics 'social relations'. for example, berthelsen et al. [85] measured perceived social support in the workplace among danish dentists, and agervold et al. [68] measured social contact, social climate and management style in a danish manufacturing company. intragroup conflict and social support were assessed by three japanese studies [62, 65, 66] . the work characteristics 'social relations' in general were measured by four studies [57, 60, 70, 82] and one study referred to siegrist's effort-reward-imbalance model (eri) [71] , in which the eri questionnaire was used to asses occupational stress with items on esteem and job promotion. the work characteristics 'working environment' and 'new forms of work' have received less attention. only three studies measured physiochemical factors: 'lighting' was measured by two studies [29, 75] , 'climate' was measured only by isahak et al. [75] . rhee et al. [76] assessed the variables "hazardous work condition" and "handling of hazardous materials". three studies investigated physical factors: "physical occupational activity" [84] , "poor physical working conditions" [90] and "heavy physical work, repetitive activities, forced postures and risks of falls" [75] . the factor 'workplace and information structure' was only studied by díaz-chao et al. [29] ("workspace"). the factor 'work equipment' was taken into account by sonnentag et al. [89] looking at "situational constraints associated with malfunctioning, missing, incomplete, or outdated equipment, tools, or information". myers et al. [83] investigated "staff and technical problems", including 'equipment breakdown and defective materials'. the characteristics 'new forms of work' was taken into account by eight studies. job insecurity was measured by five studies [29, 63, 67, 75, 91] . flexible work hours and work-life-balance was taken into account by three studies [29, 59, 92 ]. cooper's [80] theoretical exploration focused on several aspects of new forms of work, including free-lancers, flexible working hours, and work-family-conflicts related to new forms of work. there was only one study [98] focusing on accessibility and expected availability of workers due to new communication methods. another study investigated the work-home interference and well-being of self-employed entrepreneurs [59] . mental health risks and resources were the most commonly studied outcomes. as shown in table 2 , many studies examined mental health outcomes (n = 19), with general (work-related) stress outcomes being the second most studied (n = 15), followed by outcomes representing business-related outcomes. coping self-efficacy [77, 95] seven-item coping self-efficacy scale (cse-7) depressive symptoms [62] japanese version of the center for epidemiologic studies depressive symptoms scale (ces-d) depressive episodes [30] hospital anxiety and depression scale (hads-d) training needs [90] unspecified questionnaire (probably self-developed) importance of work [59] mow's question on the centrality of work general stress and mental health outcomes were assessed more often as risks, business outcomes were assessed almost equally as risks and resources and social relations were examined more often as resources. the only non-self-reported outcomes were blood pressure and the observed rate of violent crimes; all other outcomes were assessed as self-reported outcomes, mostly via questionnaires. several studies (n = 17) examined manufacturing enterprises (please see supplementary table s3 ). each of the other economic sectors was considered by less than six studies. eight studies [30, 57, 59, 63, 67, 92, 93, 98] included several sectors; for example; lai et al. [63] used data of a nationwide survey with participants of all industrial sectors, godin et al. [59] and cocker et al. [57] examined entrepreneurs of several sectors. seven studies [58, [79] [80] [81] 86, 88, 90] did not provide information on particular economic sectors. in this review, we evaluated the current evidence on work-related psychological stress in smes, also summarizing the type of outcomes investigated as well as the economic sectors considered. the majority of the 45 studies were cross-sectional (n = 37). we also identified qualitative studies (n = 2), narrative reviews (n = 3), and intervention studies (n = 3). as most studies applied a cross-sectional design investigating relationships between various outcomes and psychosocial factors in the workplace, the number of studies investigating causal relationships was relatively low. only one study conducted an intervention specifically developed for the sme setting [71] indicating a lack of studies applying a high quality research design (e.g., randomized controlled trials) with a focus on psychosocial stress in the smes. all studies were published from 2004 to 2019 considering very heterogeneous populations of smes. we did not find suitable studies from 2000 to 2004. as the definition of smes was inconsistent at the beginning of the century, articles from this period may not have met the inclusion criteria of the european definition of smes. since smaller enterprises have become a focus for researchers and policy makers in recent years, this may have resulted in an increased number of publications from 2017 onwards. only one study [29] investigated all five dimensions of work-related psychosocial demands as defined in the gda recommendations [39] . although these recommendations are based on the european council directive [21] and have great similarities with other international classifications (e.g., [12] , p. 14), we appreciate that the gda recommendations have been developed with a focus on the german context and may not be transferable entirely to other settings. nevertheless, since the job-demand-control model [36, 99] and the extended job-demand-control-support-(jdcs-) model [100] have been used for decades, work characteristics based on these models, have found their way into many study-designs and work-stress questionnaires. this resulted in a frequent examination of the work pattern 'work content and task' although some of the subcategories were hardly considered ('variability) or not mentioned at all ('completeness of task'). the work patterns 'organization of work' and 'social relations' have also been considered frequently. although the psychosocial health effects of the working environment have been studied as a cause for work-related illness for decades e.g., [101] , this work characteristics including 'physicochemical factors' or 'physical factors' have received little attention from the psychosocial perspective in the studies we identified in this review. in terms of advancing digitalization, topics such as 'workplace and information structure' are important fields of research as they involve the risk of information and stimulus satiation [47] . their psychosocial impact on employees in small and medium-sized enterprises appears to have been poorly researched to date. this may be related to the slow pace of digitization in smes requiring financial and human resources, frequently exceeding the means of small enterprises [102] . the european agency for safety and health at work (eu-osha) recognized job insecurity, precarious work, intensification of work and higher requirements for flexibility and mobility from workers as emerging risk for health and safety [2] ; nevertheless, 'new forms of work' also have received little attention in the setting of smes [103] . we also classified the outcomes considered according as defined by the baua-project [35] including these subcategories: general (work-related) stress outcomes, health, well-being, factors affecting cardiovascular health, mental health, musculoskeletal system, social relations, and business-related outcomes. most outcomes were related to the fields of general stress, mental health and business. there appears to be a gap in the investigation of outcomes on physical health, particularly those representing cardiovascular health, even though the link between work-related stress and physical illness is well established [18, 19] . promoting and maintaining the health of employees is the fundamental purpose of occupational medicine. prevention is therefore particularly important. work-related resources were, however, less frequently studied than risks, and most outcomes considering resource were related to the fields of 'social relations' and 'business-related outcomes'. factors considering the prevention of work-related stress and the promotion of a healthy working environment should therefore be considered more frequently in sme-research. finally, we categorized the studies identified to the international standard industrial classification of all economic activities (isic) [51] . with few exceptions, the number of studies allocated to economic sectors corresponded to the frequency with which smes in europe and asian countries are represented in these sectors [27, 104] . in the eu-28, for example, employment was highest in smes active in the sectors 'construction' (13,9%) and 'wholesale and retail trade; repair of motor vehicles and motorcycles' (13,6%) between 2016 and 2018 [105] . although we identified a number of studies (n = 9) investigating psychosocial demands in these sectors, the highest number of studies looked at smes active in 'manufacturing' (n = 17) where the percentage of employment was considerably lower (6.7%). this may be related to differences in lobbying activities which have been shown to be relatively high in the manufacturing sector compared to other sectors including 'wholesale trade' [106] . the other economic sectors were partly more typical for large enterprises (e.g., electricity suppliers, oil companies, insurance companies) or public and civil-service institutions (hospitals, schools, public offices) and, as expected, fewer studies were identified here. within the sector 'human health and social work activities' four studies were identified looking at psychosocial factors in the setting of general dentists' practices, pharmacies and a welfare and assistance agency for professionals. although the setting of myers et al. [83] and magola [78] was not clearly in the sme area (myers et al. examined dental practices that were also partly funded by the british national health service (nhs), magola included pharmacists who also worked for larger chains), the studies were included in this review because they represented the sector of healthcare and the organizations under review (dental practices and pharmacies) which ultimately had the structure of an sme. we also expected family or medical practices to be characterized as small businesses, but no corresponding study was identified also indicating that these "enterprises" were unlikely to be categorized as a typical sme [107] . this review also has some limitations. our broad research objective was very useful to give an overview of the current state of research and to detect gaps in knowledge. however, it was not suitable for a "classic" systematic review design and resulted in a heterogeneous sample of included studies and a high number of irrelevant hits, particularly in the database "business source premiere" which identified numerous entries on "financial stress" not related to our research objective. the bias risk has been minimized by a strict application of review methods like systematic literature search and reviewing by independent reviewers. the broad question and the application of the peo scheme could also be the reason for finding only three intervention studies. it may be appropriate to search particularly for intervention studies in smes; however, a recent review from 2019 examined health interventions in smes and only one of the included studies met our inclusion criteria [82] . the reasons for this may be related to applying different sme definitions, the inclusion of public institutions or the exclusion of work-related psychological factors as most of the interventions considered in the review [108] dealt with physical fitness or work-safety-interventions. to provide a more manageable and specific set of results, we have refrained from using 'family business terms' in our search string. apart from using a european definition of smes, this could also be a reason why we did not identify studies from south america and africa. to study psychosocial factors in the family business setting, a new search with a more specific search term would be required. the same may be true for micro-enterprises which may not be covered by our search string in all databases. furthermore, the consideration of "grey literature" (e.g., governmental reports) may provide further evidence in this context. finally, we aimed to categorize all studies identified in this review according to well-established frameworks [35, 39, 50, 51] . nevertheless, the process of systematization was partly subjective since psychosocial factors are mostly interdependent or interrelated; hence, we could have also chosen different categorizations for some of the studies identified. psychosocial risks differ between large companies and smes [30, 31, 67] . since smes represent the majority of all companies worldwide, it is important to conduct research specifically focused on smaller enterprises, also including micro-enterprises. as early as 1997, cooper et al. [109] called for further studies to investigate the long-term effectiveness of stress intervention strategies. with the findings of this review we can renew this demand for the sme setting. in order to offer smes effective interventions for the primary prevention of psychosocial risks, the long-term effects of the interventions should be examined applying high-quality study designs. for the development of interventions, it would be desirable to measure all dimensions in which psychosocial risks may occur and resources can be established and consolidated, rather than limiting the assessment of psychosocial stress to single factors. hereby, the dimensions of psychosocial risks defined by the gda [39] proofed to be a good framework for the classification of the studies identified. furthermore, we would like to point out that about one third of the outcomes identified were measured using self-developed items, scales or questionnaire, or by using adapted preexisting questionnaires. on the one hand this complicates the comparison of results with prior research and may impact the validity and reliability of previously established measures. on the other hand, it may be necessary to develop new instruments suitable for a particular research question or setting. previous research has provided valuable context for the development of new research instruments, also emphasizing the necessity to carefully discuss the pro and cons of using preexisting or newly developed measures e.g., [110] . working conditions do not only influence the physical but increasingly impact the mental wellbeing of employees [111] . the process of transforming to industry 4.0 with the resulting digitization and emergence of new forms of work (e.g., platform work, remote work, freelancers, home office) has been researched for the last decade and provided valuable insight in central issues or smes adapting to the accelerating change of the working environment (e.g., lack of operational capacity for systematic reorganization) [102, 112] . moreover, the trajectory of climate change and the current covid-19 pandemic have accelerated these processes [113, 114] . particularly the necessity of infection control has resulted in an even greater necessity of work-related mobility and flexibility and is expected to impact the working environment in the long term. as a result, employers and employees are facing new psychosocial risks, e.g., social isolation, increasing technical and social challenges related to electronic communication [115] which has been related to the development of depression, anxiety, self-reported stress, and sleeping disorders [116] . especially in smes, where frequently fewer (financial) resources are available compared to larger companies, the redesign and adaption of a continuously changing working environment is particularly challenging. new evidence of the effectiveness of workplace enhancements in the sme setting could facilitate necessary changes. the results of this review highlight that the various psychosocial factors in smes have been researched with varying intensity. as chirico [103] pointed out in 2017, the new work-related risks have not received sufficient attention from the scientific community. this can be recognized here by a lack of studies for the work characteristics "new forms of work" and "working environment". within the context of the current covid-19 pandemic, the relevance of these aspects becomes even more evident. smes from the economic sectors "professional, scientific and technical activities" and "wholesale and retail trade, repair of motor vehicles and motorcycles" should also be subject to more research, as they appeared to be underrepresented. due to the lower financial and human resources available in smes and a lower awareness of the resulting costs of inadequate health and safety management [12] , research for cost-efficient and effective interventions to improve mental health in smes is of high relevance to 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in small-and medium-sized enterprises: a meta-analytic review an intervention strategy for workplace stress measurement instruments and data collection: a consideration of constructs and biases in ergonomics research institute of world health organization. health impact of psychosocial hazards at work: an overview; world health organization back to the future: policy pointers from platform work scenarios new forms of employment series, new forms of employment series; publications office of the european union living, working and covid-19 data european agency for safety and health at work. covid-19: back to the workplace the work is associated with, but not funded by a project funded by the federal ministry of education and research of the federal republic of germany (bmbf 01gl1751a improvejob). many thanks to the four reviewers for their constructive and valuable suggestions during the review process of this manuscript. many thanks to anke wagner for her advice regarding the development of the search strategy and quality assessment of the papers. we would also like to thank the members of the seminar "health services research" (coordination centre for health services research, university hospital tuebingen) for their constructive feedback during the development of the research questions and the search string, and benjamin lee for his linguistic advice on the manuscript. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-272406-h22atwd4 authors: diotaiuti, pierluigi; mancone, stefania; bellizzi, fernando; valente, giuseppe title: the principal at risk: stress and organizing mindfulness in the school context date: 2020-08-31 journal: int j environ res public health doi: 10.3390/ijerph17176318 sha: doc_id: 272406 cord_uid: h22atwd4 background: in recent years the role of school principals is becoming increasingly complex and responsible. methods: this study was voluntarily attended by 419 italian school principals who were administered the psychological stress measurement (msp), mindfulness organizing scale (mos), polychronic-monochronic tendency scale (pmts), and the scale of emotions at work (sew). results: the study has produced a path analysis model in which the relationships between the main predictors of principals’ work discomfort were explained. the effect of depressive anxiety on perceived discomfort (ß = 0.517) found a protective mediator in the mindfulness component that recognizes the sharing as a fundamental operational tool (ß = −0.206), while an increasing sense of effort and confusion could significantly amplify the experience of psychological discomfort associated with the exercise of school leadership (ß = 0.254). conclusions: the model developed in this study suggests that focusing on organizing mindfulness can be a valuable guideline for interventions. research on work-related stress has long identified the school as one of the risk environments, and various studies and publications deal with the stress and burnout of school workers. most of the work produced devotes attention to teachers, and there are few reflections on the psychophysical resistance of the principal, especially considering all the legislative and organizational changes that have taken place [1, 2] . these continuous changes, necessary in a rapidly changing society (well described by the "liquid society" metaphor coined by zygmunt bauman) become a cause of work-related stress for all school staff, especially principals. the ongoing reform of the education system in italy involves various professionals and individuals who have a role in the complex world of school: principal (dg-dirigente scolastico in italian); director of general and administrative services (dsga-it. direttore dei servizi generali e amministrativi), all administrative, technical and auxiliary staff (ata-it. personale amministrativo, tecnico e ausiliario), teachers, students and their parents (or parental figures). each of these players must adapt to regulatory changes, both individually and collectively, with time dictated by political choices, which can be both national and supranational; the educational system, through the schools operating on the territory and the people who effectively act and put into practice the regulatory frameworks decided by the government institutions, must follow and pursue the changes in a short time period, often far from the actual physiological timing that the institution can implement. the principal is an intermediate figure and a link between the institutional organizational framework, represented by the state, the regions, and the municipalities, and the framework of school individuals, represented by the teaching staff, ata staff, parents and students. many of the skills and int. j. environ. res. public health 2020, 17, 6318 2 of 14 competences required of professional staff, but also students, are not part of the training acquired over time and especially put into practice and enriched by personal experimentation and experience, so that even people with a ten-year work experience are forced to become new students learning subjects and skills hitherto untested, and therefore they are unaware of how gifted, capable and effective they are in resolving tasks [3, 4] . the role of principal requires new skills and competences to meet the requirements and objectives defined by the proliferation of school reforms, regulations, and cultural changes [3, 5, 6] . extensive research in various countries has already focused on principal burnout [7] [8] [9] [10] [11] [12] [13] [14] . as sari [8] notes, there may be curiosity and desire to learn and experience new skills and abilities, but the stress related to possible administrative errors and criminal consequences of errors or omissions, or low performance in failing to achieve results, remains high, with emotional drain and possible burnout consequences. moreover, implicitly, the principal may also experience the feeling that his own cultural and cognitive skills, as well as tools and resources, have become obsolete or are no longer suitable for current needs, thus increasing personal insecurity [15] . these elements are sources of elevated stress levels in principals, because they become the point of reference and the catalyst of the stress of all professional and non-professional figures. not only must the principal solve his/her own problems, but he/she must also deal with those of his/her subordinates, in most cases without having adequate leadership experience and training. some countries have experienced a lack of candidates for the role of principal or an increase in resignations, because the teachers know they are not actually capable of fulfilling all the requirements the role calls for [16] [17] [18] . the principal experiences a drastic reduction in his/her educational and pedagogical function and must acquire technical, administrative, cognitive and what we could call management engineering skills: as a principal he/she becomes responsible for the safety of staff and students and a manager of economic resources, with budgetary obligations. to carry out these tasks, he/she must have medical-legal and managerial skills which the teacher who is promoted to the role of principal does not possess. moreover, the change in commitments, as well as responsibilities, can be a source of great stress and a health risk, since the individual might not be able to manage, but above all to identify, the point where he must ask for help and activate a course of treatment. this applies both at the individual level and as the head of the safety and health of subordinate staff, having to decide when it is appropriate to subject a member of staff to a medical examination, especially when the latter does not want to or cannot deal with personal problems. the principal is not alone and employs ata and dsga personnel, but as the person responsible for their work, he/she still must have the ability to evaluate and correct any errors. the above concerns only the legal-administrative part and the school is viewed as a business that must be run efficiently [19] . in addition to this, however, there is the management of the team in the areas of human resources and coordination. principals can contribute to shaping the school climate as they should promote and support students and teachers [20] . this involves managing aspects related to students, social change and the management of a multi-ethnic society in continuous evolution, with changes in customs and processes [21, 22] . for example, the new migratory flows have led some educational institutions to deal with the task of managing adult "students", independent adults and adults with a "different" culture, who are normally obliged to attend school: however, this type of "student" requires cultural and relational skills and management skills that the school staff may not have. the aim of this research is to draw attention to the figure of the principal as a person at risk of burnout and work-related stress for both exploratory, diagnostic and preventive purposes. the principal has to focus on the limit between being engaged in school life and avoiding workaholism, in order to live in a state of health and well-being [23] . chronic stress stimulates negative behaviors and thoughts and problems related to emotions, feelings and physical health that can hinder effective school administration. according to scholars as langer [24] , davidson et al. [25] , vogus and sutcliffe [26] , weick and putnam [27] , and weick and sutcliffe [28] , many positive benefits can be associated with individual and organizing mindfulness, such as health improvement, stress reduction, increased 3 of 14 creativity, and less risk of burnout. more specifically hoy et al. [29] define "school mindfulness" as the extent to which teachers and administrators in a school carefully and regularly look for problems, prevent problems from becoming crises, are reluctant to oversimplify events, focus on teaching and learning, are resilient to problems, and defer to expertise. therefore, we also hypothesized in this work that components of the organizing mindfulness could have a significant role for the school principal in increasing or limiting his/her perceived work discomfort. (1) monitor the stress levels of a large sample of principals belonging to different levels of italian schools; (2) verify the incidence of specific pathologies associated with high levels of school stress; (3) evaluate the relationships between the perceived stress, work discomfort and dimensions of organizational mindfulness; (4) test the fit of a general path model illustrating the influence of the predictors on principals' work discomfort; and (5) identify the role of organizing mindfulness on principal's perceived work discomfort. the study was voluntarily attended by 419 school principals (131 males and 288 females) with an average age of 53.93 (sd = 6.46). they were invited to participate by means of an email indicating the purpose of the study, declaring the guarantee of anonymity and the use of the data collected for scientific purposes only, and therefore were requested to fill in a questionnaire online. approximately 2500 contact e-mails were sent, extracting the addresses from a special national list. the sample size determination was made by setting a 1-alpha confidence level at 95%, therefore with z normal value at the confidence level of 1.96. the following formula was applied: x o = z 2 (p × q)/b 2 , with p as the proportion to be estimated and q the proportion of complementary character and b the desired precision set at 5%. hence: 3.8416 (0.31 × 0.69)/0.0025 = 329. the response rate recorded was 1:6, compatible with the fixed sample size (419 > 329). the average completion time was about 15 min. tools administration took place upon the release and signing of the form for an informed consent of participation in accordance with the declaration of helsinki. the study was approved by the institutional review board of the university of cassino and southern lazio. in order to collect the data necessary to carry out the study, a questionnaire was built up and articulated into the following sections: (1) socio-demographic information: gender; age; type of degree obtained; (2) school environment: (a) how long (in years) have you been a school principal? (b) how many students altogether attend the school(s) you currently manage? (c) how long (in years) have you been a principal at the current location? (d) type of school(s) related to the management (primary/secondary school, technical school, high school, etc.); (3) information on the current health of the subjects: body mass index, high/low blood pressure, possible presence of diabetes, cholesterol, heart disease, respiratory problems (e.g., asthma, bronchitis), migraines, stomach problems, back, neck or joint pain; (4) psychometric measurements: (a) the test m.s.p. (psychological stress measurement) [30, 31] . the test measures the state of subjectively perceived stress, and consists of 49 items with likert response scale 1-4 (from not at all to very much) on the individual's perception of his cognitive-affective, physiological, and behavioral state. the overall test score provides a global index of the psychological stress state. in addition to the overall score, it is possible to calculate six other values that correspond to six different articulations of the way one perceives himself as being stressed: loss of control and irritability (i.e., "i am irritable, my nerves are on edge, i lose patience with people and things."); psychophysiological sensations (i.e., "i feel tense or strained."); sense of effort and confusion (i.e., "i feel overwhelmed, overpowered and overloaded."); depressive anxiety (mixed depression and anxiety symptoms: i.e., "i review the same ideas several times, i brood, i have the same thoughts over and over again, i feel my head full of thoughts."); pain and physical problems (i.e., "i have physical pains: back pain, headache, neck pain, bellyache."); hyperactivity and acceleration (i.e., "i walk quickly"); (b) mindfulness organizing scale (mos) [32, 33] . this is a self-report measure that investigates the safety of the organization, or rather how the worker perceives that safety. it is based on concrete behavior that reflects the employee's relationship with the organization and his colleagues. the measurement is carried out on a three-point likert scale (from not at all to very much). the dimensions included in the items are: concern about failure, reluctance to simplify interpretations, sensitivity to operations, commitment to resilience, and deference to expertise. the s9, s5, and s3 scales were used for this study. a total of 9 items make up these three scales. the s9 scale measures the degree of application of group decision-making and comparison with colleagues (awareness of the value of shared problem analysis: i.e., "when a crisis occurs, we rapidly pool our collective expertise to attempt to resolve it"), the s5 scale is designed to assess the organization's reluctance to simplify in the face of critical issues (awareness of the value of a non-rigid climate: i.e., "when discussing emerging problems with co-workers, we usually discuss what to look out for"), the s2 scale is intended to assess the level of organizational awareness (awareness of the value of mutual knowledge: i.e., "we discuss our unique skills with each other so that we know who has relevant specialized skills and knowledge"); (c) scheda per la rilevazione funzionale delle aziende (functional survey module for companies) [34] . for this study, within the module, the scale of emotions at work was used; it consists of 10 items with true/false answers to assess the emotions that prevail during work. the direction of the scale is oriented to specifically assess the work discomfort perceived by the person; (d) polychronic-monochronic tendency scale (pmts) [35] . this measures the subject's ability to perform several tasks during the same time interval (i.e., "i feel at ease when i do several activities at once"). the measurement is carried out on a five-point likert scale (from strongly disagree to strongly agree) including five items. the data were processed using the statistical software spss version 22 (ibm corporation, armonk, ny, usa) and amos ibm version 22 (ibm corporation, armonk, ny, usa). the main analyses performed were: descriptive statistics to illustrate socio-demographic information; pearson and spearman bivariate and partialized correlations for all main measures (psychological stress, organizing mindfulness, work discomfort, polychronic-monochronic tendency) significant at p < 0.005 and at p < 0.001, 2-tailed); kendal's point-biserial correlations between msp, work discomfort and reported physical ailments; cronbach's alpha as scale reliability coefficient; t-test to explore significance in stress score and polychronic tendency relating to gender; anova univariate test with p < 0.05 to explore significances between work discomfort, stress and organizing mindfulness; hierarchical regression to identify the predictors of work discomfort and stress; cohen's d and eta squared as measures of effect size; sem analysis to explore predictors' effects on work discomfort. to test the adequacy of the model the following eight indices were considered: (1) the chi-square; (2) the relationship between the value of the chi-square and the degrees of freedom; (3) gfi (goodness of fit index); (4) agfi (adjusted goodness of fit index); (5) rmsea (root-mean-square error of approximation); (6) rmsr (root mean square residual); (7) cfi (comparative fit index); (8) nfi (normed fit index); (9) rfi (relative fit index); (10) pnfi (parsimony adjustment to nfi); (11) pcfi (parsimony adjustment to cfi); (12) pclose (testing the null hypothesis that the population rmsea is no greater than 0.05). the main characteristics of the sample are illustrated in table 1 below, while table 2 presents the bivariate correlations between the measures used in the study. the corresponding dataset is available as supplementary material s1. it can be observed in table 2 that stress resulted inversely correlated to age, strongly correlated to work discomfort and inversely correlated to organizational awareness. among the components of stress most associated with the perception of work discomfort were depressive anxiety (0.660 **), the sense of effort and confusion (0.601 **), and irritability (0.503 **). as the number of years of service increases, organizational awareness also improves (141 **), while stress (−0.173 **) and the perception of work discomfort (−0.162 **) decrease; at the same time, awareness of the value of mutual knowledge increases (0.149 **) and the person's level of hyperactivity decreases (−0.150 **). the level of discomfort and perceived stress was not associated with the number of students in the administered institution. as seniority increased, there was also an increase in the number of students and therefore in the size of the school administered (0.138 **). partialized correlations with the perception of work discomfort, showed a decrease in the association between the variables (determined by the control variables), which however remained statistically significant with depressive anxiety (0.333 **), the sense of effort and confusion (0.264 **), awareness of sharing analysis (0.151 **), but no longer with irritability (0.041), awareness of no-stiffness (0.144), awareness of mutual knowledge (0.337). partialized correlations with the general measure of stress (msp) showed a reduction of associations with age variables (−0.125 **) and work discomfort (0.098 **), and non-significance with awareness of sharing analysis (0.061), awareness of no-stiffness (0.008), awareness of mutual knowledge (−0.028). table 3 shows the distribution of stress levels among the sample, after the transformation of the raw score into t points and the relative comparison with the italian percentile calibration values of the scale. it can be noted that 27.7% had high levels of stress and almost 60% of the principals were affected by moderate and high levels of stress. figure 1 reports the distribution of stress scores compared to the type of school managed. it can be observed that the regency of the comprehensive schools with multiple age levels (5-13) was accompanied by the highest level of stress for the principals, although the univariate anova and tukey's post-hoc comparisons did not show significant differences between this level of stress and those associated with the other four types of school (p = 0.415). figure 1 reports the distribution of stress scores compared to the type of school managed. it can be observed that the regency of the comprehensive schools with multiple age levels (5-13) was accompanied by the highest level of stress for the principals, although the univariate anova and tukey's post-hoc comparisons did not show significant differences between this level of stress and those associated with the other four types of school (p = 0.415). univariate anova reported, as illustrated in table 4 , a significant inverse association between organizational mindfulness and stress scores; in addition, there was a significant association of the two variables (stress and organizational mindfulness) with the measure of perceived work discomfort. univariate anova reported, as illustrated in table 4 , a significant inverse association between organizational mindfulness and stress scores; in addition, there was a significant association of the two variables (stress and organizational mindfulness) with the measure of perceived work discomfort. table 5 reports point-biserial correlations between msp, work discomfort and the physical ailments the principals claimed to have. it can be noted that the disorders most associated with stress and the perception of work discomfort are: migraine, stomach problems, back and/or cervical pain. stomach problems are sometimes also associated with respiratory problems (e.g., asthma, bronchitis). in order to identify among the components of stress and mindfulness the predictors influencing work discomfort perceived by principals, a hierarchical regression analysis was carried out. hierarchical multiple regressions were run to determine if the addition of the stress and mindfulness components improved the prediction of work discomfort. the preliminary verifications of the regression assumptions excluded the presence of multivariate outliers. mardia's multivariate kurtosis index (160.51) was in fact below the critical value [p (p + 2) = 168]; therefore, the relationship between the variables can be considered substantially linear. low co-linearity was indicated by the low vif values (variance inflation factor) < 2 and high tolerance values >0.60. for verification of the assumptions on the residuals, the average between the standardized and raw residuals was equal to 0; the durbin-watson test had a value of 1.97 and was therefore indicative of the absence of autocorrelation. influential predictors have been identified in depressive anxiety the model is displayed in figure 2 , where it is shown that work discomfort was mainly affected by depressive anxiety (standardized estimate of the regression weight of 0.517 for p < 0.018). the second influential predictor turned out to be the sense of effort and confusion (standardized estimate of the regression weight of 0.862 for p < 0.001), which in turn receives a major influence precisely from depressive anxiety (standardized estimate of the regression weight of −0.248 for p < 0.010). the model has identified the awareness of the value of shared problem analysis as a significant negative predictor of work discomfort (standardized estimates of the regression weights −0.206 for p < 0.001), furthermore this was negatively affected by depressive anxiety (standardized estimate of the regression weight of 0.517 for p < 0.018). subsequently, a sem analysis was performed, combining into one explanatory model the variables that previously revealed significant association with work discomfort. the model showed overall good fit measurements: χ 2 = 271.41 df = 171 p = 0.000; cmin/df = 1.587; rmr = 0.016; gfi = 0.940; agfi = 0.918. baseline comparisons nfi = 0.922; ifi = 0.970; cfi = 0.969. parsimony-adjusted measures pnfi = 0.751; pcfi = 0.789; rmsea: 0.037; pclose: 0.995; rmsea 90% 0.029-0.046. the model is displayed in figure 2 , where it is shown that work discomfort was mainly affected by depressive anxiety (standardized estimate of the regression weight of 0.517 for p < 0.018). the second influential predictor turned out to be the sense of effort and confusion (standardized estimate of the regression weight of 0.862 for p < 0.001), which in turn receives a major influence precisely from depressive anxiety (standardized estimate of the regression weight of −0.248 for p < 0.010). the model has identified the awareness of the value of shared problem analysis as a significant negative predictor of work discomfort (standardized estimates of the regression weights −0.206 for p < 0.001), furthermore this was negatively affected by depressive anxiety (standardized estimate of the regression weight of 0.517 for p < 0.018). table 6 below summarizes the maximum likelihood estimates and regression weights estimates. the corresponding sem with amos is available as supplementary material s2. the state of chronic stress leads to an inability to manage events, both in the sense of not being able to solve the conditions of difficulty that arise and in the sense of inability to prevent them and even of unconscious tendency to intensify and proliferate obstacles and stressful events [36] . numerous studies have demonstrated that chronic stress also directly produces innumerable illness conditions, influenced by the inability to manage and improve one's own health [37] . studies on work-related stress are increasing, as are publications on the difficult condition of school workers [38, 39] . while the focus is on teachers, there are few reflections on the psychophysical fitness of the school manager, especially in light of the latest legislative and organizational changes. school leaders are also exposed to health risks related to work-related stress, but there are different aspects of the problem with respect to teachers and employees in general [40, 41] . this involves an institutional figure thrown into the continuous proliferation of reforms, legal norms, structural changes, conflict management, and radical changes in customs and processes [42] . we are substantially dealing with a figure who is at risk and to whom more research attention should be paid for exploratory, diagnostic and preventive purposes. our study first of all showed that about half of the sample of principals who participated in the research had moderate to high stress levels. this immediately emphasizes how current and critical the problem is. the widespread tendency to merge different school cycles (elementary and junior high schools) in the so-called "istituti comprensivi" (comprehensive schools, ages 5-13), for administrative reasons, was associated with a greater load of tension and pressure for principals, who probably find it difficult to manage with a single approach the problems and differences that arise from educational orientations and professional profiles traditionally characterized by a plurality of visions, different approaches to teaching and evaluation of students, diversity in the level of involvement of families in school life, different propensity and habit of teachers of different cycles (primary and secondary) to work on shared projects. it would seem that the management of these differences, rather than the size of the school (in terms of number of students, and therefore of teachers), is a reason for greater pressure and tension at work [43, 44] . women principals showed significantly higher levels of stress than their male colleagues. in this regard, according to the data, their greater tendency to engage simultaneously in the resolution of several tasks, could indicate a greater resistance to delegation, a strong sense of personal responsibility that would lead them to a total (psychological) involvement which over time can overload the person, limiting the time of physical and mental recovery. in line with these data, studies by kiral [45] have shown that women principals have higher levels of stress than male principals, as women have to reconcile the responsibility for domestic work with the official and public work they do in school. the first analyses of our study have indicated that the general perception of the principal's working discomfort presents on the one hand an association with the level of stress, which contributes to increase the value of the discomfort, and on the other hand an equally significant association with organizing mindfulness, which can substantially limit the negative effects of stress on perceived discomfort. this measure of discomfort includes de-motivational aspects, negative mood, disappointment for the unreliability of the context, negative balance between efforts and results, weight in conducting mediations, pessimistic view of the future, perception of others' insensitivity, doubts about one's self-efficacy. an interesting reflection that emerged from the observation of the data was that relating to the age and period of service of the principals. both the stress level and the perceived general discomfort had an inverse correlation with age and years of service. this suggests that experience can play a significant role in the development of the management and coordination skills required to best perform the complex functions of school leadership. the critical aspect concerns the italian situation where in the last decade there has been a substantial turnover of principals. therefore, only a few years ago, a large number of principals started their service role, in a context of extensive administrative changes imposed by the ministry of education. it is not difficult to hypothesize that in this situation the youngest principal may feel the weight and responsibility of an assignment that no longer finds a frame of reference in past experience. the results of the study confirmed the association between high levels of stress and somatization, which was already evident in the literature on the health implications of a chronic occupational stress condition: migraine, stomach problems, back and/or cervical pains, respiratory problems (e.g., asthma, bronchitis). these data were in line with mariammal et al. [46] who stated that stress manifests itself in the form of chronic disorders or diseases such as hypertension, stroke, headache, and diabetes, as well as regular physical pain. some principals experience symptoms such as suppression of the reproductive system, anxiety, aggression, indigestion, stomach-ache, pain, dizziness, and rapid heartbeat. in addition, chronic stress creates muscle tension, fatigue, constipation, and arthritis [47] . principal stress has even been associated with severe problems such as ischemia and heart problems [48] . further results of the study have identified the predictors of work-related discomfort with greater accuracy in the components of stress and organizational mindfulness. through a regression analysis and then through sem, the effects and influence relationships between the predictors were identified. among the components of the msp, it seemed that depressive anxiety had the main role of influence. the anxiety component is characterized by aspects of recurrent ruminative cognition that amplify the sense of isolation, incomprehension, discouragement, and worry [49] . this negative interpretative framework activates another significant component: the sense of effort and confusion perceived by the person, who develops thoughts of inadequacy, the impression that everything involves a considerable effort and that everything falls on his shoulders. this attitude can naturally encourage a lack of clarity in ideas and decreased attention and concentration. within the model, one of the three components of mindfulness, the awareness of the importance of sharing problem analysis, found a significant place. the effect of the variable limited the dimension of the perceived discomfort. if, on the one hand, the increase in pressure and tension drives the person to intensify their efforts by closing and defensively stiffening themselves, on the other hand, the ability to recognize the value of sharing and involvement of other collaborators and colleagues in order to deepen the understanding of the problems and the identification of the most appropriate management methods, can help one to come out of isolation and discover the value of confrontation and the functional exercise of delegation, reducing the sense of oppression and distrust. the anxiety component that characterizes the principal's stress can be mitigated by training and refresher courses focused on a model of organizing mindfulness. acquiring awareness of the value of sharing practice is the main theme, but two other aspects to be investigated should not be underestimated and which in this study have however shown strong positive correlations with the practice of sharing, and which perhaps constitute the necessary operational preparatory basis: awareness of the value/advantage of a non-rigid climate and awareness of the value of mutual knowledge. a lack of awareness of one's own way of acting can lead to behaviors that are not functional to the work context, to the quality of the interaction with one's colleagues and to the nature of the task required at the time [50] . by investing in one's mindfulness, one can expect to break the old automatisms in favor of new behaviors, effective even in difficult times, as indicated by weick and sutcliffe, who believe it is necessary to rely on a mindfulness-oriented approach when there is a need to make a quick and important decision, giving priority to one's competence (or that of one's co-workers), rather than relying on one's authority. the study by beausaert et al. [51] emphasized the influence of social support in the containment of stress and the burnout of principals. our study points out as a priority, above all, the awareness to which the principal must be individually accountable, i.e., the indispensability of a practice of sharing and mutual recognition of specificities and competences. in this way they move on to a proactive attitude that promotes social support in the first person, before expecting it (in due form) from others. when mindfulness becomes a shared social practice in an organization and permeates the routines, processes and practices among people and teams, and thus affects the organization as a whole, the organization itself becomes more resilient and proactive. even the educational institution has a vital need to promote responsible leaders at all levels, capable of maintaining self-control, a sense of balance and self-determination, despite the informational overload they have to deal with today. data collection through self-reporting measures should be expanded with a methodological design in which judgments about principal stress could be provided also by the staff and teachers of the school, in order to have a more balanced representation by an outside perspective. the cross-sectional approach of the study involved collecting data at a single point in time; instead, the extension of the study could include repeated administration at different times of the school year (beginning, middle, end). the reference hypothesis is that the level of stress and the perception of discomfort could vary in relation to different significant institutional moments, such as the opening and closing of the school year. in this regard it could be useful the novel use of neuroscience-based approaches in education, namely neurodicatics, which is directed to address the educational and psychological well-being of students and staff involved in education as part of the education environment [52] . a further important contribution could be a specific focus on how principals cope with emergencies and on the functionality of their strategies to manage individual and collective stress triggered by the exceptional nature of the problems that the situation entails (e.g., ensuring teaching activities in safe environments after the spread of the covid-19 virus). at the moment, there are also no longitudinal studies that have monitored the evolution of the principal's leadership ability over medium-long intervals. it would therefore be important to understand if and how more mature and functional patterns for the containment and management of pressure in moments of personal tension and discomfort are learned and modeled over the course of his/her career. the results of this study have shown that the levels of stress and work discomfort perceived by principals are high and require both empirical investigation and targeted support and prevention interventions. in fact, the stress experienced by a principal is associated with various physical disorders and serious health risks, such as ischemia and heart problems. the study has produced a path analysis model in which the joint effects between the main predictors of principals' work discomfort were explained. the effect of depressive anxiety on perceived discomfort found a protective mediator in the mindfulness component that recognizes sharing as a fundamental operational tool, while an increasing sense of effort and confusion could significantly amplify the experience of psychological discomfort associated with the practice of school leadership. supplementary materials: the following are available online at http://www.mdpi.com/1660-4601/17/17/6318/s1, file s1: principals stress.sav (dataset); file s2: model work discomfort.amw (model). author contributions: p.d., g.v., and s.m. designed the study. p.d., g.v., and s.m. analyzed the data and discussed the results. f.b. and s.m. drafted the manuscript. g.v. and f.b. revised the manuscript. all authors approved the final manuscript. finally, the authors have agreed to be accountable for all aspects of the manuscript in ensuring that questions related to the accuracy or integrity of any part of it are appropriately investigated and resolved. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. symptômes et caractéristiques du burn out [symptoms and characteristics of burnout school burnout and heart rate variability: risk of cardiovascular disease and hypertension in young adult females il nuovo dirigente scolastico preparing leaders to work with emotions in culturally diverse educational communities manuale per dirigenti scolastici e staff di direzione principal burnout: the concept and its components an analysis of burnout and job satisfaction among turkish special school headteachers and teachers, and the factors effecting their burnout and job satisfaction burnout among iranian school principals burnout in school principals: role related antecedents stress and job satisfaction among primary head teachers: a question of balance? a survivor's guide to the principalship: overcoming the challenges occupational stress and job satisfaction among school administrators principal self-efficacy: relations with burnout, job satisfaction and motivation to quit teachers' approaches toward cultural diversity predict diversity-related burnout and self-efficacy principal preparation programs: perceptions of high principals unaccepted accountability: the looming shortage of principals and vice principals in ontario public school boards; 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gregersen, tammy; mercer, sarah title: language teachers' coping strategies during the covid-19 conversion to online teaching: correlations with stress, wellbeing and negative emotions date: 2020-08-22 journal: nan doi: 10.1016/j.system.2020.102352 sha: doc_id: 356131 cord_uid: 3at94sle abstract teaching often is listed as one of the most stressful professions and being a language teacher triggers its own unique challenges. response to the covid-19 pandemic have created a long list of new stressors for teachers to deal with, including problems caused by the emergency conversion to online language teaching. this article examines the stress and coping responses of an international sample of over 600 language teachers who responded to an online survey in april, 2020. the survey measured stressors and 14 coping strategies grouped into two types, approach and avoidant. substantial levels of stress were reported by teachers. correlations show that positive psychological outcomes (wellbeing, health, happiness, resilience, and growth during trauma) correlated positively with approach coping and negatively with avoidant coping. avoidant coping, however, consistently correlated (rs between .42 and .54) only with the negative outcomes (stress, anxiety, anger, sadness, and loneliness). in addition, anova showed that although approach coping was consistently used across stress groups, avoidant coping increased as stress increased suggesting that there may be a cost to using avoidant coping strategies. stepwise regression analyses using the 14 specific coping strategies showed a complex pattern of coping. suggestions for avoiding avoidance coping strategies are offered. the typical days for language teachers around the world were stressful enough, given their typically heavy workloads, time pressures, and difficulties juggling roles (macintyre et al., 2019) . the sources of teacher stress have multiplied with the advent of covid-19. workloads that were once perceived as substantial have been complicated by a rapid conversion to online delivery for which many language teachers had not been prepared but whose effects seem likely to last for years to come. balancing personal and professional roles that is a challenge for many teachers, but online delivery of courses with work-at-home protocols and ubiquitous online work-related activity creates a lack of physical, temporal and/or psychological boundaries between school and home. in many cases, teachers have significant others sharing the same space and/or have their own children who also need their attention. in particular, the nature of the global pandemic brings a particular set of covid-19 difficulties to the mix: health concerns for oneself and loved ones, social and physical distancing, travel restrictions, closed borders, shortages of daily necessities, restricted services, and uncertainty as to when life will return to 'normal.' faith has been shaken in the certainty of what 'normal' even means now or what it will be in the future; the long-term consequences for language teachers and teaching are unknown. even as we write this manuscript in late april 2020, these challenges persist for many across the globe and the passing of time is itself becoming a stressor that challenges people's ability to persist under conditions described as trauma in the making (salas, 2020) . there is no denying that life with covid-19 has suddenly and unavoidably become more difficult and complicated for everyone, including language teachers. how are language teachers coping in the midst of the crisis as much of the world is locked down and education systems are disrupted? 2.0 literature review stress, a much-studied concept in psychology, generally refers to a psychological reaction to environmental conditions (stressors) that produce a variety of effects including physical arousal and threats to well-being (lazarus, 1996; . the stress-appraisal process j o u r n a l p r e -p r o o f includes identifying what is at stake and the resources available to deal with the situation, socalled primary and secondary appraisals respectively, in an iterative process that most often generates behavioral efforts to cope (lazarus & folkman, 1984) . coping is the process of responding to a stressor using one or more available techniques or strategies. a teacher's use of coping responses to stressors is an important determinant of their psychological adjustment and well-being (gustems-carnicer & calderón, 2013; pyhältö et al., 2020; . the degree to which a teacher experiences stress in a situation hinges on a variety of factors, including: appraisal of demands and the teacher's strategies to manage them; expectations of possible upcoming demands and the teacher's readiness to handle them; and the teacher's degree of skill preparedness and practice in handling demands effectively (bottiani, duran, pas, & bradshaw, 2019; dunham, 1992) . herman, rinke & eddy (2020) proposed a "3c theory of teacher stress" that describes three interconnected pathways leading to teacher stress, integrating individual differences in teachers' (1) coping, (2) competence in executing practices that effectively manage the teaching-learning process, and the systemic (3) context in which teaching occurs, including policies, practices, and administrative support. teacher stressors extend well beyond the teaching context. response to the covid-19 pandemic crisis has produced no shortage of stressors as it is most definitely a period of heightened psychological stress. language teachers, their learners, and parents around the globe are having to cope with the astonishing chain of events brought on by covid-19 as schools close and officials direct diverse levels of shelter-in-place orders, from social distancing to strict quarantines, in order to cope with the global pandemic. for many language teachers, this has required switching to teaching under unfamiliar and difficult circumstances with little warning and preparation, and barely, if any, training. there has been an expectation that teachers will simply carry on and do their best by adapting, adjusting and continuing to aim for effective communicative language teaching using a range of online resources. how realistic it is for educators to meet these demands has not been at the forefront of most (any) policy decisions, and the stress being produced is something of an afterthought -if it is considered at all. in addition to a long list of practical pedagogical stressors, educators are also contending with the strains of the pandemic itself, from health concerns for self and others, changes brought on by working from home, threats to the current job and future career, new family and domestic responsibilities, and often being confined to their home. j o u r n a l p r e -p r o o f 2.1 stress: teacher + language + pandemic as of the end of march 2020, the global pandemic already had affected an estimated 1.54 billion children and youth throughout 185 countries in asia, europe, the middle east, north america and south america through school and university closures (schleicher, 2020) . in many parts of the world, the education systems have responded by stopping face-to-face in-classroom instruction and making a rapid conversion to online or remote teaching. in many cases, teachers were given mere days or even hours to convert face-to-face classes to online teaching via synchronous and/or asynchronous methods, often in spite of challenges concerning the availability of necessary digital devices, prior training in online teaching techniques, and/or effective online learning support platforms. in most cases, teachers have not been trained in the necessary technical and pedagogical skills to integrate digital technology instruction (schleicher, 2020) . according to tuominen and leponiemi (2020, p. 7) , "the ongoing covid-19 crisis has been, and will continue to be, both a massive challenge and a learning experience for the global education community. practically no one saw (or wanted to believe) something like this coming." the situation has been described as 'emergency online homeschooling' (guzdial, 2020; lehmann, 2020; milligan, 2020) . even pre-pandemic, teaching was often listed as one of the most stressful professions (johnson, et al, 2005) . some of the 'normal' (i.e., pre-pandemic) stressors that teachers in general (not just language teachers) suffer from include pressure inflicted by heavy workloads, time constraints, unbalanced work-life integration, limited autonomy, excessive administrative obligations, strained relationships with colleagues and school leaders, role conflict/ambiguity, managing innovation and change, emotional labor, dread over losing control of the class, fear of evaluation, and low professional self-esteem, among others (mercer & gregersen, 2020) . being a language teacher triggers its own unique challenges resulting from the specificity and the emotional character of foreign-language teaching: self-doubts about one's own language ability; coping with the emotional anxieties of learners; heterogeneous proficiency in learner groups; threats to sense of self and identity; energy-intense teaching methodologies; intercultural components to teaching; and precarious working conditions (borg, 2006; cosgrove, 2001; gkonou & miller, 2017; gkonou, dewaele & king, 2020; horwitz, 1996; mercer et al., 2016; wieczorek, 2016). few if any of these stressors has disappeared; they still exist in combination with the litany of new unique stressors emerging from the global pandemic and the reaction of education systems to it. petrie (2020) identifies additional stressors for educators that include among others: 1) teachers without resources to discuss pandemics and uncertainties with their learners and who are scrambling to go digital without much support and training; 2) learners who are highly anxious and lonely, unable to focus and worried about having an unnatural closure to the academic year; 3) parents who are overwhelmed and unprepared to coach their kids about digital tools and innovative educational practices, also trying to balance work and homeschooling; and 4) the relationships among all these players, including the maintenance of positive studentteacher-parent rapport and encouraging collaborative learning and socialization that is so challenging online. considering the myriad challenges and stressors, how are language teachers coping and are there any reasons for positivity among their experiences? in their influential 'transactional model' of stress and behavioral self-regulation, lazarus and folkman (1984) divided coping strategies into emotion-focused and problem-focused strategies. whereas problem-focused coping is aimed at solving the perceived problem or doing something to alter the source of stress, emotion-focused coping is aimed at reducing or managing the emotional distress that is associated with or triggered by the situation (lazarus & folkman, 1984) . early research suggested that problem-focused was preferable to emotion-focused coping for long-term, healthier psychological functioning, though it is now recognized that most stressors elicit both types of coping (boniwell & tunariu, 2019) . carver and scheier (1998) argued that the distinction between problem-focused and emotion-focused coping was too simplistic. they developed a multidimensional model of coping and an accompanying measurement instrument, the cope inventory (coping orientation to problems experienced) with 60 items (carver, scheier & weintraub, 1989) . the cope was the predecessor to the brief-cope (carver, 1997) , a streamlined version designed for ease of administration with reduced redundancy among items. the brief-cope includes 28 self-report items divided into 14 subscales of two items, each designed to measure potentially effective and ineffective ways to cope with a stressful life event. although carver does not recommend viewing the brief-cope as a single scale to measure a general construct, it is possible to group j o u r n a l p r e -p r o o f coping strategies into larger constructs such as 'approach' and 'avoidant' coping styles (see table 1 ). approach strategies actively work to change the stressor or accept its presence in one's life; avoidant coping strategies tend toward more dysfunctional responses such as denial, distraction, or substance use. carver et al. (1989) emphasized that it is important not to preordain certain strategies as better than others. rather, it is advisable to consider the fit between an individual's coping strategies and the constraints of a given situation. different types of events require different responses as they evolve over time, and there may be moments when denial, distraction, venting, and so on are the best available responses as a short-term strategy. table 1 . subscales of the brief-cope scale (carver, 1997 a concern throughout the literature on stress and coping is how relatively successful different coping strategies are in producing more positive outcomes and leading to fewer negative outcomes. when coping strategies fail, individuals may find themselves feeling stress, anxiety, and a suite of negative emotions. among teachers, burnout has been a prominent concern (bottiani et al., 2019; herman et al., 2020) . stress-related outcomes can contribute to increased maladaptive behaviors such as denial, withdrawal, alcohol or substance abuse, and anger or aggression (carver & connor-smith, 2010) . teacher coping is particularly important given the potential for teacher stress to negatively affect students, or so-called stress-contagion (oberle & schonert-reichl, 2016) . to explore the effectiveness of coping strategies among language teachers, we have it is essential to include measures of both positive and negative outcomes because "… well-being is not simply the absence of negative function, but rather is something more. that is, a lack of negative affect, depression, loneliness, insecurity, and illness is not the same as the presence of positive affect, happiness, social connection, trust, and wellness" (butler & kern, 2016, p. 2) . to assess the psychological health of teachers enduring the stress of the pandemic, perhaps the most obvious concepts to assess are happiness and well-being. unfortunately, happiness is one of the most troublesome and difficult-to-define concepts in all of psychology and philosophy (lazarus, 2003) . empirical work in this domain has been aided by the development of the perma framework to specify a multidimensional approach to defining happiness (seligman, 2011) . perma is a five-pronged model of psychological well-being and happiness that characterizes a sense of fulfillment, happiness, and meaning. it can be measured j o u r n a l p r e -p r o o f using the 23-item perma profiler (butler & kern, 2016) , which uses three items to asses each of the five perma dimensions. along with core items, the perma profiler provides additional measures of key negative emotions and general health originally designated as 'filler items' but which provide useful information on their own (discussed below). in the present study, the 15 items of perma will be used as a composite index of teacher happiness and well-being. an additional measure well-being that has been used more widely in the literature than perma and for a longer period of time is the who-5 index of well-being (who, 1998) . used in over 200 empirical studies who-5 is among the most often used questionnaires for assessing psychological well-being; it has been translated into 30 languages (topp, østergaard, søndergaard & bech, 2015) . low scores on the who-5 index of well-being can be used to screen for depression and the versatile measure has been used as an outcome measure for intervention studies. a concept related to well-being is psychological resilience, which reflects the capacity to withstand and recover from experiences of psychological adversity or maintain effective functioning despite adverse circumstances (masten, 2001) . in essence, resilience is the ability to withstand stress or bounce back from a traumatic event to recover previous levels of functioning, along multiple pathways (bonanno, 2004) . beyond the notion of maintenance or recovery of previous function implied by resilience is the possibility of development, enhancement, and growth after trauma (tedeschi & calhoun, 2004) . therefore, the final measure of positive functioning that we will use is derived from the literature on post-traumatic stress disorder (ptsd). since its introduction as a diagnosis in 1980, ptsd has become a prominent public concern connected to traumatic events. however, reactions of individuals to trauma vary considerably and it has been argued that, in the long-term, people may be more likely to react to trauma with a sense of growth than with ptsd (rendon, 2016; seligman, 2011) . therefore, to assess the possibility of perceived growth during the current trauma, we include a measure of post-traumatic growth (ptg, taku et al., 2008) . ptg includes developing enhanced appreciation of life, relationships with others, new possibilities in life, personal strength, and spiritual change (tedeschi & calhoun, 1996) . however, given that, as we conduct the research, the pandemic is very much a current issue and we are not yet entering a 'post'-pandemic phase, we had to modify the measure to reflect the perception of growth during trauma. this study will be the first to use this revised measure to evaluate whether, while still in j o u r n a l p r e -p r o o f the midst of the pandemic, teachers perceived any sense of growth or positive change as a result of their experiences thus far. in addition to measures of stress, health and well-being, the potential negative correlates of coping include levels anxiety, anger, sadness and loneliness. anxiety has been a prominent concern in second-language acquisition (sla) research, the most frequently studied emotion in our field (dewaele & macintyre, 2016) , but has been considered primarily among learners -not teachers. teacher anxiety is becoming a more pressing concern given the difficulties they face (hiver, 2016) . the covid-19 pandemic considerably elevates the concern for teachers' anxiety as they find themselves confronted with demanding new teaching methods, extraordinary contexts, and unfamiliar media. anxiety is a fear-related, full-body reaction to an amorphous threat (as opposed to a specific, tangible threat such as a snake or fire) that includes physiological effects, unpleasant arousal, cognitive distractions and avoidant behaviors (reeve, 2015) . another potentially maladaptive emotion is anger, which is one of the most intense and potentially dangerous emotions given that it is associated with a strong urge to act out (harmon-jones & harmon-jones, 2016). it has been associated with lower levels of well-being, anxiety disorders, and ptsd (barrett, mills, & teesson, 2013) . sadness is a more passive type of emotion related to feelings of loss and although it is important to recognize a full range of emotions, in measures of subjective well-being, it is the perceived balance between positive and negative emotions that can generate a sense of hedonic well-being (schimmack, 2008) . finally, loneliness is an emotion most often expressed when access to social settings is denied, where one is feeling socially emotionally isolated. given social restrictions, changes in workplaces, and disruption of interpersonal connections during the pandemic, it is likely that there is an increased sense of loneliness (barreto et al., 2020) . in theory all emotions, even unwelcome ones, have adaptive attributes (reeve, 2015) and under certain conditions each of the emotions studied here -anxiety, anger, sadness, and loneliness -has a role to play in successful functioning: anxiety alerts us to potential dangers, sadness is associated with preventing loss, loneliness promotes social interaction by motivating us to regain connections with other people, and anger is useful in removing obstacles thereby restoring pursuit of an important goal. however, each of these emotions, if sustained over a long a total of 634 language teachers participated in the study. the use of an online survey tool with snowball sampling produced a large data set but it cannot be considered random or representative and we have no way to know what the response rate was or how many people saw the invitation but chose not to respond. a sampling frame that captures the population of teachers worldwide, which would allow for random sampling, is likely impossible to produce in practice and representativeness cannot be assessed. almost 75% were teachers of english, and approximately 80% were female. the age range of the sample is presented in table 2 . approximately half of the respondents came from europe (51.4%) and almost a quarter came from north america (23.5%), with smaller percentages coming from asia (8.8%), south america (6.9%), and the middle east (5.2%), and smaller numbers from elsewhere in the world. overall, the sample had considerable teaching experience: over 50% of respondents had been teaching for 15 years or more and only 3% were in their first year of teaching (see table 3 ). there was a wide variety of institutions and levels of instruction represented in the sample, most respondents, however, had very little time to prepare for the transition to online teaching: approximately three-quarters of the sample had less than a week to convert their courses (see table 4 ). the materials used to assess positive and negative outcomes are short forms of longer instruments, designed to be brief measures, or single-item indicators. the choice to use briefer materials results from a trade-off between depth of information and the breadth of topics that can be covered. the materials for the study included the following scales administered as part of an the survey was administered via google docs between april 5 and april 19, 2020. the survey was shared via social media networks and through personal email contacts to generate a snowball sample. respondents gave informed consent before participating and were advised on the consent page not to take part, or quit at any time, if they felt especially vulnerable or uncomfortable talking about their well-being because there was a concern that reflecting on wellbeing could act as negative trigger for those feeling fragile at the time. after 14 days in the field, the web survey received 634 responses. the first research question (rq1) asked 'what do teachers find most stressful?' figure 1 shows the mean ratings of the 15 stressors listed in the survey, all of which were rated above the midpoint of the stress scale (2 out of 4). the most stressful experience reported by teachers was workload followed by family health, which were rated as significantly more stressful than all other items. loss of control over work also rated above 3 out of 4 (m = 3.07). other teaching given the high levels of stress revealed in the above analysis, the second research question asked 'what coping strategies do teachers use most often?' figure 2 shows the ratings of the frequency mean rating j o u r n a l p r e -p r o o f of the 15 coping strategies used in the brief-cope inventory. the most frequently used coping strategy was acceptance, followed by advanced planning, re-framing, actively doing something about the situation, and using work or other activity as a distraction. all of these are considered approach coping strategies (carver et al., 1989) . the least frequently used coping strategies included disengagement, substance abuse, and denial, all of which are considered avoidant in nature. a planned comparison of the mean total scores for the six approach (m = 31.9, sd = 6.35) and six avoidant types of coping (m = 20.8, sd = 4.95) revealed a substantial difference in how often they were being used (t [585] = 35.4, p < .001). based on these data, we see that the predominant tendency in the sample was to use active coping to first accept the situation and attempt to deal with it through activity, reframing, and seeking emotional support. this finding helps to reinforce the notion that seeking emotional support is an active, approach-oriented coping strategy and not opposite of 'problem-focused' coping. figure 2 . language teachers' frequency ratings of coping strategy use during the covid-19 pandemic. as recommended by carver (1997) , we performed an analysis to assess the appropriateness of the grouping of active and avoidant coping in the present sample. using principal components analysis, we requested two factors and rotated using direct oblimin to allow for factors to correlate. the resulting structure matrix (see table 5 ) of factor loadings shows that the six approach scales loaded substantially (>.50) on the first factor and the six avoidant scales loaded substantially on the second factor, with only a small correlation between them (factor correlation = .09). the religion and humor scales did not load substantially on either factor. this analysis supports the use of categorizing coping strategies as approach (factor 1) and avoidant (factor 2) in the present sample. note: religion and humor subscales did not load significantly onto either factor. bolding indicates substantial factor loadings (>= .50). the above results, taken together, show a group of teachers encountering a large number of stressors and feeling the strain of the situation, but more often choosing active coping rather than avoidance. however, that is not to say that everyone is coping equally well. the third research question (rq3) asked about individual differences in the relationship of stress with approach and avoidance coping. results show that the total score for the six avoidant-coping styles was significantly correlated with total stress (r = .52, p < .001), as was the total of the approach-coping strategies (r = .13, p < .01) but they correlated to a significantly lower degree (z = 7.54, p < .001). to display the trend in this data, the sample was divided into quartiles based on the total stress scores, creating four groups (low stress, below average, above average, and high stress) in . comparing language teachers' mean ratings of approach vs. avoidant coping strategy use during the covid-19 pandemic across stress quartile groups. the above analyses show that individual teachers who were engaging more often with avoidant coping were also experiencing progressively higher levels of perceived stress. although cross-sectional data such as these cannot conclusively tease apart the causal sequence, coping is a reaction to stress that takes place in an iterative process of appraisals and coping, triggered by the ongoing presence of stressors and adaptations to them (lazarus & folkman, 1984) . reporting higher scores for stress indicates that, to some degree at least, coping strategies are not working successfully to mitigate the perceived stress. as these processes settle into a stable pattern, the results suggest that teachers under the most stress are relying most on avoidant coping, compared to the teachers under less stress. a similar pattern of relying on avoidance coping was also observed for the other negative outcome variables. the correlations of avoidance coping with anxiety (r = .52, p < .001), anger (r = .42, p < .001), sadness (r = .54, p < .001), and loneliness (r = .45, p < .001) all were significant. however, the correlations of those variables with approach coping (r = .003, r = -.024, r = .047, and r = .020) all were non-significant (see quartile groups j o u r n a l p r e -p r o o f addition to stress reported above, teachers who are using the avoidant coping strategies more often are experiencing increased negative emotions, but that these emotions are not correlated significantly with use of approach coping strategies. these results are consistent with the notion that there is an emotional cost of coping only when it comes to using the avoidant style. table 6a . pearson correlations between approach and avoidant coping strategies and negative outcome variables. note: * p < .01; ** p < .001. the second part of rq3 asked about correlations of approach and avoidant coping with the positive outcome variables (see table 6b ). results show that approach coping total scores correlated significantly (all p < .001) with the positive outcomes. avoidant coping correlated negatively with the positive outcomes, but not with growth (r = .01). in these data, when examining well-being and its correlates, we see approach and avoidant coping acting in similar but opposing manners, like opposite ends of a seesaw. in the case of positive outcome measures, more approach and less avoidant coping both were associated with more positive outcomes. the correlation patterns observed for positive versus negative outcomes shows that positive and negative outcomes cannot be assumed to operate in tandem. whereas rq3 provided an overview of the patterns, rq4 asked about the ability of the 14 specific strategies of the brief-cope inventory to predict positive and negative outcomes. table 7 shows the results of a stepwise multiple regression with each of the five positiveoutcome variables. stepwise regression retains only predictors that add significant levels of unique prediction to the equation. a noteworthy caveat with stepwise regressions is that each beta weight in the equation must be interpreted in conjunction with the other beta weights in the equation because the value of beta changes each time a predictor is added or deleted. in addition, the analyses to follow have produced several equations with a large number of predictors whose contribution tends to be small. the pattern of results shows that a variety of approach-coping strategies are predictive of positive outcomes and avoidant-coping strategies tend to negatively contribute to predicting those outcomes. the exceptions to this general rule are a negative contribution of advice-seeking to resilience and planning to who-5 well-being. it is unclear whether advice-seeking is generally negatively associated with feeling more resilient. however, in the case of planning, the uncertainty of the covid-19 crisis means that making plans is more problematic than usual as a coping strategy, and this produces a weak negative relationship with overall well-being. j o u r n a l p r e -p r o o f the regressions show the value of positive reframing of the situation and avoiding selfblame, strategies implicated in all the positive-outcome variables. on the one hand, although very few people likely accept blame for the covid-19 virus, the reaction to the emergency conversion to online teaching might be at play here. the skillset required for online teaching and developing courses that work well in the online environment takes time. teachers in the present study were not give much time to move online -less than a week in most cases -and those who feel that they are not performing in their online teaching role well might be blaming themselves. table 8 presents the prediction equations for the negative outcomes including stress, anxiety, anger, sadness, and loneliness. three of the strategies, self-blame, venting and disengagement, are consistently implicated in all of these negative outcomes, they significantly predict all the negative outcomes. results suggest that these three strategies are to be avoided as their increased use consistently contributes to the negative outcomes. j o u r n a l p r e -p r o o f there were also a few unexpected predictors. planning appears to be working in a negative way, contributing positively to increasing stress and anxiety and negatively to wellbeing. if planning in an uncertain and uncontrollable situation has a negative effect on wellbeing, it makes sense that the effect also emerges for stress and anxiety. uncertainty abounds globally and in local education contexts, and those who prefer to rely on advance planning may be finding themselves suffering because of the difficulty of making plans at this time. responses to covid-19 may be rewarding those teachers who show greater flexibility. the beta weight that runs counter to expectations venting which appears to contribute positively to growth, despite consistently contributing to the negative outcomes. the effect on growth during trauma might be a feature of the way the measure is conceptualized -as a way of using negative experiences in the future. although venting is consistently associated with negative emotions, the effect on growth might suggest that there is a weak tendency for respondents to pair expressing those emotions with a hope for a better future. as a note of caution for interpretation, in addition to the above caveats for stepwise regression, the large sample size used here can detect significant predictors that might not be declared significant in studies with a smaller sample. we present the results both for completeness and for possible hypotheses for future research. overall, the regression analyses show that the many stressors associated with covid-19 implicate a variety of coping responses in language teachers. in most cases, approach-oriented coping produces more-favorable outcomes and avoidance-oriented coping produces lessfavorable outcomes. coupled with the correlation and anova analyses for rq3, these results show the potentially damaging effects of avoidant coping strategies. not all coping strategies are to be recommended, and it would appear that some (accepting self-blame, disengagement and venting) work against successful coping. perhaps the major implication emerging from the regression analysis of specific coping strategies, is to 'avoid avoidance. ' boyce (2013) , writing for the psychology today blog, lists seven tips to reduce avoidant coping including: 1. develop tolerance for uncertainty. 2. take inventory of what one is avoiding during uncertain times. 3. learn to delegate. 4. recognize when one is taking on too much responsibility for other people or trying to do too much to protect others from negative outcomes. 5. learn to take action even under uncertain conditions. 6. recognize procrastination, especially when uncertainty is producing paralysis. 7. recognize when jumping from one idea to the next is being driven by uncertainty. the above advice points to the need for teachers to be realistic and moderate in their expectations, especially when dealing with the uncertainty of the present situation. the dataset in the present study included an open-ended question asking for each respondent's advice for other teachers. data analysis is just beginning and we are working on a companion report to this one that features the results of the qualitative analysis now underway. no teacher training program includes a topic such as 'how to deal with a pandemic' and switching to online teaching was done under emergency conditions. the use of a cross-sectional method of data collection, using online questionnaires, does not allow for a detailed assessment of cause-and-effect between stress and coping or how their relationships change over time as the j o u r n a l p r e -p r o o f pandemic drags on. the data used in the present study was collected at a specific period during which the pandemic was unfolding. how long the pandemic might last, how stable these patterns are post-pandemic, and whether avoidance coping continues to be as problematic as shown here, or possibly becomes even more so, is an open question. ongoing changes in language pedagogy, including but not limited to the expanded use of online learning in place of face-to-face instruction, have been accelerated by the covid-19 pandemic. it is important to study how these trends play out in the longer term both for language teacher stress and well-being. the above results emphasize that future research should not assume that stress, coping and well-being simply are opposites; their relationship is complex and continually adaptive. further, the pattern of relationships among teacher variables is no longer being neglected, it is part of a rapidly expanding literature (gkonou et al., 2020; herman et al., 2020; mercer & gregersen, 2020) . the findings emerging from these studies can be used to inform the future training of teachers, with an emphasis on enhancing their psychological health and reducing burnout and the number of teachers quitting the profession. this study shows that during the covid-19 pandemic, the worldwide response has created a number of difficulties for language teachers. the rapid conversion to online teaching, the blurred lines between work and home coupled with the omnipresent concern for the health of family and oneself, has produced high levels of stress. teachers are coping as best they can using a variety of techniques. in terms of psychological health and well-being, coping techniques that can be considered more active and approach-oriented, ones that more tackle the issues created by the situation including the emotions aroused, are associated with more positive outcomes. perhaps more importantly, increased use of avoidant-coping strategies is associated with negative psychological outcomes. in particular, it is the increased use of avoidant coping that is associated with increasing levels of stress and a variety of negative emotions (anxiety, anger, sadness and loneliness). within the approach and avoidant categories of coping are a number of specific strategies being used by the participants, most of which produced results consistent with the category in which they appear. the multidimensional nature of the stressors requires multidimensional coping strategies, but clearly some strategies are better than others. our hope is that this study can cast light on the effectiveness of coping strategies used by language teachers during the crisis. such insights are not only relevant in the present but will offer us valuable lessons on how best to support teachers in the future who may find themselves in situations of heightened stress such as during transitions across schools, during periods of educational reform, or when facing intense work phases such as end-of-year exam seasons. being a teacher is stressful at the best of times, learning how to cope with the stress is therefore an invaluable skill that all pre-service and in-service teacher education programs should be integrating as a fundamental professional competence. j o u r n a l p r e -p r o o f development of a resilience item bank and short forms. rehabilitation psychology. advance online publication loneliness around the world: age, gender, and cultural differences in loneliness mental health correlates 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teachers' emotional well-being and emotional regulation in the united states, japan and austria the posttraumatic growth inventory: measuring the positive legacy of trauma posttraumatic growth: conceptual foundations and empirical evidence the who-5 well-being index: a systematic review of the literature a learning experience for us all. spotlight: quality education for all during covid-19 crisis high inhibitions and low self-esteem as factors contributing to foreign language teacher stress wellbeing measures in primary health care/the depcare project. copenhagen: who regional office for europe key: cord-309273-gtvi37gh authors: flesia, luca; monaro, merylin; mazza, cristina; fietta, valentina; colicino, elena; segatto, barbara; roma, paolo title: predicting perceived stress related to the covid-19 outbreak through stable psychological traits and machine learning models date: 2020-10-19 journal: j clin med doi: 10.3390/jcm9103350 sha: doc_id: 309273 cord_uid: gtvi37gh the global sars-cov-2 outbreak and subsequent lockdown had a significant impact on people’s daily lives, with strong implications for stress levels due to the threat of contagion and restrictions to freedom. given the link between high stress levels and adverse physical and mental consequences, the covid-19 pandemic is certainly a global public health issue. in the present study, we assessed the effect of the pandemic on stress levels in n = 2053 italian adults, and characterized more vulnerable individuals on the basis of sociodemographic features and stable psychological traits. a set of 18 psycho-social variables, generalized regressions, and predictive machine learning approaches were leveraged. we identified higher levels of perceived stress in the study sample relative to italian normative values. higher levels of distress were found in women, participants with lower income, and participants living with others. higher rates of emotional stability and self-control, as well as a positive coping style and internal locus of control, emerged as protective factors. predictive learning models identified participants with high perceived stress, with a sensitivity greater than 76%. the results suggest a characterization of people who are more vulnerable to experiencing high levels of stress during the covid-19 pandemic. this characterization may contribute to early and targeted intervention strategies. sars-cov-2 (severe acute respiratory syndrome coronavirus 2; henceforth referred to as covid19) is a strain of coronavirus that can infect humans, attacking the lungs and causing symptoms ranging from those of the common cold to those of severe acute respiratory syndrome (sars) [1] . while approximately 80% of those who are infected recover with no special treatment (i.e., they are either asymptomatic or suffer from mild pneumonia) [2] , recent data have confirmed that older persons (60+ years old) [3] and persons with certain pre-existing medical conditions are more likely to develop serious respiratory distress that can lead to death (3-4% of the population) [4] . covid-19 spreads very easily between persons and, at the time of writing, no drugs or biologics have proven effective for preventing or treating the virus [5, 6] . covid-19 was first identified in the chinese region of wuhan in december 2019 [6] . between december 2019 and april 2020, the virus spread throughout the world, causing more than 5,000,000 infections and over 300,000 deaths [7] . on 11 march 2020, the world health organization (who) declared covid-19 a pandemic [8] . to contain the number of victims and prevent the collapse of the healthcare system, most national governments imposed strict restrictions on residents' freedom, forcing those infected with the virus to self-isolate and requiring all residents (infected or not) to stay at home and move through public areas only for reasons of absolute necessity. among western governments, the italian government was the first to apply such restrictions [9] . a few weeks after the spread of covid-19 in china, the first scientific studies investigating the psychological impact of the outbreak highlighted a mild to severe negative psychological impact of the event within a significant proportion of the chinese population (53.8%); specifically, this impact on mental health was reported to include symptoms of anxiety, depression, and stress [10, 11] . more broadly, the literature on the impact of infectious outbreaks on mental health shows that pandemics are extremely stressful events that force people to cope with totally unexpected, ambiguous, and uncertain situations [12] . specifically, two main aspects of pandemics have been found to affect people's mental state. the first relates to danger (i.e., the fear of contagion), which can increase perceived threat and sometimes lead to panic, behavioral contagion, and an emotional epidemic [13, 14] . the second regards the multiple and rapid changes to social, working, and familiar habits, due to self-isolation and social distancing measures [15] [16] [17] [18] [19] . the longer the duration of self-isolation, the more people experience frustration and boredom, along with concerns about infection [15] . well-documented psychological reactions to epidemics include emotional distress, anxiety behaviors, sleeping disorders, fear, anger, depression, health concerns, a sense of powerlessness, and uncertainty [13, 16, [20] [21] [22] [23] [24] . furthermore, studies examining the long-term consequences of infectious epidemics have shown that some individuals may even develop symptoms of post-traumatic stress disorder (ptsd) [12, 25, 26] . one review indicated that those who develop ptsd may experience the symptoms for 3 years following the end of the epidemic [15, 23] . stress is defined as an adaptive psycho-physical reaction to a physical, social or psychological stimulus, called a stressor [27] . stress-related responses may be cognitive, emotional, behavioral, or physiological. depending on the type, timing, and severity of exposure to a stressor, the resulting stress may become a risk factor for a number of illnesses, including those of a psychiatric or cardiovascular nature [28] [29] [30] [31] [32] . an emergency such as the covid-19 outbreak might rightly be considered a severe stressor, as it is a new and unexpected situation with a potentially serious impact on health (experienced both personally and through loved ones) that also involves social restrictions [13] . nevertheless, no event, in and of itself, is the precipitating cause of pathology and illness. rather, it is the perception of stress (i.e., the degree to which one considers the event stressful) that accounts for the varying physical and mental responses to the situation [33] . in this sense, it is important to detect vulnerable persons early, and to promote effective preventive programs in order to treat such persons rapidly and limit negative psychological outcomes. the identification of psycho-social risk and resilience factors for psychological distress during the covid-19 emergency comprises a significant step in this direction [34] . to date, studies on the psychological impact of covid-19 have mainly focused on the role of sociodemographic variables (e.g., gender, age, education level, and social connections) in moderating reactions to the outbreak [10, 11, 24, 35, 36] . the identified sociodemographic risk factors for psychological distress include gender (female), age (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) or 60+ years old), student status, education level, perception of the public health system, specific physical symptoms (e.g., coryza, cough, sore throat, headache), and a low reported level of health [10, 24, 35] . for chinese students, living in an urban area, having a stable family income, living with parents, and having good social support were also found to protect against anxiety [11] . in addition to investigating sociodemographic factors, many studies have also outlined the role of certain dispositional traits in modulating responses to stressful events. however, these constructs have been poorly investigated in relation to the psychological impact of epidemics and, specifically, covid-19. more generally, research has shown how individual differences, including dispositional traits, can explain life outcomes [37] . according to the theory of traits (or dispositional theory), individual differences may be explained by certain predispositions (traits), which are expressed in a relatively stable way across situations and time. traits comprise a person's manner of thinking, feeling, perceiving, and relating to others [37] . based on these considerations, dispositional traits might play a relevant role in predicting perceived stress in relation to coping is one of the most widely studied dispositional traits, and it has been found to be significant in modulating responses to stressful events. coping is defined as the effort to solve personal and interpersonal problems in an attempt to master, minimize, or tolerate stress and conflict [38] . distinct coping strategies have been found to be differentially associated with specific emotional responses [39] , physiological stress responses [40] , and self-efficacy [41, 42] . one investigation into the role of coping strategies during a virus outbreak (2009 h1n1 flu pandemic) found coping style to influence the perceived risk of contagion and vaccination intentions among canadian adults [43] . another study, based in singapore, found coping strategies to be associated with post-traumatic outcomes within visitors to community health care services during the national outbreak of sars [23] . as regards emotional self-regulation and adaptation to the world, self-control may represent a significant protective factor. there is empirical evidence that people with high dispositional self-control have better psychological adjustment and impulse control [44] ; this suggests that good self-control may mitigate the influence of a negative environment. similarly, perceived control over life outcomes has been shown to be positively associated with well-being and health-related quality of life, and negatively associated with emotional distress, in the context of stressful events [45, 46] . in this regard, locus of control [47] is a relevant concept, describing the degree to which an individual believes that they have control over the outcome of life events, as opposed to feeling that their life is subject to external forces beyond their control. finally, among the many individual difference variables that might influence reactions to covid-19, personality traits merit significant attention. several studies have highlighted an association between the big five personality traits [48] and various health behaviors, such as sedentary behavior [49] , sexual health behavior [50] , physical activity [51] , and alcohol consumption [52] . the first aim of the present study was to investigate the impact of the covid-19 pandemic and the related government-imposed restrictions on perceived stress in a western country (i.e., italy). as reported above, most studies on the psychological impact of covid-19 have related to the chinese population. however, countries differ from one another in many important aspects (i.e., social, cultural, political, and economic aspects, to name only a few); consequently, psychological responses may also vary between contexts and communities, revealing unique qualitative and quantitative psychological reactions and psychological needs. our sample of italian adults, collected during the covid-19 outbreak, would show higher levels of perceived stress compared to italian normative values. the second aim of the study was to confirm the role of certain sociodemographic factors in modulating stress responses to the covid-19 pandemic, as reported in the recent literature. (a) participants who were female, younger, and students, with a lower level of education and lower income, would report higher levels of stress, and (b) participants who were living with others would report lower levels of psychological distress. the third aim of the work was to investigate the association between certain stable psychological traits and psychological distress relating to the current situation. to this end, participants were tested for coping strategies, self-control, locus of control, and select personality traits. (a) participants with positive coping strategies, higher levels of self-control, an internal locus of control, and higher levels of emotional stability would report lower levels of stress, and (b) participants with negative coping strategies, lower levels of self-control, an external locus of control, and lower levels of emotional stability would report higher levels of stress. finally, with the goal of anticipating persons in need of treatment and improving the targeting and overall effectiveness of preventive programs, we aimed at developing machine learning models to predict individual psychological responses to the covid-19 pandemic, based on sociodemographic and psychological variables with maximal sensitivity in classifying subjects with high versus low levels of perceived stress. to summarize, the study was novel in the following two respects: first, it considered the role of not only sociodemographic variables, but also stable psychological traits, as predictors of a stressful reaction to covid-19; and second, it leveraged machine learning techniques to identify people at the greatest risk of developing severe and negative psychological outcomes due to the pandemic. to test the abovementioned hypotheses, we implemented a cross-sectional study. using google forms, we designed an ad hoc online questionnaire to collect data on participants' stress reactions to covid-19, demographical variables, and psychological traits. the questionnaire also assessed the following sociodemographic factors: gender, age, education, number of family members and/or others living in the household, monthly household income, and student status. subsequently, we administered five standardized questionnaires, as follows: • the italian version of the 10-item perceived stress scale (pss-10; cronbach's alpha = 0.74) [53] . the pss-10 is a frequently used psychological instrument to measure perceived stress [54] . respondents are asked to answer 10 questions pertaining to the frequency of experiences of stressful situations during the last month on a five-point scale ranging from 0 (never) to 4 (very often) [55, 56] . example items include "in the last month, how often have you been upset because of something that happened unexpectedly?" higher scores indicate higher levels of perceived stress. moreover, in the present study, the score corresponding to 1.5 sd above the italian normative score [53] was used as a cut-off to divide participants into two classes: low perceived stress (males: pss-10 score <24.35; females: pss-10 score <24.55) and high perceived stress (males: pss-10 score ≥24.35; females: pss score-10 ≥24.55); • the italian shortened version of the coping orientations to the problems experienced (cope-nvi-25; cronbach's alpha of factors range 0.68-0.95) [57] . the cope-nvi-25 is a multi-dimensional inventory that assesses individual differences in coping styles. it is comprised of 25 items, which are rated on a 4-point scale ranging from 1 (i usually don't do this at all) to 4 (i usually do this a lot) [58] . the instrument includes five subscales corresponding to five different coping styles: social support, avoidance strategies, positive attitude, problem solving, and turning to religion [59] . an example item is "i admit to myself that i can't deal with it, and quit trying" (avoidance strategies). a higher score on a particular subscale indicates a greater use of that specific coping strategy. the italian translation of the brief self-control scale (bscs; cronbach's alpha = 0.85) [44] . the bscs measures individual differences in dispositional capacity for self-control. the scale is comprised of 13 items that are rated on a five-point scale ranging from 1 (not at all) to 5 (very much). an example item is "i do certain things that are bad for me, if they are fun." higher scores on the bscs indicate a greater capacity for self-control, and they are also correlated with better psychological adjustment, interpersonal skills, and emotional responses [44] ; • the italian short version of the locus of control (loc) scale [60] . this 20-item questionnaire is used to measure generalized expectancies relating to an internal versus external locus of control, rated via dichotomous options ("yes" vs. "no"), similar to rotter's original internal-external locus of control scale [47] . an example item is "to do well in life, luck is more important than commitment." respondents with an internal locus of control (i.e., a high score on the internal loc scale) tend to attribute life outcomes-and general life events-to their own behavior, whereas those with a prevalent external locus of control (i.e., a high score on the external loc scale) tend to attribute life events to fate, others, or external causes beyond their control [47, 61] ; • the italian version of the 10-item big five inventory (bfi-10; spearman-brown coefficients ≥0.50) [62] . the bfi-10 assesses personality traits according to the five-factor approach [63] . it is comprised of 10 items rated on a five-point scale ranging from 1 (strongly disagree) to 5 (strongly agree), measuring five dimensions of personality, which are extraversion, agreeableness, conscientiousness, neuroticism (or, if reversed, emotional stability), and openness [64] . an example item is "i see myself as someone who is outgoing, sociable" (extraversion). the higher the score on a particular subscale, the more that specific dimension represents a characteristic trait of the respondent's personality. the complete list of 18 variables that were extracted from the responses to the questionnaire is provided in the supplementary materials. the present research was designed in accordance with the declaration of helsinki and approved by the ethics committee for psychological research at the university of padova (protocol number 3576, unique code 189b46fe116994f1a8d1077b835d83bb). data were collected during the period of 20-31 march, 2020. participants were recruited online through an invitation posted on social media (facebook and whatsapp). this approach of online recruitment was selected primarily due to the lockdown situation, which prevented us from collecting data in the laboratory. according to the aim of the study, it was necessary for us to capture the psychological state of participants at the time of the pandemic; thus an a posteriori study would not have provided useful and reliable information. participants were invited to complete an anonymous online questionnaire to report their personal experiences with the covid-19 emergency and their mental state. the inclusion criteria were the following: (a) living in italy at the time of data collection and (b) being aged 18+ years (18 years is the legal age in italy, defined by the capacity to act and be emancipated). participation was voluntary. all participants were required to read and provide informed consent before beginning the online questionnaire. they received no compensation for their participation. in total, 2072 volunteers took part in the study. of these, 19 were excluded on the basis that they responded to the questionnaire twice (we kept only their first response). thus, the final sample was comprised of 2053 participants, of whom 1555 were female, 480 were male, and 18 were reported as "other." the participants' average age was 35.81 (sd = 13.19; range: and their average education level was 15.35 years (sd = 3.43; range: 5-21). a more detailed description of the sample's demographic characteristics is provided in the supplementary materials. it has been calculated that a sample size of 2053 is sufficiently large to achieve at least a statistical power (1-β) = 0.90 in a linear multivariable regression analysis involving 18 predictors, given a significance level α = 0.05 and an effect size of 0.356 [65] . data are provided in the supplementary materials. data analysis was conducted using the jasp software [66] . a single sample t-test (t, two-sided) was performed in relation to the pss-10 score, in order to determine whether the sample's true mean (µ) was statistically different from that of the known population (m 0 ). a multivariable regression analysis was run to investigate the relationship between the pss-10 score and the independent variables that were hypothesized to impact the level of perceived stress. the collinearity assumption was checked prior to running the model, using the tolerance and variance inflation factor (vif). as a rule of thumb, if vif >10 and tolerance <0.1, the assumption is greatly violated, whereas if vif >1 and tolerance <0.2, the model may be biased [67] . the results indicated that the collinearity assumption was not violated by any of the independent variables entered in the regression model. the analysis was performed using the stepwise variable selection method, which identified predictors with a significant (p < 0.05) individual association with the outcome (pss-10 score). the results were reported using unstandardized coefficients, as recommended by friedrich [68] . recently, researchers in different scientific fields, including the clinical and social sciences, have emphasized the utility of focusing on prediction, rather than explanation, during data analysis [69] [70] [71] [72] . this increased attention to predictive models may be largely attributed to the significant spread of machine learning (ml)-a branch of artificial intelligence that trains algorithms on data samples (i.e., training sets) in order to make predictions on completely new data (i.e., test sets) without being explicitly programmed to do so [73] . as regards psychology, ml techniques have been shown to be particularly useful for predicting human behavior, including high-risk behavior; thus, they may be applied to improve the effectiveness and targeting of preventive programs and interventions [74] . in brief, ml models are capable of predicting the behavior of individual subjects, allowing greater attention to be paid to those considered most critical [69] . in the present study, ml algorithms were trained on psycho-social data to identify subjects who were more likely to present high levels of perceived stress during the covid-19 emergency, and who were consequently at the greatest risk of developing psychological symptoms, including those of ptsd. for this purpose, participants were split into two classes: high perceived stress and low perceived stress. the high perceived stress class included participants with a pss-10 score of more than 1.5 sd above the italian population mean (n = 393) for men and women, respectively. conversely, the low perceived stress class included participants whose pss-10 did not exceed 1.5 sd above the italian normative value (n = 1642). it should be noted that participants who reported their gender as "other" (n = 18) were excluded from this analysis, as the italian normative values were available for males and females only [53] . as ml models are built to fit particular data, it is important to test how each model fits new (i.e., unseen) data. for this reason, part of the data (the training set) is generally used to train and validate the model, while another part (the test set) is used to test the model's accuracy on new examples [73, 75] . this procedure guarantees the model generalization and increases the replicability of the results [76, 77] . in the present study, 20% [73, 75] of the participants were randomly chosen and retained as the test set. accordingly, the training set consisted of 1628 participants (314 with high perceived stress and 1314 with low perceived stress), and the test set consisted of 407 participants (79 with high perceived stress and 328 with low perceived stress). in the first step, feature selection was performed to remove redundant and irrelevant features and to increase model generalization by reducing overfitting and noise in the data [78] . a good strategy for feature selection is to identify the subset of features that are highly correlated with the class to predict, but not correlated with each other [78] . this procedure was performed in the present study using the correlation-based feature selector (cfs) in the weka 3.9 software [79] . the problem of class imbalance was addressed while running the classification algorithms. the ratio between participants with high perceived stress and those with low perceived stress was approximately 1:5. as ml methods work best with balanced datasets, it is necessary to account for any class imbalance, especially when training examples are limited-a condition that is frequently met by datasets in health and clinical psychology [80] . at the same time, it is equally important for ml models to be built on samples that are representative of the population, reflecting real distribution [80] . one strategy to overcome these two limitations consists of altering the relative costs associated with misclassifying the minority and majority classes, in order to compensate for the class imbalance [81] . in the present study, ml algorithms were set in such a way that any algorithmic error made in classifying the minority class (high perceived stress) was weighted four times more than any error in classifying the majority class (low perceived stress). this cost-modifying strategy has been shown to provide better results than other methods in addressing the class imbalance problem [81] . moreover, it should be noted that, for the goal of the present task, it was more beneficial to minimize false negatives than to minimize false positives (i.e., to have a model with high sensitivity rather than high specificity). in other words, it was more important to identify people who were truly at risk than to avoid misclassifying people who were not truly at risk. ml models were trained and validated on the training sample (n= 1628) through a 10-fold cross-validation procedure using the weka 3.9 software [79] . the different algorithms (i.e., logistic regression [82] , support vector machine (svm) [83] , naïve bayes [84] , random forest [85] ) were chosen as representatives of different classification strategies, to ensure that the results would be stable across classifiers and not dependent on specific model assumptions (details on the parameters of the ml classifiers are reported in the supplementary materials). k-fold cross-validation is a resampling procedure that seeks to reduce the variance in model performance relative to the performance that may be obtained from a single training set and a single test set. the procedure consists of portioning the sample into k subsets (i.e., folds; in the present study, k = 10), and using k-1 (i.e., 9) subsets to train the model and the remaining subset to validate the model's accuracy. this is repeated k (i.e., 10) times [86] . the final model metrics are obtained by averaging the metrics obtained in all validation subsets. in the present study, the models developed from the 10-fold cross-validation procedure were tested on the test sample (n = 407). the main results of the data analysis are reported in this section. a more complete descriptive analysis of each variable, including the composition of high perceived stress versus low perceived stress samples, is reported in the supplementary materials. the average pss-10 score of the entire sample was 18.81 (sd = 6.25). analyzing the responses of males and females separately (note that participants who reported a gender of "other" were excluded from this analysis due to a lack of normative data), males obtained an average score of 16.71 (sd = 6.91) and females obtained an average score of 19.44 (sd = 6.79). to determine whether the sample mean statistically differed from that of the italian normative population (males: average = 15.2, sd = 6.1; females: average = 16.3, sd = 5.5) [53] , a one-sample t-test was run separately for each gender. a first multiple regression analysis was run, including sociodemographic variables that have been shown to potentially impact the level of perceived stress during a pandemic [10, 11, 24, 35, 36] . the pss-10 score was set as the dependent variable, while gender (male), age, education, monthly income, number of family members, and student status (student) were entered as covariates. the final model accounted for a significant proportion of the variance in the level of perceived stress (r 2 = 0.103, adjusted r 2 = 0.101, f-change (1,2029) = 12.009, p < 0.001). all of the aforementioned variables, with the exception of student status, were found to contribute to the level of perceived stress. results are reported in table 1 . to better understand the role of stable psychological traits in predicting the level of perceived stress (pss-10 score), a second multiple linear regression was run, adding to the previous model the scores of the five coping styles measured by the cope-nvi-25 (cope positive, cope problem, cope avoidance, cope religion and cope support), the bscs total score, the internal loc score, and the scores for the five personality traits measured by the bfi-10 (bfi-10 agreeableness, bfi-10 conscientiousness, bfi-10 emotional stability, bfi-10 extraversion and bfi-10 openness). this second model accounted for a larger proportion of the variance in the level of perceived stress (r 2 = 0.356, adjusted r 2 = 0.352, f-change (1,2022) = 5.908, p < 0.05) compared to the previous model. bfi-10 emotional stability, cope positive, age, bcsc total score, gender (male), cope avoidance, internal loc, number of family members, cope support, monthly income, and bfi-10 conscientiousness were identified as significant predictors of the level of perceived stress during the covid-19 epidemic (see table 2 ). education, cope religion, cope problem solving, bfi-10 agreeableness, bfi-10 extraversion and bfi-10 openness were excluded. ultimately, the 18 questionnaire variables were considered predictors of perceived stress. the entire list of predictors, along with their descriptions, is provided in the supplementary materials. of these 18 variables, the following 7 were identified as the best set of predictors using correlation-based feature selection: age, monthly income, cope avoidance, cope positive, bscs total score, bfi-10 emotional stability, and bfi-10 agreeableness. using these predictors, ml algorithms were trained and tested according to the procedure described in the "data analysis" section. classification results for the test set are reported in table 3 , which quantifies predictive performance according to the following metrics: receiver operating characteristic curve (roc) area, precision, recall and f-measure (f1 score). it is worth noting that the classifiers showed an roc area ranging from 0.70 to 0.78 in the test set. however, the random forest algorithm highlighted the lower sensitivity (recall) of the high perceived stress class compared to the other classifiers, making it a weaker model for the purposes of prediction. the present study measured the impact of the covid-19 emergency on perceived levels of stress, taking into account sociodemographic variables and stable psychological traits. the results confirmed that participants perceived the covid-19 crisis as a stressful experience; in the present sample, the level of perceived stress was higher than that of the general population in a non-emergency condition. indeed, almost 20% of the sample scored above the results from the normative data on measures of perceived stress. these results are in line with the findings of recent studies on the psychological impact of covid-19 [10, 11, 87] and the international literature on epidemic outbreaks [13] . the mean values of the single items of the pss-10 suggest that, in addition to nervousness and stress, feelings of being unable to control one's personal life accounted for the majority of participants' perceived stress. this suggests that the unpredictability and uncontrollability of the pandemic may play a significant role in determining levels of perceived stress during the crisis. moreover, it may reflect participants' attitudes toward the significant lifestyle changes demanded of them due to the lockdown and other restrictive measures. as regards sociodemographic variables, the results suggest that the female gender is associated with higher levels of stress. this is consistent with the literature indicating gender differences in the psychological response to covid-19 [24, 88] and other epidemics [89] ; it is also in line with the normative data for the general population. consistent with other studies (20, 30) , the present study found an association between higher incomes and lower levels of perceived stress. one explanation for this is that higher incomes might be related to less concern about the economic effects of self-isolation and/or with more comfortable housing solutions (e.g., larger living spaces, access to outdoor spaces (such as gardens), and access to leisure activities). moreover, people with higher incomes may be more likely to perform work that can easily and fully transition to the online environment, thereby reducing some sources of stress. in the present sample, older age was found to be associated with lower levels of stress. this finding might appear surprising, since it contradicts both the results of studies on the chinese population [24, 35] and the association between older age and higher covid-19 mortality. however, the result is in line with recent italian data [87] . several studies have indicated age-related differences in coping and locus of control, with older adults presenting greater self-control and emotional self-regulation relative to younger adults [90] [91] [92] . considering the current pandemic, older people may be more used to staying at home, so their daily routines might be less impacted by mandatory self-isolation measures. data from previous investigations on age differences in stress responses to the sars epidemic reflect inconsistencies [93, 94] , but sociopolitical and cultural aspects, such as differences in elder care services and policies, might account for these discrepancies. the present study did not find education to be a significant predictor of the level of perceived stress. the large percentage of highly educated participants in the sample might partially explain this finding. however, prior research on this subject has generated mixed results-recent studies on the chinese population [24, 35] have found that education does not seem to affect mental health, while data from a spanish sample [88] and from previous studies on psychological adjustment to sars [95] have confirmed an association between a higher level of education and better mental health. the present study also found that living alone or with few family members was a protective factor against perceived stress. we might argue that this condition both conveys a sense of protection from contagion and offers continuity with pre-epidemic economic and social conditions. moreover, when cohabiting with family members, concern for loved ones might contribute to increasing perceived stress. as regards psychological variables, emotional stability was found to be an important protective factor. according to the five-factors model [63] , people with high emotional stability remain calm in response to stressful situations, and view problems in proportion to their importance. as a result, they tend to worry less about problems than do people with low emotional stability [96] . many studies have found that emotional stability is able to buffer stress responses to adverse events [97, 98] . in the present study, conscientiousness and agreeableness were found to predict psychological distress. generally speaking, individuals who score high on agreeableness tend to dislike conflict and be less suspicious of others; generally, they seek to pacify and mediate. in this sense, agreeable people might be more flexible and accepting when faced with unexpected and undesired situations, such as restrictions and changes to daily routines. conscientious people are more likely to perceive lower levels of stress (see correlation analysis). according to the literature, they are more aware of their actions and tend to exhibit more goal-oriented behavior. in this sense, in times of self-isolation, conscientious people might have a greater tolerance for frustration and imposition relative to less conscientious people, who might engage in more impulsive behavior [96] . moreover, previous research has indicated that conscientiousness may influence adaptive behavior, especially in health-related programs [99, 100] . the present study found higher levels of dispositional self-control to predict lower levels of psychological distress in response to the covid-19 emergency. personal self-control skills may play a role in determining tolerance to restrictions to personal freedom during self-isolation. this result further suggests that self-regulatory processes may have a strong influence on responses to the outbreak. the results regarding dispositional self-control are consistent with those relating to the emotional stability trait of the big five model. in fact, these dimensions are often correlated [101] , and this specific pattern may indicate the importance of personal skills, such as the ability to remain calm, and maintain emotional balance and a sense of acceptance. in this sense, practices that enhance emotional stability and acceptance, such as mindfulness, could be useful in reducing the stressful impact of the emergency [102, 103] . the results concerning coping styles are also in line with this. besides confirming the protective effect of functional coping styles and the adverse impact of dysfunctional coping styles, the results of the present study suggest that people who use a positive attitude as a coping strategy may be much less likely to experience psychological distress during the present emergency. such persons may appraise the emergency as a unique opportunity, and feel less need for psychological support. in contrast, the present findings suggest that people who use avoidance strategies may be more likely to experience higher levels of stress during the emergency. these results are consistent with the findings of previous investigations into the relation between coping style and response to an epidemic [23, 43, 104, 105] . the results relating to dispositional locus of control indicate that people with an internal locus of control may be less likely to feel stressed. again, these results suggest that the more people are inclined to confidently rely on themselves, the better they will cope with uncertainty and change. several studies have indicated an association between an internal locus of control and self-efficacy and emotional stability [106, 107] . furthermore, previous studies have found a relation between an internal locus of control and the positive appraisal of an emerging infectious disease outbreak [93] . it could be hypothesized that people with an internal locus of control interpret self-isolating as something that they determine and enact for themselves as a protective behavior, rather than something that is imposed on them; this might account for their lower levels of perceived stress. overall, the results of the present study identify some population subgroups that may be more vulnerable to experiencing stress during the covid-19 emergency. specifically, a set of seven psycho-social variables may identify a high percentage of people experiencing high stress during the covid-19 pandemic, with sensitivity approaching 0.759 (roc area of predictive models ranging between 0.70 and 0.78). according to this model, we may develop targeted preventive interventions. furthermore, self-regulatory skills (including emotional stability, an internal locus of control, and self-control) were shown to be a protective factor, indicating the importance of raising awareness of these skills during the emergency and offering training and education to increase personal abilities in these areas (e.g., mindfulness programs). the present research aimed at improving our understanding of the possible risk and protective factors for high perceived stress during the covid-19 outbreak. it is worth noting that all data were collected from an italian population. therefore, the findings were inevitably influenced by specific contextual and socio-cultural aspects. further investigations involving people of different ethnicities and residents of other countries would deepen our understanding of the generalizability of these results, and the effective influence of psychological traits. in this regard, the open access data reported in the supplementary materials may contribute to facilitating comparisons between ethnicities, countries, and specific traumatic events. some further limitations should be considered when interpreting the findings of this research. first, participants were recruited via an online link posted on social networks. while online recruitment guarantees large samples, it does not guarantee sample representativeness. for this reason, very vulnerable groups, such as homeless or low-income persons, may not be well represented in this study. similarly, the average age of the sample was young, predominantly female, and largely well educated, as indicative of a sample that is more likely to participate in an online survey. second, the use of self-report measures did not enable us to verify the reliability of the responses, or to ensure that participants correctly understood the questions. future research should aim at overcoming these shortcomings. finally, future research should also investigate the interplay and mutual interrelationship between protective and risk factors, to improve the targeting and overall effectiveness of preventive programs and interventions. indeed, the literature suggests that, during a pandemic, it is extremely important for people to sustain their use of psychological services, either online or in the context of social distancing [108, 109] . this is particularly essential for those who are more vulnerable to experiencing high levels of stress, and it is important that we ensure that such persons can access timely and high-quality psychological services in order to prevent the development of chronic outcomes, including ptsd. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3350/s1, table s1 : descriptive statistics, table s2 : item by item analysis of the pss-10, table s3 : list of predictors, table s4 : details on ml classifiers parameters. funding: this research received no external funding. the authors declare no conflict of interest. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak characteristics of and important lessons from the coronavirus disease 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interventions to reduce diabetes distress: the role of personal conscientiousness and diabetes self-efficacy higher-order factors of the big five let's conquer covid-19 and sustain our abilities mindfulness mediates the physiological markers of stress: systematic review and meta-analysis coping responses of emergency physicians and nurses to the 2003 severe acute respiratory syndrome outbreak the importance of coping appraisal in behavioural responses to pandemic flu relationship of core self-evaluations traits-self-esteem, generalized self-efficacy, locus of control, and emotional stability-with job satisfaction and job performance: a meta-analysis perceived behavioral control, self-efficacy, locus of control, and the theory of planned behavior 1 psychological interventions for people affected by the covid-19 epidemic the role of telehealth in reducing the mental health burden from covid-19 key: cord-353592-gsvobusu authors: luceño-moreno, lourdes; talavera-velasco, beatriz; garcía-albuerne, yolanda; martín-garcía, jesús title: symptoms of posttraumatic stress, anxiety, depression, levels of resilience and burnout in spanish health personnel during the covid-19 pandemic date: 2020-07-30 journal: int j environ res public health doi: 10.3390/ijerph17155514 sha: doc_id: 353592 cord_uid: gsvobusu the number of health workers infected with covid-19 in spain is one of the highest in the world. the aim of this study is to analyse posttraumatic stress, anxiety and depression during the covid-19 pandemic. associations between burnout, resilience, demographic, work and covid-19 variables are analysed. cross-sectional data on 1422 health workers were analysed. a total of 56.6% of health workers present symptoms of posttraumatic stress disorder, 58.6% anxiety disorder, 46% depressive disorder and 41.1% feel emotionally drained. the profile of a health worker with greater posttraumatic stress symptoms would be a person who works in the autonomous community of madrid, in a hospital, is a woman, is concerned that a person he/she lives with may be infected, and thinks that he/she is very likely to be infected. the risk variables for anxiety and depression would be a person that is a woman, working 12or 24-h shifts, and being worried that a family member could be infected. high scores on emotional exhaustion and depersonalization are risk factors for mental health, with resilience and personal fulfilment being protective variables. data are provided to improve preventive measures for occupational health workers. the world health organization declared the covid-19 outbreak as a pandemic on march 11, 2020 . in europe, italy and spain were the first to report a high number of deaths, as well as a rapid increase in admissions to intensive care units (icu) of patients with symptoms associated with the disease. in may 2020, spain is one of the top five countries with the highest number of people infected, registering over 242,707 cases as of 12 june 2020, and more than 27,136 deaths [1] . in critical pandemic-related situations, research indicates that individuals experience a stress response associated with their fear of contracting the virus from contact with other people or objects. they also have symptoms of posttraumatic stress, such as intrusive thoughts, insomnia or nightmares [2] . during the epidemic of severe acute respiratory syndrome (hereinafter, sars), a high prevalence of symptoms of posttraumatic stress, anxiety and depression was identified in emergency service professionals including hyperarousal, anger, loss of motivation at work, difficulty concentrating or trouble falling asleep [3] . however, not all individuals exposed to high negative impacts or crisis situations develop such symptoms, with resilience being relevant as a protective factor [4, 5] . resilience, the individual's ability to deal with adversities as challenges, has been shown to reduce the impact of traumatic events, decreasing the likelihood of developing posttraumatic stress disorders [6] . resilience can be understood as a process of positive adaptation to a stressful situation, in which an interaction between personal resources and the environment is established [7] . resilience varies from person to person and depends on several factors, such as personality or interpersonal and social backgrounds. the strategies to cope with the current pandemic that have been identified are optimism, social support, staying actualized, avoiding information overload and maintaining online communication [8] . in healthcare personnel, a key factor for promoting resilience is to increase the sense of control over the adverse situation. for example, perceiving that disease prevention measures can be managed or controlling the possibility of protecting oneself with the resources that health care providers have around them to care for infected patients are some of the strategies that have been adopted in this pandemic [9] . in similar critical situations, such as the severe acute respiratory syndrome (sars) epidemic, nurses who have shown higher levels of confidence in infection protection and control equipment have shown lower levels of anxiety, negative mood and emotional fatigue [10] . individuals with high levels of resilience have less irritability, less concern for environmental stimuli, better interpersonal relationships, fewer headaches and musculoskeletal pains, and lower levels of depression [11] . if these symptoms persist over time, the feeling of a lack of control and uncertainty at work may increase, leading to burnout. this syndrome is related to work. it is characterized by high emotional exhaustion, high levels of depersonalization and low personal accomplishment [12] . the person may experience dysphoric symptoms, such as tiredness or emotional exhaustion. the symptoms appear in relation to work situations in individuals who previously did not show psychosocial alterations. in addition, burnout is associated with a decrease in work performance due to negative behaviors towards work [13] . in particular, in health workers exposed to traumatic situations during this pandemic, the presence of burnout has been detected, as well as a reduction in the ability to apply coping strategies or negative attitudes towards work. in addition to the symptoms of exhaustion, related to anxiety, depression or other symptoms related to physical pathologies (e.g., cardiovascular problems), burnout can lead to intention to leave the post, which would cause high costs [14] . the speed with which the disease has spread, as well as the state of confinement, has led some researchers to analyse psychological variables resulting from the situation. for example, in a recent study of the general spanish population, in which 3480 people participated, more than 20% were found to suffer from anxiety, 18.7% revealed symptoms associated with depression, and approximately 16% suffered from posttraumatic stress. in addition, female gender was associated with greater symptomatology in anxiety, depression and posttraumatic stress, while being in the older age group was related to fewer symptoms [15] . this data are similar to those obtained in a survey carried out in the general population in china, in which 1210 people participated, of which 16.5% reported moderate to severe depressive symptoms and 28.8% moderate to severe anxiety [16] . in other european countries, such as italy, the general population has shown high levels of anxiety, depression and stress, highlighting a higher prevalence in women, people with negative affect and individuals who had family members infected or had to work away from home [17] . however, despite the state of confinement, certain professional groups, as in the case of health care personnel, have performed their jobs under great stress for weeks. these professionals, together with security forces, funeral staff and others, have been highly exposed to the virus and situations with high emotional impact. they are thus more likely to suffer mental problems, especially in the first three months in which symptoms of posttraumatic stress, affective disorders, burnout or others may increase [18] . spain leads the ranking of the number of health professionals infected with covid-19 during their work. more than 40,000 health workers have tested positive for covid-19 [19] . the critical situation requires the study of the psychological state of health professionals, and the potential harm to mental health caused by their exposure during the pandemic. a recent study on stress in 958 health workers from the city of wuhan indicates that more than half had symptoms related to anxiety and depression. specifically, 54% of the total sample experienced symptoms of anxiety, and 58% of depression, with the prevalence of stress being higher than that previously detected in health professionals who had to deal with the sars virus [20] . in a study involving 1257 health workers from china, of which 760 were from wuhan, 71.5% also showed symptoms of distress, 44.6% of anxiety, 50.4% of depression and 34% of insomnia. these symptoms were more severe in nursing staff, front-line professionals and those who worked at the epicentre of the covid-19 outbreak (wuhan) [21] . similar results have been found in other european countries such as germany, where health workers, specifically nurses, have reported high levels of stress, emotional fatigue and depressive symptoms [22] . the impact of the situation on health workers may even produce symptoms of psychotic disorder, even when this is not evident in their clinical history [23] . additionally, the stress generated by the possibility of being infected with the disease also adds to the rest of the stressful conditions of these professionals. during the influenza a (h1n1) pandemic, health workers were reported to be twice as likely to be infected through contact with patients [24] . working on the front line with infected people increases the likelihood of becoming infected, especially in this group [25] . health professionals must work in extreme conditions, in situations where resources can be scarce. for example, they must take care of a high number of patients in disaster or epidemic situations, often without sufficient beds or staff [26] . in addition to their jobs in hospitals, primary care or intensive care units, they also work in nursing homes, where the disease has had a major impact in spain. some authors indicate that risk factors for infection may include: (a) factors related to organization, such as the rapid development of new tasks and procedures, a shortage of protective material, frequent equipment changes or the high risk of increased demand for care by other different pathologies, in addition to covid-19; (b) watching patients die alone; (c) fear of infecting loved ones or having to practice social distancing for an indefinite period to protect them; and (d) prioritization of care for certain patients [27] . on the basis of the above, health professionals must deal with possible psychological, work-related consequences during the covid-19 crisis, such as posttraumatic stress, anxiety, depression or burnout [28] .the aim of this study is to assess the symptoms of posttraumatic stress, anxiety, depression, levels of burnout and resilience in the spanish health workers during the covid-19 pandemic. it also aims to evaluate the relationship between each of the variables (demographic, work, covid-19, burnout and resilience) and the symptoms of posttraumatic stress, depression and anxiety. it is equally intended to identify which variables have the most weight in each of the three categories (posttraumatic stress, anxiety and depression). the main hypotheses of this study would be the following: (a) health care workers evaluated will have high levels of post-traumatic stress, anxiety, depression and burnout; (b) resilience factor will be associated with lower burnout and with symptoms related to the above three categories; (c) the female gender will be associated with symptoms of the three categories; (d) older health care personnel would have fewer symptoms; (d) health care workers in contact with other infected patients, who are highly likely to become infected and have fewer resources or protective equipment, will have more symptoms of post-traumatic stress disorder, anxiety, depression and burnout. the sample includes 1539 subjects, recruited by non-probabilistic sampling. as criteria of exclusivity, the participants had to be in contact with patients of covid-19. finally, 117 were eliminated because they were not health personnel in contact with these patients. the sample of the study was made up of 1228 women (86.4%) and 194 men (13.6%). the mean age was 43.88 (sd = 10.82, ranging between 19 and 68). the following instruments were used: demographic, job-related and variables specific to covid-19. due to the importance of understanding how the disease affects these professionals, the researchers of this study collected information related to demographic variables, associated with the job, changes of residence, possible contact with people during work, covid-19 tests, hospitalization, isolation, protective equipment, concern over becoming infected, concern that a family member and/or someone with whom they are living may be infected. posttraumatic stress: the impact of event scale-revised (ies-r) was used [29] . this scale was used to assess the emotional distress that accompanies a stressful life event. it is made up of 22 items distributed in three scales: intrusion (7 items, an example of this scale would be "i thought about it even when i did not want to"); avoidance (8 items, an example is "i tried not to think about the event"); and hyperarousal (7 items, a sample item is "i was easily startled and scared"). in relation to posttraumatic stress, a score of 20 was considered as the cut-off point. a total score greater than or equal to 20 on the ies-r is associated with a diagnosis of psychiatric disorder and a mean score less than or equal to 14 is associated with a non-diagnosis of a psychiatric disorder [30] . it shows adequate psychometric properties in its spanish adaptation, confirming the solution of the three factors mentioned and a reliability greater than 0.70 in all subscales. anxiety and depression: the spanish adaptation of the hospital anxiety and depression scale (hads) instrument was used [31, 32] . consisting of 14 items that correspond to two subscales: anxiety and depression, with 7 items each, on a likert 0-3 response scale. an example of an item in the anxiety scale is "i feel tense and nervous" and "i feel slow and awkward" in the depression scale. it evaluates symptoms of anxiety and depression in patients and in the general population. the cut-off values are between 7 and 13 possible or probable presence of a mood disorder, and greater than 14-15 for severe disorder (the range ranges from 0 to 21) for both anxiety and depression scales. accordingly, to analyse the prevalence of symptoms in this study, the variables have been categorized as follows: < 6.99 no disorder, 7-13.99 possible or probable, > 14 severe disorder. the higher the score, the greater the prevalence of symptoms of anxiety and depression. in its spanish adaptation, it has shown adequate psychometric properties, confirming the validity of two factors and an internal consistency of 0.77 in anxiety and 0.71 in the depression subscale [32] . burnout: the spanish adaptation of the maslach burnout inventory-mbi-hss instrument was applied, which assesses burnout syndrome [33, 34] . it consists of 22 items of seven response options on a likert scale from 0 (never) to 6 (every day). the cut-off points for health personnel in the spanish sample were used to analyse the prevalence of the different components of burnout in this study: emotional fatigue (low < 22, medium 22-23, high > 31); depersonalization (low < 7, medium 7-13, high > 16); and personal accomplishment (low < 30, medium 30-35, high > 35). it presents adequate psychometric characteristics, showing an appropriate fit for the three-factor solution and an internal consistency greater than 0.71 in all subscales [35, 36] . an example of an item in the emotional exhaustion scale is "i feel emotionally drained for my work", "i think i treat some people like impersonal objects" in the depersonalization scale, and "i easily understand how people feel" in the personal accomplishment scale. resilience: the spanish adaptation of the brief resilience scale (brs) was used [37, 38] . it evaluates the resilience construct, understood as the subject's ability to deal with environmental obstacles and recover from stressful circumstances. it is made up of 6 items that are answered on a likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). the higher the score, the greater the degree of resilience the person shows to deal with adversities. an example of an item in this scale is "i tend to recover quickly after going through difficult times". the spanish adaptation presents adequate psychometric properties, corroborating the single-factor solution and an internal consistency of 0.83. the approval of the deontological committee of the faculty of psychology of the complutense university of madrid (ref. pr_2019_038; 01/04/2020) was obtained before beginning the study. researchers contacted both the coordinators and trade unions of health centres to inform them of this study. due to lockdown, data were collected by means of an online survey, from 1 to 30 of april 2020, which included the instruments described above. before starting the survey, participants had to give their informed consent in order to continue. informed consent included the purpose of the study, those responsible for it and information on the confidentiality of the data, anonymity and the legal clause on personal data protection. before completing the survey submission, participants were required to respond to all items. the completion time for all items was approximately 15 min. the analyses were carried out with the spss 26 statistical package. the proportion of cases with symptoms of the disorders mentioned above was analysed. descriptive analyses (frequencies, mean, standard deviation) were performed for symptoms associated with posttraumatic stress disorder, anxiety, depression, burnout and resilience. linear regression equations were used to evaluate the relationship between each of the variables (demographic, work, covid-19, burnout and resilience) irrespective of the symptoms (posttraumatic stress, anxiety and depression), using the r 2 value and the standardized β coefficient. the objective was to calculate the impact of posttraumatic stress, anxiety and depression on each of the variables. dummy variables were used for this. finally, linear regression models were used to see which variables (demographic, work, covid-19, burnout and resilience) were jointly related to symptoms of posttraumatic stress, anxiety and depression. the model was estimated by least squares, using the forward extraction method. analyses were carried out to evaluate the proportion of health care cases with symptoms associated with possible posttraumatic stress disorder, anxiety, depression and burnout at their different severity levels. the results of both genders are shown in table 1 . there are gender differences in symptoms of post-traumatic stress disorder, anxiety and depression. in addition, there are differences between men and women in depersonalization scale of burnout. the data of internal consistency and correlations between the factors evaluated with the instruments used are represented in table 2 . regarding gender and age, being a woman is positively and significantly associated with posttraumatic stress, anxiety and depression, while age is negatively and significantly associated with symptoms of posttraumatic stress and anxiety. working outside the autonomous community of madrid and in any centre other than primary care, hospital, nursing home or day centres (other category) is negatively and significantly related to posttraumatic stress. possessing doctoral and postgraduate studies is negatively and significantly associated with anxiety and posttraumatic stress, respectively. being a member of the non-supervisory staff is positively and significantly associated with posttraumatic stress, just as being a doctor is negatively and significantly associated with symptoms of all the evaluated categories. the caregiver position is negatively and significantly associated with anxiety and depression. working a fixed shift in the afternoon is positively and significantly associated with posttraumatic stress, anxiety and depression, while working a night shift is only positively and significantly associated with posttraumatic stress. with respect to rotating shifts, the late-night shift and on-call, or 12/24 h shifts are positively and significantly associated with posttraumatic stress and depression, respectively. working part-time is positively and significantly associated with all three categories (posttraumatic stress, anxiety, and depression). having a permanent statutory (civil servant) contract and having a training contract are negatively and significantly associated with posttraumatic stress and depression, respectively. finally, the number of hours worked per week is positively and significantly associated with depression, and the number of on-call hours performed per month is positively and significantly associated with posttraumatic stress and depression (see table 3 ). changing address or living with people who are at risk is positively related to symptoms of posttraumatic stress, anxiety or depression. on the other hand, having personal protective equipment and not being very concerned that family members are infected is negatively related to symptoms of posttraumatic stress, anxiety or depression. hospitalization for symptoms of covid-19 and isolation due to possible contagion of the disease is positively related to posttraumatic stress. thinking that becoming infected with covid-19 is highly unlikely is negatively related to symptoms of posttraumatic stress and anxiety. emotional fatigue and depersonalization are positively and significantly related to symptoms of posttraumatic stress, anxiety and depression, while personal accomplishment is negatively and significantly related to symptoms of anxiety and depression. finally, resilience is associated in a negative and significant way with symptoms from all the evaluated categories. on the other hand, these professionals present moderate levels of resilience, while the highest possible score on resilience is 6, the mean score for these individuals is 3.02 (sd = 0.39), therefore indicating moderate levels. all associations can be seen in table 4 . as shown in table 5 , the posttraumatic stress symptom model was significant, explaining 39.6% of the variance (f(17, 1405) = 54,022, p < 0.001). it was also significant for anxiety symptoms, explaining 40.2% of the variance (f(12, 1412) = 78.593, p < 0.001). in relation to the depression model, it was significant, explaining 39.3% of the variance (f(14, 1408) = 64.932, p < 0.001). the variables common to the three models were emotional fatigue, depersonalization, resilience, gender, and concern that someone with whom they live could be infected. the variables positively related to posttraumatic stress are emotional exhaustion, depersonalization, working in the autonomous community of madrid, having a primary education, working in a hospital, being very concerned that someone with whom they live may become infected and thinking that there is a high risk of also becoming infected with covid-19. on the other hand, the variables negatively related to posttraumatic stress are resilience, being a man, having a doctor's degree, living with an unmarried partner, being a doctor or having another profession (mainly pharmacist or psychologist, but not a nurse, nursing assistant or caregiver), having protective equipment at work, not being concerned that someone you live with can be infected with the disease and the number of people you live with. the variables positively and significantly related to anxiety would be emotional exhaustion, depersonalization, 12-or 24-h shifts or on-call hours and being very concerned that someone with whom they live could be infected. the variables negatively and significantly related to anxiety would be resilience, being a man, being separated, working in nursing homes or day centres, being a doctor, having a rotating morning-afternoon shift and not having been isolated due to covid-19. finally, having symptoms of depression is positively and significantly related to: emotional exhaustion, depersonalization, 12-or 24-h shifts or on-call hours, the number of guards per month, being very concerned that someone with whom you live may be infected, not having a family and thinking that it is very likely that you will be infected with covid-19. the variables negatively and significantly related to depression would be personal fulfilment, resilience, being a man and having a fixed or training contract. the gender-differentiated posttraumatic stress models were significant, in both men and women, explaining 35.2% of the variance in women (f(13, 1210) = 50,667, p < 0.001) and 53.7% in men (f(9, 183) = 23,548, p < 0.001). on the anxiety scale, the models were significant, explaining 36.3% of the variance in women (f(11, 1212) = 64,280, p < 0.001) and 62.7% in men (f(6,168) = 52,066, p < 0.001). for the depression scale, the models were significant, explaining 37.73% of the variance in women (f(14, 1223) = 52.310, p < 0.001). tables 6 and 7 show the regression models of post-traumatic stress, anxiety and depression, differentiated by gender. in relation to the gender-differenciated models, in women (see table 6 ): the symptoms of posttraumatic stress are positively and significantly related to emotional exhaustion, depersonalization, working in a hospital, being very concerned that someone with whom they live may become infected and thinking that becoming infected with covid-19 is very likely. on the other hand, the variables negatively related to posttraumatic stress are personal accomplishment, resilience, living with and unmarried partner, working in nursing homes, being a doctor or having another profession (mainly pharmacist or psychologist, but not a nurse, nursing assistant or caregiver) and thinking that is very unlikely to be infected with covid-19. the variables positively and significantly related to anxiety would be emotional exhaustion, depersonalization, being very concerned that someone you live with can be infected with the disease and change of residence through fear of infecting family members. the variables negatively and significantly related to anxiety would be resilience, being separated, working in nursing homes and not being at all concerned that someone you live with can be infected with the disease. having symptoms of depression is positively and significantly related to emotional exhaustion depersonalization, being a nurse, 12-or 24-h shifts or on-call hours, those who live with people who are at risk and being very concerned over a possible infection of a family member they do not live with. the variables negatively and significantly related to depression would be personal accomplishment, resilience, being a doctor, having a fixed or training contract. in relation to the gender-differentiated models, in men (see table 7 ): the symptoms of posttraumatic stress are positively and significantly related to emotional exhaustion, having a primary education and being very concerned that someone with whom they live may become infected. on the other hand, the variables negatively related to posttraumatic stress are resilience, having a doctor's degree, not having been isolated due to covid-19 and not being concerned that someone with whom you live with can be infected with the disease. the variables positively and significantly related to anxiety would be emotional exhaustion, being a nurse and having been isolated due to covid-19. the variables negatively and significantly related to anxiety would be resilience, having a training contract and having a statutory fixed-term employment. having symptoms of depression is positively and significantly related to emotional exhaustion depersonalization and being very concerned that someone with whom they live may become infected. the variables negatively and significantly related to depression would be personal accomplishment, resilience, having a doctor's degree, having a training contract, not having been isolated due to covid-19 and not having been hospitalized for symptoms compatible with those of coronavirus. table 6 . regression models for posttraumatic stress, anxiety and depression in women (n = 1228). table 7 . regression models for posttraumatic stress, anxiety and depression in men (n = 194). this research aimed to assess the symptoms of posttraumatic stress, anxiety, depression, burnout and resilience in spanish health workers during the covid-19 pandemic. it was also aimed at evaluating the relationship between each of the variables independently (demographic, work, covid-19, burnout and resilience) and the symptoms of posttraumatic stress, depression and anxiety, as well as the variables that (together) carry more weight in each of the three categories (posttraumatic stress disorders, anxiety and depression). the results show that 56.6% of health workers present symptoms of posttraumatic stress disorder. the number having a possible anxiety disorder is 58.6%, with 20.7% having a severe disorder. equally, a high percentage, specifically 46%, would have a possible depressive disorder and 41% feel emotionally drained. in this sense, the first hypothesis would be fulfilled, although it would be necessary to make a thorough evaluation to determine a clinical diagnosis. most workers present probability of developing a posttraumatic stress disorder, anxiety or depression. during the middle east respiratory syndrome (mers) or ebola crises, among others, health professionals reported a higher number of symptoms related to posttraumatic stress [39] , so it is necessary to pay attention to the increase in these symptoms, even more so in the situation of the covid-19 pandemic that has not yet subsided. the demographic variables show that having a doctoral or postgraduate degree represent protective variables of anxiety and posttraumatic stress, respectively. in addition, lower-level workers show more symptoms of posttraumatic stress. this result may be due to the fact that, in lower-level jobs, control over procedures and decision-making capacity is lower than in other higher-level positions. some authors have shown that the main differences among professions regarding the symptomatology evaluated during the covid-19 pandemic apply to nurses and other positions, such as doctors. nurses present more symptoms of anxiety and depression [40] . these differences may be associated with the contact of these professionals with infected patients. on the other hand, being a woman is associated with greater symptoms of posttraumatic stress, anxiety or depression in the sample of health workers evaluated. younger health workers show greater levels of posttraumatic stress and anxiety. this may be due to a lack of work experience in similar stressful situations. another possible reason is that, during the current pandemic, the lack of health care staff has required that senior students or people with fewer experience have had to deal with the demands of the covid-19 patients. the data obtained in this study on the gender and age variables coincide with the findings of other studies in health personnel from different countries [41, 42] . some authors suggest that, both in the current situation due to covid-19 and in similar previous situations, symptoms of stress, anxiety and depression generally increase in health professionals and also coincide in pointing out that women present more symptoms than men do. regarding gender differences, the data obtained in this study may be due to the high number of women in positions such as nurse or nursing assistant. on the other hand, in mood disorders, which have a high comorbidity with those of anxiety, there is a high prevalence of women compared to men. for example, women present more rumination and there are hormonal differences that can explain these results [43] . variables related to jobs show that those health workers who have part-time jobs have more symptoms of posttraumatic stress, anxiety and depression. the shifts most related to psychological problems are the night, afternoon and afternoon-night rotating shifts. in this regard, similar results have been identified with health personnel that indicate the association between working the night shift and having gastrointestinal problems, hormonal problems, and changes in mood and cognitive state, among others [44] . regarding the work shift, other authors specify that there is a greater risk of having symptoms of depression as the number of days worked in the night shift increases [45] . doctors have fewer symptoms of posttraumatic stress, anxiety, or depression, and caregivers have fewer symptoms of anxiety and depression. health workers with a lower job category have more symptoms of posttraumatic stress, while those who work more hours a week have more symptoms associated with depression. health workers who do more on-call hours a month have more symptoms of posttraumatic stress and depression. therefore, the hours of rest for these professionals must be respected. in relation to the information collected on covid-19, it should be noted that the health workers who have had to change their residence due to the pandemic have been isolated due to possible contagion, and those who live with people who are at risk or think they may infect other people have more symptoms of posttraumatic stress, anxiety or depression, although these differences were not statistically significant in depression for the two variables: being isolated due to possible contagion and the likelihood of becoming infected with covid-19. regarding the hypothesis related to these variables, it would be partially fulfilled, since the association between the possibility of infection and the symptoms of depression would not be significant. recent research indicates that one of the greatest concerns of health personnel is the possibility of infecting others, especially family members [46] . believing that they are very unlikely to be infected with covid-19 is related to fewer symptoms of posttraumatic stress and anxiety. on the other hand, as proposed in the study hypothesis, those health workers who have personal protective equipment to cope with the disease have fewer symptoms of posttraumatic stress, anxiety or depression. these results may be due to the fact that, on the one hand, contact with people who may be infected is a risk factor for imminent contagion among health workers, as a result of the high transmission of the disease [47] ; on the other hand, the use of personal protective equipment is essential to be able to work and treat patients with covid-19 [48] . the profile of a health worker with greater symptoms of posttraumatic stress would be a person who works in the autonomous community of madrid, in a hospital, is a woman, has primary studies, worries that their family members may become infected and thinks that they are very likely to be infected with covid-19. the protective variables of suffering posttraumatic stress symptoms are being a man, having a doctor's degree, living with a partner (not married), being a doctor or working in "another position" (a category made up mainly of pharmacists and psychologists), having protective equipment at work, not being concerned about infecting the people with whom they live and not living alone. the risk variables associated with anxiety symptoms are being a woman, having a 12-or 24-h on-call shift and being worried that a person he/she lives with could be infected. additionally, the following protective factors have been identified: being a man, being separated, working in nursing homes or day centres, being a doctor, having a rotating morning-afternoon shift and/or not being isolated by covid-19. the profile of the health worker with greater symptoms of depression is being a woman, working 12-or 24-h shifts or on-call hours, the number of on-call hours per month, thinking that they are very likely to become infected with covid-19, being worried about infecting someone with whom they live and having no family. the protective variables of depression are being a man, having a fixed-term or training contract, feeling professionally accomplished and not living with people who are at risk. presenting high scores in emotional exhaustion and depersonalization are risk factors for posttraumatic stress, anxiety and depression. however, resilience would be a protective variable that reduces symptoms in all three disorders, and personal accomplishment would be a protective variable against depressive symptoms. one study points out that, in 2003, during the sars epidemic, health personnel showed symptoms related to posttraumatic stress disorder and, in general, higher levels of psychological stress [49] . recent research has indicated that, during the influenza a (h1n1) outbreak, resilience levels had a direct influence on the psychological health of health personnel [50] . regarding the gender-differentiated models, being a nurse is associated with symptoms of mental disorder, specifically depression in women and anxiety in men. in men, depersonalization and personal accomplishment are only associated with depression (although this relationship is not significant), while in women it is associated with the three scales of burnout. that is to say, women who have high scores in depersonalization would have more symptoms of mental disorder than men. in men, personal accomplishment would not be a protective variable, while for women it would be a protective factor for posttraumatic stress, anxiety and depression. having a primary education would be a risk variable for posttraumatic stress and having doctoral studies would be a protective variable of posttraumatic stress and depression, but only in men. in relation to the type of contract, having a statutory fixed-term employment contract is negatively related to anxiety in men, not being a relevant variable for women. the type of shift is a relevant variable for women, but not for men. specifically, 12-or 24-h shifts are positively related to depression in women, but not in men. having a fixed contract is a more relevant variable for women than for men, since it is a protective variable of depression in women. with regard to the foregoing, one of the main results of this study is that the levels of resilience of the healthcare workers evaluated are moderate. taking into account that resilience is presented as a possible protective factor of symptoms of posttraumatic stress disorder, anxiety and depression, the need to promote resilience among health personnel is highlighted. different studies have pointed out various measures to promote resilience among these professionals during the covid-19 pandemic. these include the following: providing psychological training to healthcare workers so that they can help patients and encouraging support within the organization by the network of personnel and train communication [51] . the model of intervention in psychological resilience based on peer support (battle buddies) developed by the us military should be highlighted. this model requires a close support partner as well as a designated mental health consultant to facilitate training in stress inoculation methods and to coordinate referral to the outpatient psychological consultation [52] . it might be interesting to introduce the elements mentioned in the spanish health care system to establish measures to promote resilience in possible future waves of covid-19. in this sense, the hypothesis proposed at the beginning would be fulfilled, since resilience would be a protective factor for such symptoms. in relation to burnout, it should be noted that a large percentage of these professionals have high scores in emotional exhaustion, but low in depersonalization and very high in personal accomplishment. the first study hypothesis, therefore, would be partially fulfilled, since it was expected that the workers would have low levels of personal accomplishment. this could be explained by the fact that health professionals have been intensely involved with patients in this situation, have felt valued by patients and society, and have realized the great importance of their profession, which may have had a very positive influence on their personal fulfilment. the variable with the greatest weight in the regression models is emotional fatigue. hence, preventive measures to reduce this should be implemented for these workers. emotional fatigue is the dimension that has the greatest relevance compared to depersonalization and personal fulfilment, within the burnout construct [53] . other authors have also identified high levels of fatigue and negative emotions in health personnel from emergency teams in coping with the covid-19 situation [54] . the study does have some limitations. the data were obtained using an online tool and people not familiar with the web could not be included in this study. in addition, the survey was carried out at the peak of the pandemic in spain: the continuous exposure to negative stimuli and the constant information in the media about the state of health care workers and those infected and deceased by covid-19 may have had an influence in the perception of anxiety and depression levels, due to the feeling of fear experienced [55] . furthermore, there is a high proportion of women compared to men. other studies have shown the same limitation [56, 57] . in this case, one of the main reasons for this difference is that, in many positions, such as nurses and nursing assistants, the majority of the positions are occupied by women. another limitation has to do with the cross-sectional design of the study: the pandemic has not yet finished and its influence on mental health cannot be reflected in this research, so it would also be advisable to carry out a longitudinal study that evaluates the evolution over time of the symptoms assessed in this work. on the other hand, there has not been a previous situation in spain in which there has been a lockdown, and it is likely that after its ending, the levels of experienced symptomatology will be lower. however, during the sars crisis, other authors have found that the symptoms of psychological problems after the quarantine period of the disease have lasted up to three years later [3, 58] . in the long term, the effects of posttraumatic stress disorder, anxiety and depression will depend on the possible outbreaks of covid-19. the measures currently being taken in order to adapt the work place to the new situation (such as providing protective equipment or increasing the number of healthcare professionals) are relevant for mitigating these symptoms. if the appropriate actions to protect health care providers are not taken, they may make medical errors in the future, present higher burnout levels associated with depressive symptoms, anxiety, suicidal ideation, have poorer interpersonal relationships or develop substance abuse [59] . therefore, a follow-up study along the next few months becomes necessary. this research presents a detailed description of the association between different variables and symptoms of posttraumatic stress, anxiety and depression. previous studies for spanish health professionals to evaluate these characteristics, including resilience, and its associations with the described variables, have not been found. among the main uses for this study is the description of the profiles of spanish healthcare providers that present a greater risk of suffering from post-traumatic stress, anxiety and depression, so more specific intervention measures can be designed to reduce these symptoms. on the other hand, resilience is a protective factor of the mentioned symptoms, so it would be advisable to include the promotion of resilience in the design of interventions to reduce stress, as other authors point out [60] . the information presented is relevant in order to protect the health of those who care for patients in future waves of covid-19 or 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professionals during the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-352465-n746e8qt authors: wang, fei; li, juan; fan, shengjie; jin, zhigang; huang, cheng title: targeting stress granules: a novel therapeutic strategy for human diseases date: 2020-08-16 journal: pharmacol res doi: 10.1016/j.phrs.2020.105143 sha: doc_id: 352465 cord_uid: n746e8qt abstract stress granules (sgs) are assemblies of mrna and proteins that form from mrnas stalled in translation initiation in response to stress. chronic stress might even induce formation of cytotoxic pathological sgs. sgs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, sgs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. emerging evidence has shown that small molecules can affect sg dynamics, including assembly, disassembly, maintenance and clearance. thus, targeting sgs is a potential therapeutic strategy for the treatment of human diseases and the promotion of health. the established methods for detecting sgs provided ready tools for large-scale screening of agents that alter the dynamics of sgs. here, we describe the effects of small molecules on sg assembly, disassembly, and their roles in the disease. moreover, we provide perspective for the possible application of small molecules targeting sgs in the treatment of human diseases. a large portion of mrna in mammalian eukaryotic cells completes transcription in the nucleus and is then transported to the cytoplasm for translation and expression. when eukaryotic cells are stimulated or disturbed, the mature mrna in cells cannot be translated into proteins immediately. these temporarily untranslated mrna or translation-stalled mrna then polymerize with rna-binding proteins (rbps) to form messenger ribonucleoprotein (mrnp) granules without a membrane structure, known as cajal bodies, stress granules (sgs), processing bodies (p-bodies), rna transport granules, or germ granules [1, 2] . while mrnp granule types are complex and diverse, there are three commonalities between mrnp granules [3] : first, mrnp granules usually contain non-translated or poorly translated mrna, and these mrna can re-enter polysome for translation after cellular adaption or environmental recovery. second, different mrnp granules may contain the same mrna or rbp and these components can be relocated from one mrnp granule to another granule. third, different mrnp granules can interact dynamically, involving docking, fusion, and becoming another mrnp granule after maturation. mrnp granules have a very important effect on mrna function and cell signalling, and are also closely related to diseases [4] . one of the most studied mrnp granules is sgs. sgs are a type of dynamic granular substance formed of mrna of stagnant translation and rbps in the cytoplasm of eukaryotic cells, the formation of which is stimulated by various stresses including oxidative stress, heat shock, hypoxia, or viral infection (fig. 1) . it is an adaptive regulatory mechanism that protects cells from apoptosis under adverse conditions [5, 6] . besides, sg formation and dynamic can affect mrna localisation, translation, and degradation as well as signalling pathways and antiviral responses [7] . the assembly of sgs is closely related to the translation initiation of eukaryotes. the canonical translation initiation in eukaryotes begins with circularization of mrna by eukaryotic initiation factor 4f (eif4f) complexes and assembly of 43s preinitiation complexes (43s pic) [8, 9] . the 43s complexes start searching for the initiation j o u r n a l p r e -p r o o f codon along mrna, and once the initiation codon is identified, eukaryotic initiation factor 2 (eif2) hydrolyses gtp to gdp, which triggers dissociation of eifs from ribosome and allow the join of 60s ribosomal subunit to form a complete 80s initiation complex. a key step in this process is the formation of ternary complex comprising eif2, gtp and met-trnai met and mammalian activating transcription factor 4 (atf4). the translation-stalled mrnas are then recruited by rbps to promote the formation of sgs [10] [11] [12] [13] [14] [15] . however, eif2α phosphorylation cannot certainly cause sg formation. for example, doxorubicin (dox) efficiently triggers phosphorylation of eif2α while fails to promote sg formation [16] . the inactivation of eif4f complexes through the disruption of eif4e and eif4g binding or inhibition of eif4a activity by hippuristanol [17] , h2o2 [18] , pateamine a [19] can also lead to translation inhibition and sg formation [20] . in addition to mrna, sg components include various rbps, translation initiation factor and non-rbps such as signalling proteins. sg composition is different in the same type of cells subjected to different types of stress or in different cells subjected to the same type of stress. sgs also share some ubiquitous rbps to facilitate sg aggregation and nucleation, such as ras gtpase-activating protein-binding protein 1 and 2 (g3bp1/2), cell cycle associated protein 1 (caprin1) and t cell restricted intracellular antigen-1 (tia-1) [21, 22] . these proteins are often used as protein j o u r n a l p r e -p r o o f markers of sgs to facilitate sg research (fig. 1) . with the deepening of research, sg proteins and proteins that regulate sg assembly/disassembly have been discovered. sgs are not of a uniform structure; rather, there are two distinct layers: the core and the shell [23] . these two layers of sgs may have different components, functions and dynamics. two models for discrete phases of sgs assembly have been proposed, the cores first model and the liquid-liquid phase separation (llps) first model. in the cores first model, the core is surrounded by a less concentrated and more dynamic shell, and the core size and dynamics of sgs do not change over time [9, 24] . in the llps first model, the condensation of the liquid inside the cell can drive the assembly of sgs. in some special circumstances, liquid aggregates of sgs are transformed into solid-like states, which may causes fibrosis or devastating protein aggregation diseases [25] . as the stress disappears, the clearance of sgs from the cytoplasm of cells occurs rapidly, i.e., within minutes (fig. 1) . the sg depolymerisation process is opposite to the aggregation process. first, the less stable shell is dissolved and then the core is removed [26] . sgs are generally believed to be sites of temporary mrna storage and triage. the mrnas in sg will have at least two directions after depolymerisation. the first is that, after chaperone or autophagy-mediated clearance of sgs upon recovery from stress or stress adaptation, specific mrnas reversely mobilise back to the polysome and translation is resumed. the second is that specific mrnas are translocated from sgs to p-bodies for storage or degradation [26] [27] [28] [29] [30] . the disassembly of sgs depends on the activation of heat hock proteins and the dephosphorylation of eif2α, as well as the regulation of the microtubule system [31, 32] . increasing evidence suggests that sgs are involved in many human diseases (fig. 2) , including cancers, neurodegenerative diseases, viral infections and autoimmune disease, well-documented by elegant reviews [33] [34] [35] [36] [37] . sgs affect multiple pathways j o u r n a l p r e -p r o o f by intercepting and sequestering signalling components, such as rack1 (p38/jnk signalling), traf2 (nf-b signalling), raptor (mtor signalling) and rhoa/rock1 (wnt signalling) [33] [34] [35] [36] [37] . when exposed to stress environments, cells may arrest the cell cycle and repair stress-induced damage, or proceed to apoptosis. acute stress-induced sgs selectively recruit and exclude mrnas, thereby promoting the translation of stress-related response genes and enabling cells to quickly return to a normal state after stress. conversely, if sg formation is blocked during stress, the cell survival rate is significantly reduced. however, sgs induced by pathological chronic stress (neurodegeneration, nutrient starvation) lack several classical sg components that contribute to the pro-survival functions of canonical sgs (rack1, small ribosomal proteins), and conversely have a pro-death function [38, 39] . the tumour microenvironment is full of stresses, such as a high concentration of ros, hypoxia and hypoglycaemia, which can strongly trigger sg formation. sgs are observed in many human cancers, such as pancreatic cancer, hepatocellular carcinoma, glioma/glioblastoma, sarcoma, mantle cell myeloma, colorectal cancer, head and neck cancers and prostate cancer [36] . sgs regulate the expression of oncogenes, tumour metabolism, and adapt tumour cells to the microenvironment, which are directly related to the development of tumours and the efficiency of anticancer drugs. several anti-tumour drugs have been shown to induce the formation of sgs. on the one hand, sgs are partially involved in apoptosis regulation. for example, eif2α phosphorylation-dependent sgs (type i) induced by sodium arsenite (sa) and bortezomib [40] may protect cells in the stress response, inhibit apoptosis and promote cell survival by the sequestration of signalling molecules, such as rack1 [41] , rock1 [42] and raptor [43] . disturbing the formation of antiapoptotic type i sgs not only influences tumour progression but also sensitises cancer cells to chemotherapeutic agents. on the other hand, eif2α phosphorylationindependent sgs (type ii), induced by sodium selenite and silvestrol, may impair cell resistance to stress, reduce cell survival and promote apoptosis [39, 44, 45] . induction j o u r n a l p r e -p r o o f of pro-apoptotic type ii sgs might be one mechanism underlying their anti-tumour effect. viruses are another type of adverse stress to host cells. viruses interfere with the gene expression of infected cells, and antiviral responses drive the formation of sgs to control viral rna translation and viral replication. virus-induced sgs have a unique composition (such as trim25) and function, and have been named anti-viral sgs (avsgs) to distinguish them from typical sgs [46] . based on the ways of regulating sg formation, viruses are divided into different types. the first type of virus temporarily triggers sg formation early in the replication cycle, but later limits sg formation. these viruses include poliovirus, genovirus and orthovirus [47] . mammalian orthoreoviruses (mrv) induce sg formation via eif2α phosphorylation in a strain-and cell type-dependent manner. as the infection progresses, viral infections leads to the inhibition of sgs. in addition, several strains of mrv inhibit sg formation induced by arsenite to facilitate infection [48] . the second type of virus can inhibit sg formation during the entire infection process, such as influenza a virus, which inhibits sg formation by blocking eif2α phosphorylation to avoid the repression of virus replication [48] . the third type of virus induces sg formation to help virus rna replication. respiratory syncytial virus (rsv), picornavirus and coronavirus may benefit from inducing sg formation as part of the mechanism by which they inhibit host cell protein synthesis and innate immunity [50] [51] [52] . the eif2α kinase pkr is the primary sensor responsible for the rapid inhibition of translation initiation. pkr-mediated sgs can block the translation of viral rna and interfere with virus replication in response to virus infection. some viruses, however, encode for pkr inhibitors, thus avoiding pkr-mediated eif2α phosphorylation and sg formation [53] . the drugs that induce sgs by bypassing pkr and/or eif2α phosphorylation may have therapeutic potential to control viral infection. recently, sars-cov-2 nucleocapsid (n) protein has been identified to inhibit sg formation by llps of n protein from the sg essential protein g3bp1/2, which j o u r n a l p r e -p r o o f enhances viral rna replication and translation [33, 54, 55] . sars-cov-2 suppression of sg formation may be mediated by the interaction of n protein with g3bp1/2 and/or csnk2b/csnk2a2, the subunits of casein kinase 2 (ck2) [56] . ck2 activity is enhanced following sars-cov-2 infection in cells which increase sg disassembly through the promotion of g3bp1 phosphorylation. several small molecules, including ck2 inhibitors, have been suggested to inhibit the process of viral rna replication via sg disassembly [56, 57] . sgs have been linked to several neurodegenerative diseases, such as amyotrophic lateral sclerosis (als), frontotemporal dementia (ftd) and alzheimer's disease (ad). a number of proteins in sg components, such as tdp-43, fus, hnrnpa1, hnrnpa2, tia1, matr3, ewsr1, taf15, atx1 and atx2 [58, 59] , have been shown to be associated with neurodegenerative diseases. misfolded proteins or mutations in pathogenic rbps cause abnormal protein aggregation and sg formation. when stress becomes chronic, persistent and more severe, original reversible sgs change into irreversible stable amyloid-like assemblies and insoluble fibre aggregates, leading to the loss of normal rbp function in the nucleus and the induction of neurotoxicity in the cytoplasm [58, 59] . moreover, partially mutated proteins (vcp, optineurin, p62, ubiquilin-2) may result in the dysfunction of autophagy and cause defects in the clearance of abnormal sgs, subsequently exacerbating the disease process [62] . at present, research on sgs and neurodegenerative lesions are focused on rbp biological models. rbps guide the transport, translation and degradation of intracellular rna, thereby regulating and controlling protein expression. irreversible sgs impair the function of rbps and interfere with mrna transport and transcription, and worsen neurodegenerative diseases. thus, reversing the irreversible solid-state of sgs may enables sgs to maintain reversible liquid properties, or promote sg disassembly and autophagy clearance, which is beneficial to the treatment of neurodegenerative diseases. aging is accompanied by many intracellular processes such as cellular metabolic change, mitochondrial dysfunction and abnormal protein quality control, which could result in disrupted protein homeostasis and the abnormal formation, maintenance, disassembly and clearance of sgs, consequently leading to chronic or pathological sgs [35] . these aberrant sgs may accelerate the aging process and aging-associated diseases. in aged c. elegans, the sg proteins pab-1 and tiar-2 can form aggregates, associated with shorter lifespan [63] , suggesting a connection between sg protein aggregation and shorter lifespan. an abnormal protein-rna interaction may lead these proteins to be aggregated and form irreversible aggregations [64] , leading to persistent sgs, which would cause the pathogenesis of aging-related diseases [65] [66] [67] . in addition, the sg components can also disturb sg dynamics, i.e., tdp-43 can increase amyloid-β oligomeric aggregates and accelerate aging and the onset of neurodegenerative diseases [68, 69] . however, studies have also shown that cellular senescence can cause eif2α hyperphosphorylation and impair sg formation in the stress response in a g3bp1-dependent manner [70] . omer and colleagues found that sg assembly is defective in aged cells and sg assembly has an anti-aging effect by recruitment of the pro-aging protein, plasminogen activator inhibitor-1 (pai-1) to sgs [71] . whether preventing chronic sg formation or inducing the formation of canonical sgs could retard aging process is worth investigating. in inflammatory diseases, the inflammasome is an important component that activates proinflammatory cytokines and induces inflammatory cell death. however, sgs and the nlrp3 inflammasome compete for ddx3x molecules to coordinate the activation of innate responses and subsequent cell fate decisions under stress conditions. sg formation leads to the sequestration of ddx3x and thereby inhibits nlrp3 inflammasome activation [72] . in the immune response, the eif2α of t cells undergoing antigen presentation for the first time is phosphorylated to form sgs that wrap the mrna of cytokines inside. when antigen presentation occurs again, some mechanism is initiated to dissolve sgs and release the mrna, thus translating and j o u r n a l p r e -p r o o f secreting cytokines [73] . sgs suppress the stress-induced inflammatory response by recruiting inflammatory factors traf2 and inhibiting tnf-α-mediated nf-κb proinflammatory signalling [74] . sgs are also associated with several other diseases, such as atrial fibrillation, organ fibrosis, autoimmune disease and brain ischemia [75] . it is expected that more diseases will be linked with sgs soon. at present, many compounds have been identified to affect the process of sg assembly, maintenance and disassembly via different mechanisms. sgs transiently exist in specific stress states and are regulated by a large number of post-translational modifications, protein remodelling complexes and microtubule networks. regulating sg assembly or disassembly may be a key method of controlling cell fate or treating disease. hence, it is a promising field to develop potential therapeutic strategies by targeting sg proteins in related diseases. in this part, we will focus on the reported small molecules that affect sg aggregation or depolymerisation (table1-4), summarise them according to their functions, and discuss the potential for drug development targeting sgs. the accumulation of sgs and related pathogenesis is fundamentally regulated at three levels: formation, maintenance and clearance. the major sg formation-regulating signalling pathways include the eif4f and eif2α pathways, and the mammalian target of rapamycin (mtor) also involved in signalling. the eif2, gtp and met-trnai met ternary complex and eif4f complex are two major points for the regulation of several small molecules have been reported to induce eif2α phosphorylation and sg assembly, although different eif2α kinases are employed. the raf1/mek/erk kinase inhibitor sorafenib is approved for advanced hepatocarcinoma [76] , and has previously been reported to induce er stress by the activation of perk phosphorylation of eif2α [77, 78] . recently, it has been found that sorafenib induces sg formation in various cancer cells via perk-mediated eif2α phosphorylation [78] . sg formation reduces the sensitivity of cells to sorafenib, reduces cell death and improves cell survival. thus, the disruption of sg formation can restore cell sensitivity to sorafenib. the anti-tumour drug 5-fluorouracil (5-fu) [79] has been found to induce sg assembly, inhibit cell proliferation and promote apoptosis via pkr-mediated eif2α phosphorylation. sg assembly is associated with the 5-fu metabolite furd that is further converted into futp and incorporated into rna to promote sg assembly [80] . bortezomib is a peptide boronate inhibitor of the 26s proteasome approved by the fda for the treatment of myelomas and other haematological tumours [81, 82] . hs578t breast cancer cells. the effects of bortezomib-induced eif2α phosphorylation is mediated through the activation of hri and gcn2, leading to sg formation, subsequently resulting in resistance to bortezomib in cancer cells [40] . in contrast, several small molecules have also been reported to inhibit eif2α phosphorylation and impair sg assembly. psammaplysin f , a marine sponge-derived metabolite, has effective activity against the malaria parasite [83, 84] . psammaplysin f treatment may lead to resistance to sg inducers in many cell lines (e.g., the vero, j o u r n a l p r e -p r o o f mcf7, t47d, hela and mcf7mdr cell lines). one possible mechanism is that psammaplysin f decreases the phosphorylation of eif2α under stress. trehalose is a natural disaccharide sugar, widely occurring in various organisms including bacteria, plants, insects, yeast, fungi and invertebrates. trehalose protects cells from protein denaturation and exhibits neuro-protective effects in several neurodegenerative diseases under heat, freezing, oxidation, drying and dehydration conditions. this effect is accompanied by p-eif2α dephosphorylation, which accelerates the restoration of translation. trehalose pretreatment also decreases p-eif2α induced by er stress or heat shock, sensitizes cells to stress and impairs cell survival [85] . inhibition of chemotherapy drug-induced sgs by this type of small molecule may be beneficial to relieving drug resistance in cancer patients. the integrated stress response, mediated by eif2α, regulates protein synthesis under stress. isrib targets eif2b and is the first reported antagonist of the isr that blocks signalling downstream of all eif2α kinases [86] . in non-stress situations, 43s preinitiation complexes containing eif2-gtp-met-trnai met begin translation. to engage in a new round of initiation, eif2-gdp released during this process needs to reload gtp to form new eif2-gtp, in a reaction catalysed by the heteropentameric eif2b [87] . isrib can reverse cognitive deficits following traumatic brain injury, protect against prion-induced neurodegeneration and prevent metastasis in a subset of cancers [86] . it has been reported that isrib reverses the effect of eif2α phosphorylation and restores translation by targeting eif2b. isrib prevents the formation of sgs induced by thapsigargin (a potent er stressor that inhibits the er calcium pump [88] ) and arsenite through eif2α phosphorylation, without reducing pat-a or hippuristanol-induced sgs through eif4a inhibition [89] . in another study, isrib also inhibited sin-1-induced sg assembly in u2os cells [90] . isrib also leads to the disassembly of pre-formed sgs by loading dissociating mrnas with actively translating ribosomes [89] . isrib, with its ability to quickly disassemble sgs even in the presence of stress, is expected to develop into new therapies for sgrelated diseases. j o u r n a l p r e -p r o o f assembly of the eif4f complex requires eif4a, eif4e and eif4g as a key step in mrna circulation and translation initiation. it is tightly regulated by cell signalling, particularly through mtor signalling. in normal conditions, mtor phosphorylates eif4e-binding protein 1 (4e-bp1), which blocks 4e-bp1 binding to eif4e and forms the eif4f complex. the eif4f complex is a major target of environmental stress to induce eif2-independent sgs. in response to stresses or chemical compounds, mtor is inactivated, which results in the accumulation of hypophosphorylated 4e-bp1 and enables its binding to eif4e, causing displacement of the scaffolding protein eif4g and the rna helicase eif4a from mrna cap structures. consequently, translation initiation and polysome formation are blocked, concomitantly with sg assembly. disruption of the eif4f complex may induce non-canonical sgs, which lack some of the core components of eif2 phosphorylation-induced canonical sgs [91] . thus, targeting the eif4f complex might be a promising approach to regulating sg dynamics. eif4a inhibition and the disruption of eif4e binding to eif4g are the common mechanisms underlying the formation of eif2-independent sgs induced by several small molecules. malonate, a classic competitive inhibitor of the respiratory electron transport chain, induces mitochondrial stress and inhibits mitochondrial respiration [92, 93] . it has been reported that malonate induces sg aggregation through 4e-bp1mediated inactivation of the eif4e pathway, which may inhibit apoptosis in hela cells [94] . like malonate, selenium, an essential micronutrient that promotes the production of reactive oxygen species (ros) for targeting tumour cells [31] , induces sg assembly by destroying the eif4f complex via the dephosphorylation of 4e-bp1 [39] . interestingly, selenite-induced sgs prompts u2os human osteosarcoma cells to undergo apoptosis, probably because of the unique sg composition [39] . of note, perturbing eif4a activity induces sg assembly independent of eif2α phosphorylation, which may exert anti-cancer activity [20, 95] . 15-deoxy-delta12,14prostaglandin j2 (15d-pgj2) possesses multiple biological activities [96] , which blocks translation by targeting cysteine 264 and inactivating eif4a, without the involvement of eif2α phosphorylation, suggesting that the translation inhibitory action of 15d-pgj2 is likely to contribute to the antineoplastic activity of 15d-pgj2 [96] . pateamine a and its analogues, dmda-pata and b-pata, induce sg formation through the prevention of eif4a heterodimerisation with eif4g and stabilising the eif4a:mrna complex, consequently limiting the availability of eif4a incorporation into eif4f complexes [19, 97] . hippuristanol and silvestrol have also been found to induce sg assembly by perturbing eif4f atpase, helicase and rna-binding activities, explaining their anti-tumour and antiviral replication activity [17, 98] . some small molecules have been reported to have combined activities of eif2α phosphorylation and eif4f disruption. for example, the vinca alkaloids vinorelbine, vinblastine and vincristine promote polysome disassembly and sg assembly through simultaneous activation of eif4e-bp1 and phosphorylation of eif2α [16] . blocking vinca alkaloid-induced sg assembly by inactivating 4ebp1 or inhibiting eif2α phosphorylation decreases cancer cell viability and promotes apoptosis [16] . nitric oxide-generating compounds 3-morpholinosydnonimine (sin-1) and s-nitroso-nacetylpenicillamine (snap) are protein synthesis inhibitors that trigger sg formation through no-induced eif2α phosphorylation and 4e-bp1 dephosphorylation [90] . no plays a major role in this process. interestingly, sin-1-induced pro-apoptotic sgs are less dynamic and less mobile compared to typical arsenite-induced anti-apoptotic sgs because of the loss of eif3b over time [90] . while the dissociation of eif4e-eif4g induces eif2-independent sgs, association of eif4e-eif4g is required for the assembly of eif2-dependent sgs. therefore, disruption of eif4e-eif4g could inhibit the assembly of eif2-dependent sgs, or promote the disassembly of pre-existing eif2-dependent sgs. this was proposed by fournier and colleagues, who found that mtor inhibitor-mediated disruption of eif4e-eif4gi impairs sg formation under mild stress [91] . when the cell is treated with a mild stressor such as arsenite or bortezomib (both induce eif2 j o u r n a l p r e -p r o o f phosphorylation), eif4e binds to eif4g, but the eif4e-4gi complex is in an inactive state, accumulates gradually and eventually leads to the production of sgs. in this case, depletion or inactivation of mtor leads to hypophosphorylation of 4e-bp1, which then blocks the interactions between eif4e and eif4gi to promote the disassembly of formed sg [91] . pp242 is an atp-competitive inhibitor of mtor that blocks the phosphorylation of akt at s473 and suppresses mtor activity. pp242 directly binds to the atp-binding site of mtor and inhibits the catalytic activity of both mtorc1 and mtorc2 [99] . it also decreases the phosphorylation of 4e-bp1 to prevent the binding of eif4e to eif4gi, thus inhibiting sg formation [91] . the depletion of mtor significantly inhibits sg formation in cells treated with pp242 [91] . similar to pp242, the kinase inhibitor torin1 is also a selective atp-competitive inhibitor of mtor [100] , which inhibits mtorc1-dependent phosphorylation of 4e-bp1 and impairs sg formation [91] . these data suggest that the suppression of mtor signalling may inhibit the assembly of mild stress-induced eif2-dependent sgs and sensitize tumour cells to apoptosis. ck2 is a stress-activated protein kinase linked to the phosphorylation of several translation initiation factors, including eif3 and eif5 [101, 102] . ck2 accelerates sg disassembly through the promotion of g3bp1 phosphorylation to inhibit sg assembly [103] . thus, ck2 inhibitors may block sg disassembly and recover from the stress. tetrabromocinnamic acid (tbca) is a specific ck2 inhibitor [104] and increases residual sgs in cells during recovery from stress [103] . the another ck2 inhibitor, 5oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl acetic acid (iqa), also inhibits the disassembly of virus-induced sgs [103] . a recent study has suggested that the ck2 inhibitors tmcb and silmitasertib could interfere with sg disassembly by sars-cov-2 n protein, but this needs to be confirmed [56, 57] . the recruitment of mrnps in the cytoplasm to fuse into mature sgs requires transportation along microtubules by motor proteins; thus, microtubule integrity is essential for sg assembly. microtubules are intracellular structures assembled with heterodimers of  and  tubulin, and are responsible for various kinds of movements in cells. microtubules are involved in cell division, the organisation of intracellular structure and intracellular transportation. microtubule disruption delays sg formation. sgs are still formed, but they are smaller in size, greater in number and variable in distribution [105] . tubulin inhibitors are used in the treatment of cancer because they induce cell cycle arrest. paclitaxel, a first-line anti-cancer drug, is a β-tubulin inhibitor and promotes microtubule assembly and stabilisation [106, 107] . paclitaxel induces sg formation in many cells [16, 105] . similarly, latrunculin b, an actin filament inhibitor [108] , has been reported to accelerate microtubule-based motility via actin disruption, thus facilitating the motility of sgs and promoting the formation of large sgs [109] . in contrast, tubulin depolymerising agents could trigger sg disappearance. as a reversible microtubule inhibitor, nocodazole binds to β-tubulin and disrupts microtubule assembly and disassembly kinetics, thereby preventing mitosis and inducing apoptosis in tumour cells [110] . nocodazole inhibits sg formation but does not prevent sa-induced eif2α phosphorylation, suggesting that nocodazole acts downstream of eif2α phosphorylation [109] . vinblastine is a therapeutic drug for hodgkin's disease, lymphocytic lymphoma, histiocytic lymphoma, advanced testicular cancer, advanced breast cancer, kaposi's sarcoma and letterer-siwe disease [111, 112] . vinblastine has higher affinity for tubulin and causes microtubule depolymerisation [113] . similarly to nocodazole, vinblastine disrupts the formation of sgs and the disassembly of the formed sgs [109] . however, a higher concentration or prolonged treatment with vinblastine potently promotes translation repression and induces microscopically visible sgs [16, 109] . since the cytoskeleton also serves as scaffold and is closely associated with the polysome for the active translation of mrna [114] , vinblastine at a high dose may j o u r n a l p r e -p r o o f induce robust polysome disassembly through the collapse of microtubules and the secondary aggregation of sgs [16] . these studies have provided helpful strategies for targeting microtubules to regulate sg formation, as well as strategies for overcoming resistance to tubulin inhibitors in cancer patients. the core components of sgs includes g3bp1/2, tia-1, tiar, caprin1, tdp-43, a number of energy-driven protein/nucleic acid chaperones or remodelling complexes and untranslated mrna. targeting the components may influence sg dynamics. tia-1 has been reported to prevent cell proliferation, tumour growth and invasion [115] . under physiological conditions, tia-1 binds to an adenine/uridine-rich element in the 3'-utr of tnfα mrna and represses tnfα mrna translation. in contrast, tia-1 increases tnfα mrna translation in response to stress. boric acid exhibits anticarcinogenic and bone strengthening effects by balancing the anti-apoptotic eif2α-sg pathway and pro-apoptotic eif2α-atf4 pathway via tia-1 translocation from the nucleus to sgs [116] . similarly, troxerutin, known as vitamin p4 with multiple nutritional and pharmacological properties (antioxidant, anti-inflammatory, radioprotective and hepatoprotective activities), has also been shown to promote sg formation in a tia1-dependent manner. this effect is diminished in tia-1 suppressed cells, suggesting that enhanced sg formation by troxerutin might recruit essential stress-fighting mrnas/proteins into sgs and thereby impair the cellular machines required for survival and recovery [117] . tar dna binding protein 43 (tdp-43) is an rbp pathologically and genetically linked to als, ftd, alzheimer's disease, parkinson's disease and huntington's disease [118, 119] . several studies have shown that the accumulation of tdp-43 in sgs is mediated through activation of the stress kinase c-jun n-terminal kinase (jnk) [120] . inhibiting extracellular signal-regulated kinase (erk) could prevent the accumulation of tdp-43 and human antigen r-(hur)-positive sg formation. mutations in the tdp-43 coding gene tardbp as well as changes in the expression level of tdp-43 proteins may disrupt the dynamics of sgs [120] . mitoxantrone is a type ii topoisomerase inhibitor approved for the treatment of acute myeloid leukaemia and multiple sclerosis [122] . mitoxantrone reduces tdp-43 in [124] . emerging evidence suggests that llps-driven sg formation is associated with the pathogenesis of neurodegenerative diseases, aging, cancer and virus infections [125] because of increased irreversible hydrogelation that interferes with normal cellular functions. hyperphosphorylated tdp-43 undergoes llps to colocalise with other stress granule components and promote aberrant protein aggregation, forming chronic sgs. the small molecule inhibitors of tankyrase-1/2, xav939, g007-lk and iwr-1endo, suppress the formation of cytoplasmic tdp-43 and increase llps of tdp-43 from other sg components [126] ; this prevents the pathogenesis of neurodegenerative diseases and may be used to treat als and ftd. llps can be enhanced by poly(adp-ribosyl)ation (parylation), an important post-translational modification (ptm) involved in dna damage repair and apoptosis. additionally, a parp inhibitor was found to attenuate tdp-43-mediated neurotoxicity in an als model [127] . it has been reported that the parp inhibitor olaparib delays the recruitment of tdp-43 and hnrnp a1 to sgs and delays sg assembly, which is beneficial in als and ftd j o u r n a l p r e -p r o o f [127] . in pathologic fibrillation, sgs are unlinked from the chemical chaperone trimethylamine n-oxide (tmao), which enhances rnp liquid condensation and inhibits protein fibrillation, suggesting a therapeutic strategy for tdp-43 aggregationassociated degenerative disorders [128] . g3bp1 and g3bp2 contain rna-binding domains, and are associated with rna binding [129] . g3bp1/2 are core component of sgs and essential for sg formation. arsenite induces sg formation, probably via the induction of the dephosphorylation of g3bp at ser149 [22, 130] , and increases the interaction of g3bp with histone deacetylase 6 (hdac6) [131] . phosphatase inhibitors such as okadaic acid and vanadate inhibit the interaction of g3bp and hdac6 and increase g3bp phosphorylation level and regulate sg formation [22, 131] . since g3bp1/2 are essential for the initiation of sg formation, small molecules targeting g3bp1/2 could be expected as a new approach for cancer therapy. compound c108, 2-hydroxy-n-[1-(2-hydroxyphenyl)ethylidene]benzohydrazide, could alleviate the function of g3bp2 and inhibit the activity of tumour-initiating cells [132] , showing anti-tumour activity. a recent study showed that sars-cov-2 n protein interacts with g3bp1/2 to inhibit sg formation; this inhibits virus rna replication, suggesting a potential therapeutic target for covid-19 [54] . however, the small molecules targeting this complex need to be tested. the assembly, fusion, depolymerisation and transportation of sg components require atp. consequently, intracellular atp levels can affect sg dynamics [90] . the formation of sgs and maintenance of sg core proteins in the dynamic pool of sgs require atp driven chemical reactions rather than specific kinase activity [133] . it seems that the different atpase complexes have different effects on sg assembly and disassembly [133] . the movement of sgs along the microtubule also requires atp j o u r n a l p r e -p r o o f energy [134, 135] . thus, atpase inhibitors may affect both the assembly and disassembly of sgs. soil-dwelling microbes produce the chemical staurosporine and its analogue ro-31-8220, which are both atp-competent kinase inhibitors [134, 135] that may inhibit sa-induced sgs [138] . 5´-iodotubercidin (5´-itu), a kinase inhibitor [139] , interferes with the fusion and growth of sgs by inhibiting atpase activity, but does not affect the disassembly of sgs during recovery from sa stress [138] . the rna/dna binding protein fused in sarcoma/translocated in liposarcoma (fus/tls) is linked to als [140] . like tdp-43, fus/tls is sequestered in the cytosol of als-affected motor neurons. mutations in fus cause severe loss of motor neurons in the spinal cord associated with juvenile als. the unique feature of fus pathogenesis in als is their nuclear clearance and simultaneous cytoplasmic aggregation in affected motor neurons [118] . fus accumulation in sgs is a reversible process, which may lead to pathological aggregation of fus in sgs under chronic stress [60, 61] . fus droplets form by llps, and become gel-like forms containing amyloid fibrils and are thus less dynamic. additionally, some disease-related fus mutations can exacerbate this phenomenon and aggravate the condition [80] . lipoamide and lipolic acid have been reported to act on fus to reduce sg formation and promote the dissolution of pre-existing sgs. lipoamide and lipoic acid have been shown to improve fus condensation behaviour, prevent sg component aggregation in a c. elegans model, and recover neuron and organism fus-associated defects in a drosophila model of als [80] . [141, 142] , suggesting that targeting ptms is a therapeutic option for these diseases. ubiquitination and the ubiquitin-like proteins nedd8 and sumo, which mediate neddylation and sumoylation, regulate various cellular processes [143] [144] [145] , and involved in the disassembly of sgs. thus, the pharmacological inhibition of ubiquitination may improve the efficiency of cancer therapies. neddylation induces sg assembly under a stress state via enhanced polysomic disassembly following translation arrest. however, so far, no pharmacological agent has been reported to act on neddylation. acetylation and deacetylation, another major form of protein modification, are also involved in sg formation and disassembly. histone acetylases and histone deacetylases (hdacs) regulate the acetylation of proteins. it has been shown that hdac6, but not other hdacs, may directly interact with the sg core component g3bp1 to enhance sg formation. pharmacological inhibition or genetic ablation of hdac6 blocks sg assembly and sensitises cells to apoptosis under stress [131] . this effect of hdac6 might be linked to the deacetylation of tubulin [146, 147] , which is important for sg dynamics, since the pan-hdac inhibitor tsa results in microtubule network hyperacetylation and decreases sg formation [131] . thus, targeting hdac6 deacetylase activity or its interaction with g3bp are worth considering for sg regulation and cancer therapy. interestingly, the nad + -dependent deacetylase sirt6 interacts with g3bp to induce sg assembly in response to stress [148] , and inhibiting the activity of sirt6 impairs sg formation. thus, sirt6 antagonists may be a strategy to inhibit the formation of anti-apoptotic sgs and induce apoptosis in tumour cells. sgs are disassembled when the stress is relieved, which is accompanied by translation restoration. similar to sg formation, the disassembly of sgs is also a multi-step process and is mediated by chaperones and autophagy [26] . the roles of the hsp70 family in chaperone-mediated sg dissolution have been studied by several groups. for example, hsp70, hsp40, hsp104 and hspb8 (the small heat shock protein hsp22) participate in sg removal and relieve the accumulation of untranslated ribosomal products in sgs. therefore, it is worth investigating the effect of small molecules targeting these chaperone proteins on sg dynamics. autophagy removes sgs, in a process called granulophagy. autophagic vesicles may engulf sgs and clear the particles. the process requires valosin-containing protein (vcp), an atpase required for the autophagy disaggregation of sg components [29] . trehalose, an autophagy enhancer [149] , has been found to delay sg assembly and facilitate sg premature post-stress disassembly in neuroblastoma sh-sy5y cells, implicating that trehalose is a possible therapeutic approach in neurodegenerative diseases. however, the action of trehalose is mediated via an increase in p-eif2α rather than the autophagosome pathway. one study showed that only a minor fraction of aberrant sgs is cleared by autophagy, whereas the majority of sg disassembly requires the hspb8-bag3-hsp70 chaperone [149] . whether autophagy enhancers could accelerate sg disassembly, and thereby be developed as therapeutic agent against cancers and neurodegenerative diseases, still needs further investigation. the proteasome is also responsible for the degradation of mrnas in sgs, which was confirmed by the induction of sg assembly by pharmacological and clinically used proteasome inhibitors [150] . the proteasome inhibitor bortezomib induces sg assembly via the stimulation of eif2α phosphorylation by the kinases hri, pkr, gcn2 and perk [40, 151] . this disrupts multiple pathways in the cell and bone marrow microenvironment, leading to apoptosis and inhibiting cell cycle progression, angiogenesis and proliferation [82] ; this is a common approach in the treatment of cancers [81, 151] . in contrast, prolonged inhibition of the proteasome instead leads to partial recovery of translation and sg disassembly due to stress adaptation [40, 151] , j o u r n a l p r e -p r o o f suggesting that the assembly of sgs in response to proteasome inhibition is timedependent and reversible [40, 151] . thus, the application of proteasome inhibitors has to consider the involvement of sgs. the polysome is a complex formed by cytoplasmic rnas and ribosomes undergoing active translation. during the stress process, the polysomes originally in the translationally active state begin to dissociate, and the excess free mrna from ribosomes associate with aggregation-prone rbps or misfolded proteins, thereby promoting sg protein recruitment and sg assembly [152] . emetine and cycloheximide are protein synthesis inhibitors that stabilise polysomes by freezing ribosomes to keep ribosomes loaded with mrna; this prevents the ribosome from reaching the stop codon. mrna can shuttle between sgs and polysomes under stress. emetine and cycloheximide prevent the assembly of sainduced sgs by trapping mrna in polysomes and maintaining polysomes, preventing free mrna from accumulating in the cytoplasm [150, 153] . on the contrary, puromycin, an inhibitor of protein synthesis that destabilises polysomes by promoting translational premature termination, enhances sa-induced sgs rather than inhibiting sg aggregation, suggesting that the drugs promoting polysome stabilization or dissociation can affect sg formation. here, we have summarised recent advances in small molecules targeting sgs (fig. 3) . sgs have been identified in many biological processes and diseases. the assembly and disassembly of sgs determine further storage, translation remodelling or degradation of untranslated mrna, which affect cell death or survival under specific conditions. in cancer treatment, on the one hand, the formation of sgs can lead to cell survival and increase cell resistance to chemotherapeutic drugs. the combined use of drugs that inhibit sg formation or promote sg disassembly with chemotherapeutic drugs may alleviate drug resistance. on the other hand, some drugs may enhance the vanillin unknown [168] j o u r n a l p r e -p r o o f psammaplysin f decrease eif2α phosphorylation [83] trehalose promote eif2α dephosphorylation [85] syringic acid unknown [117] penfluridol unknown [123] gitoxigenin unknown [123] benzethonium unknown [123] pyrvinium unknown [123] quinacrine unknown [123] these compounds interfere with the fusion and growth of sg, rather than the initial assembly of sg. when sa stimulates cells that pretreated with these compounds, the proportion of cells with sgs decreases, the number of sgs in each cell increases, and the proportion of large sgs decreases. "*" represents that this drug has been reported that can both induce sgs assembly and disturb sgs assembly, depending on cell type and dosage. 5´-iodotubercidin disrupt the atp-driven chemical reactions [138] j o u r n a l p r e -p r o o f figure 1 . sg dynamics. sg dynamics is observed by fluorescence microscopy with immunostaining against sg-nucleating proteins or cells expressing gfp-tagged sgnucleating proteins including g3bp1, g3bp2 and tia-1, which are commonly used as sg markers. (a). immunostaining results show g3bp1 and tia-1 are extensively overlapped in sa (sodium arsenite)-induced sgs. (b). hela cells stably expressing gfp-tagged g3bp2 protein show sgs are efficiently assembled after 30min treatment of sa, partially disassembled after 1h recovery (rec) from sa treatment and completely disassembled after 3h rec from sa treatment. figure 2 . the physiological and pathological roles of sgs. by recruitment of a variety of rbps and signaling proteins into sgs to regulate cellular processes including stress response and adaption, sgs are important to maintain homeostasis and human health. disruption of sg dynamics is involved in the progression of aging and many types of human diseases, including cancer, chemotherapy resistance, neurodegenerative diseases and viral infection. (a). mutation in disease-associated rbps (e.g., fus, tdp-43, hnrnpa1) promotes the formation of abnormal and persistent sgs, which lead to neurotoxic protein aggregates and progress towards neurodegenerative diseases. (b). tumor microenvironment and many chemotherapeutic drugs act as stressful condition and might promote sg formation. tumor cells hijack sg property to escape from apoptosis, resulting in tumorigenesis and drug resistance. (c). agingassociated changes in cellular environment might lead to disruption of sg dynamics. during aging, downregulation of g3bp1 expression causes defect in sg formation. thus, aging-promoting protein pai-1 fails to localize to sg, which further accelerates aging. (d). in response to viral infection, sgs are induced in host cells to inhibit viral replication, via repression on viral mrna translation and stimulation on host innate immune response. however, viral proteins (e.g., protease and nucleocapsid protein) antagonize sg formation to favor viral mrna translation. alternatively, viral proteins could also induce asg (atypical sg, viral mrnas are excluded) to inhibit translation of cellular mrna while allow translation of viral mrna. biomolecular condensates: organizers of cellular biochemistry rna granules: the good, the bad and the ugly rna granules: post-transcriptional and epigenetic modulators of gene expression tia1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis stress granules and cell signaling: more than just a passing phase? cap-dependent, scanning-free translation initiation mechanisms regulation of translation initiation in eukaryotes: mechanisms and biological targets phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded rna-dependent protein kinase stress granules: the tao of rna triage regulated translation initiation controls stress-induced gene expression in mammalian cells kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure perk is essential for translational regulation and cell survival during the unfolded protein response stressful initiations vinca alkaloid drugs promote stress-induced translational repression and stress granule formation hippuristanol-a potent steroid inhibitor of eukaryotic initiation factor 4a hydrogen peroxide induces stress granule formation independent of eif2α phosphorylation eukaryotic initiation factor 2α-independent pathway of stress granule induction by the natural product pateamine a inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2α phosphorylation stress granule assembly is mediated by prion-like aggregation of tia-1 the rasgapassociated endoribonuclease g3bp assembles stress granules unravelling the ultrastructure of stress granules and associated p-bodies in human cells principles and properties of stress granules liquid phase condensation in cell physiology and disease distinct stages in stress granule assembly and disassembly p bodies promote stress granule assembly in saccharomyces cerevisiae possible roles in the control of translation and mrna degradation eukaryotic stress granules are cleared by granulophagy and cdc48/vcp function a surveillance function of the hspb8-bag3-hsp70 chaperone complex ensures stress granule integrity and dynamism stress granules: sites of mrna triage that regulate mrna stability and translatability dynein and kinesin regulate stressgranule and p-body dynamics translation inhibition and stress granules in the antiviral immune response stress granules and neurodegeneration the involvement of stress granules in aging and aging-associated diseases stress granule: a promising target for cancer treatment cytoplasmic stress granules: dynamic modulators of cell signaling and disease differences between acute and chronic stress granules, and how these differences may impact function in human disease selenite targets eif4e-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules the chemotherapeutic agent bortezomib induces the formation of formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways rhoa/rock1 signaling regulates stress granule formation and apoptosis inhibition of mtorc1 by astrin and stress granules prevents apoptosis in cancer cells the chemotherapeutic drug 5-fluorouracil promotes pkr-mediated apoptosis in a p53-independent manner in colon and breast cancer cells stress granule-inducing eukaryotic translation initiation factor 4a inhibitors block influenza a virus replication dhx36 enhances rig-i signaling by facilitating pkr-mediated antiviral stress granule formation regulation of stress granules in virus systems mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection influenza a virus inhibits cytoplasmic stress granule formation respiratory syncytial virus induces host rna stress granules to facilitate viral replication mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies picornavirus 2a protease regulates stress granule formation to facilitate viral translation stress granules (sg) and processing bodies (pb) in viral infections sars-cov-2 nucleocapsid protein impairs sg assembly by partitioning into g3bp condensate nucleocapsid protein of sars-cov-2 phase separates into rnarich polymerase-containing condensates a sars-cov-2 protein interaction map reveals targets for drug repurposing the global phosphorylation landscape of sars-cov-2 infection relation between stress granules and cytoplasmic protein aggregates linked to neurodegenerative diseases emerging small-molecule therapeutic approaches for amyotrophic lateral sclerosis and frontotemporal dementia converging mechanisms in als and ftd: disrupted rna and protein homeostasis amyotrophic lateral sclerosis-linked fus/tls alters stress granule assembly and dynamics relationships between stress granules, oxidative stress, and neurodegenerative diseases more stressed out with age? check your rna granule aggregation a systematic survey identifies prions and illuminates sequence features of prionogenic proteins a new subtype of frontotemporal lobar degeneration with fus pathology tdp-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis ubiquitinated tdp-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis polyglutamine-expanded huntingtin exacerbates age-related disruption of nuclear integrity and nucleocytoplasmic transport cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and rna dissecting the molecular mechanisms that impair stress granule formation in aging cells stress granules counteract senescence by sequestration of pai-1 ddx3x acts as a live-or-die checkpoint in stressed cells by regulating nlrp3 inflammasome activation of the integrated stress response during t helper cell differentiation sequestration of traf2 into stress granules interrupts tumor necrosis factor signaling under stress conditions presence and function of stress granules in atrial fibrillation the kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells clinical pharmacology of 5-fluorouracil 5-fluorouracil affects assembly of stress granules based on rna incorporation proteasome inhibitors in cancer therapy psammaplysin f increases the efficacy of bortezomib and sorafenib through regulation of stress granule formation psammaplysin f, a new bromotyrosine derivative from a sponge, aplysinella sp modulation of p-eif2α cellular levels and stress granule assembly/disassembly by trehalose structural insights into isrib, a memory-enhancing inhibitor of the integrated stress response protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation the unfolded protein response triggers selective mrna release from the endoplasmic reticulum the small molecule isrib reverses the effects of eif2α phosphorylation on translation and stress granule assembly nitric oxide triggers the assembly of "type ii" stress granules linked to decreased cell viability inactivation of the mtorc1-eukaryotic translation initiation factor 4e pathway alters stress granule formation role of the malonyl-coa synthetase acsf3 in mitochondrial metabolism malonate induces the assembly of cytoplasmic stress granules molecular pathways: the eif4f translation initiation complex-new opportunities for cancer treatment 15-deoxy-∆-12,14-prostaglandin j2 (15d-pgj2), an endogenous ligand of ppar-γ: function and mechanism translational dysregulation by pateamine a a comparative study of small molecules targeting eif4a active-site inhibitors of mtor target rapamycin-resistant outputs of mtorc1 and mtorc2 mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mtorc1 mass spectrometry analysis of a protein kinase ck2β subunit interactome isolated from mouse brain by affinity chromatography one-thousand-and-one substrates of protein kinase ck2? casein kinase 2 is linked to stress granule dynamics through phosphorylation of the stress granule nucleating protein g3bp1 tetrabromocinnamic acid (tbca) and related compounds represent a new class of specific protein kinase ck2 inhibitors microtubules govern stress granule mobility and dynamics taxol stabilizes microtubules in mouse fibroblast cells molecular effects of paclitaxel: myths and reality (a critical review) latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: i. comparison with cytochalasin d disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation nocodazole induced suicidal death of human erythrocytes total synthesis of vinblastine, related natural products, and key analogues and development of inspired methodology suitable for the systematic study of their structure-function properties molecular recognition pattern of cytotoxic alkaloid vinblastine with multiple targets structural insights into microtubule function emerging role for the cytoskeleton as an organizer and regulator of translation knockdown of t-cell intracellular antigens triggers cell proliferation, invasion and tumour growth boric acid induces cytoplasmic stress granule formation, eif2α phosphorylation, and atf4 in prostate du-145 cells screening novel stress granule regulators from a natural compound library tdp-43/fus in motor neuron disease: complexity and challenges tdp-43 aggregation in neurodegeneration: are stress granules the key c-jun n-terminal kinase controls tdp-43 accumulation in stress granules induced by oxidative stress reduced stress granule formation and cell death in fibroblasts with the a382t mutation of tardbp gene: evidence for loss of tdp-43 nuclear function a topoisomerase ii inhibitor, induces apoptosis of b-chronic lymphocytic leukaemia cells small-molecule modulation of tdp-43 recruitment to stress granules prevents persistent tdp-43 accumulation in als/ftd small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization prevents pathological phase separation of tdp-43 by promoting liquid demixing and stress granule localization parylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related rna-binding proteins a chemical chaperone decouples tdp-43 disordered domain phase separation from fibrillation more promiscuous than ever: a review of g3bp a novel phosphorylation-dependent rnase activity of gap-sh3 binding protein: a potential link between signal transduction and rna stability the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response stress granule-associated protein g3bp2 regulates breast tumor initiation atpase-modulated stress granules contain a diverse proteome and substructure atp enhances at low concentrations but dissolves at high concentrations liquid-liquid phase separation (llps) of als/ftd-causing fus life and work of stress granules and processing bodies: new insights into their formation and function different susceptibility of protein kinases to staurosporine inhibition staurosporine: new lease of life for parent compound of today's novel and highly successful anti-cancer drugs atp-driven reactions are required for the assembly of large stress granules identification of the glycogenic compound 5-iodotubercidin as a general protein kinase inhibitor mutations in fus, an rna processing protein, cause familial amyotrophic lateral sclerosis type 6 posttranslational control of hur function post-translational modifications of rna-binding proteins and their roles in rna granules the ubiquitin code the ubiquitin system ubiquitin-like proteins hdac6 is a microtubuleassociated deacetylase at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination the sirtuin sirt6 regulates stress granule formation in c. elegans and mammals autophagy induction by trehalose: molecular mechanisms and therapeutic impacts inhibitors of protein synthesis identified by a high throughput multiplexed translation screen inhibition of the ubiquitin-proteasome system induces stress granule formation free mrna in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules dynamic shuttling of tia-1 accompanies the recruitment of mrna to mammalian stress granules rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2α to the assembly of mammalian stress granules oxidative stress mislocalizes and retains transport factor importin-α and nucleoporins nup153 and nup88 in nuclei where they generate high molecular mass complexes evidence that gquadruplex dna accumulates in the cytoplasm and participates in stress granule assembly in response to oxidative stress tdp-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor identification of pcbp2, a facilitator of ires-mediated translation, as a novel constituent of stress granules and processing bodies malonate induces the assembly of cytoplasmic stress granules alterations in stress granule dynamics driven by tdp-43 and fus: a link to pathological inclusions in als? front mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay stress-specific composition, assembly and kinetics of stress granules in saccharomyces cerevisiae stress granules and processing bodies are dynamically linked sites of mrnp remodeling darinaparsin is a multivalent chemotherapeutic which induces incomplete stress response with disruption of microtubules and shh signaling restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress impairment of stress granule assembly via inhibition of the eif2alpha phosphorylation sensitizes glioma cells to chemotherapeutic agents therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model vanillin inhibits translation and induces messenger ribonucleoprotein (mrnp) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger rnp granules and attenuate translation activity in saccharomyces cerevisiae anti-inflammatory lipid mediator 15d-pgj2 inhibits translation through inactivation of eif4a a selective inhibitor of eif2α dephosphorylation protects cells from er stress als-associated tdp-43 induces endoplasmic reticulum stress, which drives cytoplasmic tdp-43 accumulation and stress granule formation pkcalpha binds g3bp2 and regulates stress granule formation following cellular stress inhibition of tdp-43 accumulation by bis(thiosemicarbazonato)-copper complexes the authors have no competing interests to declare. the authors declare no conflict of interest.j o u r n a l p r e -p r o o f key: cord-309161-ceahghs1 authors: epel, elissa s. title: the geroscience agenda: what does stress have to do with it? date: 2020-09-28 journal: ageing res rev doi: 10.1016/j.arr.2020.101167 sha: doc_id: 309161 cord_uid: ceahghs1 geroscience offers a counterpoint to the challenged pursuit of curing diseases of aging by focusing on slowing the biological aging process for extended healthspan earlier in life. remarkable progress has led this field toward animal trials and the next challenge lies with translation to humans. there is an emerging number of small human trials that can take advantage of new models integrating behavioral and social factors. understanding dynamic aging mechanisms, given the powerful social determinants of aging (crimmins, 2019) and human variability and environmental contexts (moffitt, 2019), will be critical. behavioral and social factors are intrinsic to aging. toxic stressors broadly defined can lead to stress-acceleration of aging, either directly impacting aging processes or by shaping poor behavioral health, and underlie the socioeconomic disparities of aging. in contrast, hormetic stressors, acute intermittent stressors of moderate intensity, can produce stress resilience, the ability for quick recovery and possibly rejuvenation of cells and tissues. although health research usually examines static biomarkers, aging is reflected in ability to recover from challenges pointing to new interventions and targets for examining mechanisms. a fuller model incorporating stress resilience provides innovative biobehavioral interventions, both for bolstering response to challenges, such as covid-19, and for improving healthspan. circumstances and substances. hormesis traditionally described a cells or organisms bi-phasic response to an external chemical or stressor. there is indeed overlap between stress processes and aging processes, and two become intertwined with the concept of hormesis. toxic stress includes traumatic or ongoing adversity for months on end, and the psychological responses--chronic high perceived stress, burnout, or depression. many large scale studies demonstrate that traumatic or chronic psychosocial adversity, including low socioeconomic status, predict higher allostatic load, whereas high levels of psychosocial resources are associated with lower allostatic load, with small but reliable effects (danese & mcewen, 2012; wiley et al., 2017) , described further under "reserve capacity" (section 3). homeostasis to allostatic load. stress research started with examination of the acute stress responses to acute stressors in rodents. cannon's stress studies led to the popular concept of homeostasis (cannon, 1932) but a simple linear model of homeostasis does not explain the range of human stress responses, and there have been many elaborations of this concept. selye described the continuum from acute stress to chronic stress (selye, 1956) . acute stress can be hormetic when there is quick recovery back to homeostasis. given the complexity of physiological regulation, and that our body mounts a response in mere anticipation of threat, sterling and colleagues have described allostasis as a more encompassing description of the body's regulation-the constant fluctuations to meet expected demands (schulkin & sterling, 2019) which in biogerontology has been called "homeodynamics." chronicity of stressor exposure reveals a "fragility in homeostasis" (ramsay & woods, 2014) when physiological signs of 'exhaustion' appear, such as, in rodents, damage in organs. mcewen and colleagues have labeled this cost of adaptation--the dysregulation and damage across systems--as allostatic load (mcewen, 2004) . the concept of allostatic load, whether it is at a systemic or cellular level, gives us an intermediate phenotype of aging, an early step toward development of diagnosable disease. this is a critical concept to geroscience, and in fact many of the actual measures of allostatic j o u r n a l p r e -p r o o f load used in from the psychology and public health literature are actually indices of aging (entringer & epel, 2020) . geroscience leaders have started to identify the biomarkers important in geroscience trials, as those that can predict aging outcomes and mortality, and are responsive to interventions, and this short list includes glucose control and inflammation (justice et al., 2018) . thus, there is potentially great overlap between geroscience biomarkers and the stress-related allostatic load markers described in section 3 (cellular, multi-system, and measures of recovery), and these fields can inform each other. 1. an integrative model of stress and aging. stress acceleration (toxic stress) and stress rejuvenescence (hormetic stress). given the important role of social stress in aging, we need a deeper understanding of types of stress exposures. an overarching model explains the range of stress exposures, from toxic stress to acute hormetic stress, and our body's diverging responses to these exposures. our stress responses are not typically thought of as basic mechanisms of aging but indeed they are actively shaping rate of aging. as shown in figure 1 , the dose and intensity of the stressor determines in part whether the organism responds with positive physiological changes or impairments in aging processes (modified from franceschi et al., 2018) . the exact timing of stressor exposure is an important consideration of a hormetic or pre-conditioning effect, as some stressors lead to sensitization across stressors, rather than habituation (i.e., belda et al., 2016) and this may differ by species, stage of development, and stressor paradigm. therefore, a general model cannot determine the exact parameters of intensity and dose for hormetic stressors. it is important area of future research to identify the boundary conditions and inflection points for the range of potentially hormetic stressors . moderate stressor exposure can lead to both housecleaning in the cells, making them appear younger or rejuvenated, as well as growth of new neural pathways. over time, the accumulation of hormetic stress can promote slowing of aging processes. j o u r n a l p r e -p r o o f 1.1 hormesis is a form of stress resilience. this paper brings together the hormesis literature with the broader stress resilience literature. the cellular biology of hormetic responses is well mapped, and has general common responses as well as stressor specific responses. the acute stress response has a common pathway of creating calcium influx, oxidative stress, and energetic stress. this increases transcription factors such as nrf-2, foxos, creb, and nf-kb, leading to many hormetic effectors, such as chaperone proteins (eg, heat shock proteins which help fold proteins efficiently and prevent protein aggregation), er stress, endogenous antioxidants (sod, glutathione), growth factors, and mitochondrial proteins (mattson, 2008a) . after moderate doses, the cells become resistant to many other types of stressors (heat, uv, oxidative stress, metals), and to death (murakami et al., 2003) . hormesis is a universally observed phenomena across types of cells and types of stressors, including psychological stress. in model organisms, short manageable stressors lead to improvements in aging, although this depends on types of stressor and species (lagisz et al., 2013; rattan, 2008) . for example, low dose gamma radiation over time can extend average lifespan up to 30% in mouse studies (calabrese & baldwin, 2000) . in humans, there is evidence of hormetic stress, such as the effects of exercise, although this is not typically labeled as hormesis. hormesis naturally applies to humans-not just to cells but to physiological and psychological regulation. a typical example is vaccinationwhich leads to enhanced immune responses later. here we expand the definition of hormetic stress to include the positive stressors that humans engage in-such as short term bodily stressors like exercise and temperature stress, but also novel challenging experiences that expand coping resources, knowledge, generativity, and feelings of accomplishment, described further below (section 1.2). since hormetic stress has traditionally been applied to cellular physiology, we use the larger concept of 'stress resilience' as the widest umbrella term for describing when humans recover quickly, in any system, from various exposures. as shown in the appendix, there are many j o u r n a l p r e -p r o o f overlapping terms that relate to the concept of stress resilience. just as the term "stress" is a multi-level construct that needs to examination in a sophisticated interdisciplinary manner, stress resilience is also a multi-level concept that encompasses the full range of human exposures, responses, and inter-related systems. the term stress resilience thus subsumes psychological resilience, physiological resilience/enhanced allostasis, and social resilience. this model of stress resilience can thus be applied to most processes--at the cellular level, physiological level, and psychosocial level. psychosocial stress resilience here refers to the dynamic recovery in psychological, behavioral and social processes and related physiological processes. high stress resilience is reflected by quick physiological and affective recovery from psychological stressors. the neurochemistry of psychological resilience has also been described, based on rodent models (cathomas et al., 2019) . whether a stressor leads to a hormetic or toxic response is not solely determined by the chronicity of the stressor. it is also determined in part by the psychological appraisals, which is shaped by the context, culture, personal history and personality of the individual. when one feels demands exceed resources, in any situation, this creates a physiological and emotional stress response (folkman et al., 1986) . if it is perceived as a threat they cannot cope with, this repeated stress response over time will last longer and be more wearing. in contrast, if they view it as positive challenge that they have the resources for, they will have a profile of quicker recovery, as summarized elsewhere . thus the appraisal of the stressor codetermines the physiological response. it is not just stress responses to major events that matter. our frequent daily stress responses have cumulative effects: the tendency to have slower recovery of negative mood or greater loss of positive mood after a daily stressor predicts inflammation and long term disease and mortality mroczek et al., 2015; piazza et al., 2013; sin et al., 2015) . short term manageable stressors, such as physical or cognitive challenge that can promote growth, learning and development can lead to protective responses. an example of this is found in studies of the experience corps. exposing elderly retired people who are often isolated to a job mentoring at-risk youth in schools is often viewed as stressful but leads to feeling more purpose in life. in men, it has been linked to better health and increases in hippocampal volume gruenewald et al., 2015; varma et al., 2015) . in the case of coping with chronic stressors, most people (around 80%) recover to baseline levels of well being after a loss or disaster (galatzer-levy et al., 2018) . resilience may develop over time, leading to more mastery, purpose, faith, self esteem, and thus more resilient responses to future stressors. in contrast, toxic exposures accumulate over a lifetime, promoting "stress-acceleration of aging" processes. chronic stressors for decades, multiple shorter term exposures over years, and stressors embedded early in life, can all have toxic effects when there is insufficient resources to cope, and nor opportunities to fully recover. developmental factors are critical for understanding when stressors can be hormetic vs. toxic. we do not know precise developmental trajectories for differential effects of toxic stress on mental and physical health and even less is known about hormesis across the human lifespan. with aging, there is a decrease in both the reproductive and anabolic hormones that are part of a salutary acute stress response (epel et al., 1998) , and also a reduction in aspects of molecular hormesis, such as a lower heat shock response to stressors (calabrese et al., 2014; epel, 2008) . we know most about the developmental impact of toxic stressors. while there are myriad individual patterns of exposures, traumatic stress or material deprivation have larger effects early in life than when they occur at later periods; early life adversity is predictive of a range of poor outcomes, including poor mental health, health behaviors, biomarkers of aging, and earlier disease onset (deighton et al., 2018; hughes et al., 2017) although plasticity is still possible (mcewen & morrison, 2013) . given the sensitive period j o u r n a l p r e -p r o o f of pregnancy, it is not surprising there is evidence of transgenerational effects of stress and pregnancy complications on systems regulating aging like telomeres and epigenetics (epel, 2020; girchenko et al., 2017; ross et al., 2020) . a careful meta-analysis of the effects of early adversity points to psychological threat, rather than material deprivation, as the factor underlying accelerated biological aging such as early puberty, telomere shortening, and brain development (colich et al., 2020) . for trauma and abuse, the earlier in life, prenatal and pre-pubertal, when the brain is most rapidly developing, the larger the imprint of lifelong effects on mental and physical health (agorastos et al., 2018) . there are many examples of early adversity with accelerated biomarkers of aging in children: in prepubertal children, early life adversity leads to greater inflammatory acute stress response, and basal inflammation several years later (slopen et al., 2014) . in prepubescent youth, exposure to violence is associated prospectively with telomere shortening (shalev et al., 2013) , and telomere shortness in early childhood predicts carotid artery thickness several years later, and during puberty (barraclough et al., 2019; skilton et al., 2016) . early adversity may accelerate aging in part through inducing early puberty which in turn is linked to earlier onset of metabolic disease (gur et al., 2019; sun et al., 2017) . early adversity may also initiate a trajectory of early aging through lower reserve capacity such as low optimism and higher stressful events in adulthood (surachman et al., 2019) . early adversity also predicts poor health behaviors such as sedentariness, smoking and substance use in youth (wiehn et al., 2018) and these habits appear to persist long into adulthood . the pillars of mammalian aging, represent fundamental and related pathways such as genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, metabolic pathways such as deregulated nutrient sensing, mitochondrial j o u r n a l p r e -p r o o f dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication, macromolecular damage, chronic low-grade inflammation, and adaptation to stress (kennedy et al., 2014; lópez-otín et al., 2013) . other hallmarks of cellular aging are being identified in the brain (mattson & arumugam, 2018) . molecular pathways are often not closely related to each other, pointing to the use of algorithms, for better prediction of outcomes, described below. several of these basic mechanisms in immune cells have been associated with aspects of social stress, including systemic inflammation and shorter telomeres (epel et al., 2004; kiecolt-glaser et al., 2011; miller et al., 2008) poor mitochondrial function (picard et al., 2018) , and accelerated epigenetic aging (park et al., 2019; wolf et al., 2018) . these associations with lifespan stress demonstrate there is no closed system of intrinsic aging, and even at these most molecular levels our aging rate is influenced by our life exposures. we cannot rule out the possibility that some of these observations are from transgenerational effects. a new practical approach already used in humans is to measure a panel of biomarkers of aging that reflect cumulative damage across regulatory systems (e.g., metabolic, immune, stress related), and reducing this a composite measure. the first of these measures was allostatic load (seeman et al., 2001) , and there are newer algorithm measures like 'pace of aging' (belsky, caspi, et al., 2017) , and lack of normal covariation among regulatory systems (belsky, huffman, et al., 2017) . these measures serve as a barometer of biological aging across the lifespan, linked to early experience, and may be useful to examine the effect of interventions (moffitt, this issue). so far, the markers used have been chosen out of convenience of availability, but there is exciting potential to develop further translational measures based more directly on the basic mechanisms of aging. this admittedly requires high intensity collaboration between basic and clinical scientists (eg, assessments of mtor activity, senescent cells, mitochondrial functioning). 2.3 speed of recovery as a novel measure of latent aging at any age. geroscience recognizes that physiological adaptation to stress stands out as a common phenotype of aging j o u r n a l p r e -p r o o f across model systems of aging. stress resilience, and its impairment, is partly an outcome of the social hallmarks of aging, and a common underlying process that in part regulates the cellular hallmarks of aging. snapshot one-time measures of aging based on blood have inherent limitations in that they do not directly test how a person responds to an acute stressor. recovery from challenge is a critical measure of stress resilience that may be important, as it assesses the latent homeostatic capacity of a system. speed of recovery is thought of as intrinsic homeostatic capacity, a latent capacity that reflects biological aging. recovery is such an important marker of aging that it is central to the emerging areas of 'physiological geroscience" and "translational geroscience." naturalistically, acute events often precede a rapid decline in function, reflecting lack of stress resilience. for example, 50% of new disabilities develop after an acute accident or illness and hospitalization (gill et al., 2004) . there are many examples of paradigms measuring recovery that have validated the importance of using a challenge, and measuring functional or biological recovery from the challenge. frailty is a measure of advanced biological age that reflects loss of stress resilience due to age related decline in physiological reserve (hoogendijk et al., 2019) . however, frailty is a final common pathway, one that is probably not reversible. stress resilience interventions will need to target people earlier in life long before frailty sets in. in contrast physiological resilience, which refers to ability to bounce back from a stressor, is measurable at any age (whitson et al., 2016 (whitson et al., , 2018 . stress resilience depends in part on the pre-existing level of reserve capacity, the positive protective factors of an organism, as well as the immediate adaptive psychological response to stressors (cognitive appraisals). in the stress literature, reserve capacity has been defined as j o u r n a l p r e -p r o o f combination of personal resources such as optimism and sense of control, and social factors such as social support. high psychosocial reserve capacity appears to buffer those from low ses backgrounds from developing cardiovascular disease (matthews et al., 2008) . in geroscience, reserve capacity refers to a broader set of resources of or buffers, social, psychological or physiological including cognitive function (e.g., high iq), physiological (e.g., aerobic fitness, sleep), and psychological assets (e.g., high optimism or positive affect). high reserve capacity increases the likelihood that one will have a hormetic protective response to a stressor. as shown in figure 2 , after diverse types of stressors (eg, chemical, physiological or psychological), an organism reacts and recovers to baseline with different speeds and this is moderated by baseline reserve capacity. for example, in response to a hip replacement surgery, the biggest predictor of good recovery was reporting good physical function at baseline (colón-emeric et al., 2019) . in response to general anesthesia, predictors of protection from dementia and other cognitive outcomes was measures of cognitive reserve such as education and vocabulary ability (cizginer et al., 2017) . indices of reserve capacity in functional abilities (such as ability to stand, gait speed, level of fitness), and glucose-insulin response to a glucose load, predict time to mortality, as reviewed elsewhere (seals & melov, 2014) . one of the most well-developed areas of reserve capacity comes from examination of individual dispositions of temperament, typically called personality traits. there are many psychological assets in adulthood that are associated with both better recovery from stressors, and with health and mortality. these include optimism, positive affect, mindfulness, coping with stress with cognitive reappraisal or active coping, high presence of social support or seeking support, purpose in life, and quality relationships. many of these assets have been associated with indices of good health, such as self-reported health and higher heart rate variability (carnevali et al., 2018) . these positive assets are both shaped by genetics and life experience. will promote further positive responses to future stressors, and may help slow the rate of aging in humans, as shown in figure 1 . how can we best translate hormetic interventions to humans? there are many potential interventions that may improve stress resilience, listed in table 1 . lifestyle interventions, such as exercise, caloric restriction, intermittent fasting, challenging cognitive activities, and response to phytochemicals in vegetables and fruits, are thought to work in part through hormesis (mattson, 2008b; radak et al., 2017) . at least one group is pilot testing a cocktail of stressors in humans to examine rejuvenation effects, using intermittent cold, heat, fasting and hypoxia, together with phytochemicals (pruimboom et al., 2016) . the hypoxic preconditioning effect demonstrates protection of neurons and cardiac cells, and is a potential area of translation (li et al., 2017 ). an interesting novel intervention inducing acute stress (through exposure to intermittent hypoxia and cold) appears to improve immune response to endotoxin at least in a small initial study, with a replication underway (kox et al., 2014) . at ucsf we are testing a similar protocol to see if it improves autonomic and neuroendocrine response profiles (including a quicker recovery from acute stress). another dramatic way to increase stress resilience is to enter periods of fasting, or fasting mimicking with low calories. in rodents, this leads to stress resistance and regeneration and rejuvenation processes through hormesis, in part by down regulating gh, igf-1. mtor, and pka signaling (longo, 2019; rangan et al., 2019) . reserve capacity is built during formative developmental experiences, such as level of education, attachment relationships, and stress exposures that shape the neural architecture of stress responding, narratives of optimism, and foster positive challenge mindsets. one can build reserve capacity by increasing physiological buffers (fitness, or antioxidant diet), or psychological stress resilience, through psychological trainings that might decreasing chronic stress arousal and bolster one's mental filter so they j o u r n a l p r e -p r o o f habitually perceive less threat. interventions that build psychological positive assets like optimism, mastery, and purpose in life need to be further developed and refined. mind-body interventions have a strong empirical base for improving self-reported wellbeing (creswell, 2017) with mixed effects on basal inflammation (bower & irwin, 2016) . mindfulness training may lead to changes in heart rate variability and telomere biology, although the evidence again varies by population and study (rådmark et al., 2019) (conklin et al., 2019) and appears stronger with clinical samples-those with high stress or early disease. there is emerging evidence that mind-body interventions improve physiological acute stress reactivity, changing stress appraisals and physiology to more of a positive challenge profile with a strong peak and faster recovery (daubenmier et al., 2019; lindsay et al., 2018) health behaviors regulate healthspan. the social hallmarks of aging shape health behaviors from an early age, which track throughout life. adverse health behaviors, such as diet, physical activity, sleep, and smoking are shaped by social stress. chronic stress both biologically drives toxic nutrition choices (sugar, fast food), impairs sleep, and promotes addictions, an indirect pathway in stress-acceleration of aging. the converse is also true, health behaviors lead to stress-slowing of aging. seventh day adventist who practice lifelong positive health behaviors, and lack the adverse behaviors of substance use, tend to have optimal longevity, living at least four years longer than the average us life expectancy and thus being the only blue zone in the us (fraser & shavlik, 2001) . exercise is the prototypical hormetic intervention. it increases the odds of healthy aging by 39% (daskalopoulou et al., 2017) . the mechanisms at the cellular level are becoming well explicated, as it can enhance mitochondrial health, telomere biology glucose, v02 max, oxidative stress, no) and upregulation stress resistance pathways, such as autophagy, and creating the opportunity for a long healthspan for all (health equity) requires improving economic and social factors. social factors are intrinsic to aging, our rate of aging depends on our social context and conditions. material deprivation and poor neighborhood quality confer psychological stress and risk of poor mental and physiological health (brisson et al., 2020) . for example, food insecurity is associated with over two fold risk of clinical anxiety or depression in adults, and confers even higher risk in college students (arenas et al., 2019; leung et al., 2020) . we now have a better understanding of how social threats lead to toxic stress. the primary motivational forces shaping human behavior are seeking safety and connection with others, and avoiding danger and anxiety. our mind is constantly seeking cues for safety or danger, even when we are not aware of this, and these social signals are transduced to biological signals, including patterns of autonomic activity and gene expression that are linked to inflammation. it is thought that exposure to or perception of frequent social threats (such as social rejection, discrimination, violence, and lack of safety) creates higher chronic systemic inflammation and sympathetic arousal, even while sleeping, and greater risk of affective disorders (brosschot et al., 2017; o'donovan et al., 2013; slavich, 2020; slavich et al., 2010) . conversely, social support, and social capital including perceived safety in neighborhoods, may be stress buffering, and are often associated with less inflammation and longer telomeres (brown et al., 2020; rentscher et al., 2020; thames et al., 2019) (geronimus et al., 2015; m. park et al., 2015) . social support and social networks can bolster healthspan interventions: our stress, emotional and physiological, is contagious to close others (carnevali et al., 2020; engert et al., 2019) , and conversely positive emotion and positive health behaviors are also socially influenced (christakis & fowler, 2013; kim et al., 2015) . the geroscience interventions that may work in mice will not be useful if they cannot be translated well to humans, taking into account our need for support and the significant challenges we have with adherence to exercise and other lifestyle changes. poor behaviors can override effects of protective pharmaceuticals. a common example of this is that people still develop diabetes while taking metformin due to overeating a western junk food diet. improving health behaviors can best be prioritized and implemented in the context where basic social needs are met. creating a supportive built environment and positive social environment are critical to promoting long-term behavior change. the science of behavior change, including the nih initiative focusing on this (nielsen et al., 2018) , has dramatically raised the sophistication of the research in this area, using the experimental medicine model to identify and manipulate the behavioral and social factors that facilitate adherence to health behaviors. behavioral or health interventions that work beyond the individual level, that can decrease loneliness and improve support will be more successful. the covid-19 pandemic demonstrates well the role of social factors in resilience to mental health disorders and infection. the pandemic led to dramatic increases in mental health disorders in the us and other countries (xiong et al., 2020) but this was not equally distributed. those with low education, income, minority status, loneliness, or low social support have significantly higher rates of mental health disorders from pandemic stress (arafa et al., 2020; holingue et al., 2020; palgi et al., 2020) . these vulnerable groups also tend to have higher rates of covid severity (adhikari et al., 2020; webb hooper et al., 2020) . any policies that improve social equity are also 'stress reducing' health policies that may contribute to healthspan, and can be incorporated into the geroscience agenda. geroscience is now more important than ever, both to our aging global demography but also to the health challenges we face going forward. in our new era we have dramatically increasing temperature extremes, wildfires and small particle pollution, and new zoonotic viruses to contend with intermittently. thus reducing social disparities, improving stress resilience and bolstering immune function have become critical public health goals. the vulnerability to covid-complications, while still largely unknown beyond older age and pre-existing diseases, clearly depends on ability of the immune system to respond robustly. the relevance of immune senescence in covid-lethality has stimulated many hypotheses about geroscience-related prevention and treatment (barzilai et al., 2020; salimi & hamlyn, 2020; sargiacomo et al., 2020) . while vaccination is essential for traditional prevention, it is not a universal solution: the elderly have poorer antibody responses to vaccination, there are many strains of the current virus, and there are expected to be many proliferations of future viral strains novel to the human body, due to climate change. therefore, geroscience interventions have unique universal importance across time. pharmacological interventions have been suggested for covid such as rapalogs, senoytics, nicotinamide adenine dinucleotide nad+, and metformin for anti-inflammation, telomere stability, or to boost vaccination response (omran & almaliki, 2020) . those with diabetes appear to benefit from metformin, which has hormetic properties, to prevent covid-related mortality (luo et al., 2020) . beyond pharmacological treatments, it is likely some of the interventions for boosting stress resilience in table 1 may enhance resistance to viral infections, from common cold to novel viruses. the malleable lifestyle behaviors like fitness, nutrition, sleep quality, and stress reduction, are important ways to reduce insulin resistance and comorbidities, and thus may help prevent immune senescence and covid complications. one pathway through which stress resilience interventions could impact immunity is through stabilizing telomere length. short telomeres predict greater vulnerability to rhinovirus infection, acute respiratory syndrome disorders, and mortality from sepsis (cohen et al., 2013; liu et al., 2020) . chronic psychological stress shortens telomeres in animal studies and impairs viral immunity (cohen et al., 1991 (cohen et al., , 1998 . short telomeres indicate lower ability to mount a robust replicative t cell response, and this may be a critical or even fatal j o u r n a l p r e -p r o o f limitation in the face of covid related lymphopenia (aviv, 2020) . in short, covid-19 presents a potent example of the potential for using indices of aging as predictors of disease and targets of intervention. the goal of geroscience is to slow aging to improve healthspan. in the next generation of research, we will benefit greatly from incorporating the large malleable factors that impact human aging-biobehavioral and social factors. the nia's intervention and testing program, a multi-institutional infrastructure to study biological agents for healthspan in animals is a model that can be extended to human trials that takes into account the social and behavioral factors (moffitt, this issue). the social hallmarks of aging shape rate of aging, in part through toxic stress processes. the understanding of toxic stress and hormetic stress as factors shaping aging will have implications for interventions. stress resilience, the ability to recover quickly and turn on rejuvenative processes, is an important dynamic endophenotype of healthy aging. it remains to be seen how much resilience is merely a characteristic of healthy aging or a causal factor, although much evidence reviewed here suggests it is at least partly causal. a better understanding of how to measure stress resilience, and to promote stress resilience at the cellular, physiological and psychosocial levels will lead to important gains in slowing aging. the science of stress is an integral part of geroscience, and offers insights on how to harness stress for optimal longevity, and implications for how to conduct the most effective interventions incorporating these stress processes as both target mechanisms and outcomes. by having an integrative paradigm that can be examined across levels, we can reduce the gap between physiological stress research in model organisms and human research on stress, resilience and adaptation. there are many ways to measure biological aging in humans that can serve as a barometer of change for interventions. this includes cellular level markers, multi-system composites, and ways of examining dynamic stress resilience, as reviewed. this j o u r n a l p r e -p r o o f can include recovery from a medical event in the elderly, but also recovery to standardized challenges, and to naturalistic stressors. geroscience offers an exciting opportunity for high feasibility impact interventions. this integrative paradigm can shape the next generation of researchers. the training models need to change to bridge the many fields as outlined by pioneers in geroscience (newman et al., 2019) . models which are focused on pharmacological interventions must expand to be inclusive of both social and behavioral interventions, the current 'big levers.' lastly, this field, like all of science, needs to actively encourage and support young investigators from diverse and underprivileged backgrounds to enter this important and growing field which has the potential to minimize socioeconomic and ethnic/racial health disparities not just for equity but for the improved science that results with diverse life experiences and perspectives. frameworks for proof of concept trials related to loss of physiological resilience have been initially outlined (justice et al., 2016) , and there are many geroscience trials in the field. these human trials can draw on the rich insights from decades of biobehavioral basic and intervention research. the science of behavior change initiative at nih is supporting the development of more effective behavioral and social interventions using the experimental medicine model using the same attention and rigor as pharmaceutical studies. by working across disciplines, with an understanding the role of lifespan experiences, and complexity of human environments, the geroscience framework has tremendous potential for breakthrough innovations in increasing healthspan. there has been a proliferation of terms from related disciplines that overlap and are differentiated here. the discipline most often using the term is noted, but these terms could be used to describe all levels of analysis, including cellular, physiological/organ systems, or psychological, behavioral and social processes. resilience has also been applied ast the systems level, to organizations, communities, societies, and ecosystems. how systems change in response to stressors, typically referring to level of hermetic or a protective adaptive responses in cell such as heat shock protein increases. (mattson) . mild stressors induce adaptive capacities that protect an organism for a short while from future stressors and may improve the physiological state of that organisms. preconditioning is a case of hormesis where exposure to a chemical agent leads to a 30-60% stronger adaptive response to subsequent exposures, across cell types and stressors (calabrese, 201) . in psychology, the tem 'stress inoculation' is used in a similar way. in terms of psychological stressors, under-exposure to the typical daily and major life events can lead to lack of development of stress buffering resources, and poor ability to quickly recover from stressors. biologically the lack of acute stressors prevents the intermittent episodes of cellular 'housecleaning' activities that slow aging. ideal exposure to sufficient numbers of manageable challenges throughout life stimulate cognitive growth, coping skills, and emotion regulation skills, as well as the need for supportive social networks. biologically, ideal exposure to acute stress can have hormetic effects, leading to rejuvenescence-functioning that is enhanced (or "younger") compared to baseline. overexposure to stress without sufficient resources (toxic stress) can lead to maladaptive neural pathways of overresponding to stress, depression, and stress related acceleration of aging from cells to regulatory systems. this figure is adapted from francheschi et al, 2018. in response to acute stressors, individuals have a kinetic trajectory of responses across psychological and physiological regulatory systems that lead to reactivity and recovery profiles. resilient stress responses (typically rapid high peak and rapid recovery) often have hormetic effects at the cellular or systemic levels. high levels of reserve capacity predict more rapid recovery, and this may lead to a positive feedback loop promoting higher reserve capacity. level of stress resilience is multiply determined by the social context and individual 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behaviours in german public university students: a crosssectional analysis relationship of psychosocial resources with allostatic load: a systematic review traumatic stress and accelerated dna methylation age: a meta-analysis impact of covid-19 pandemic on mental health in the general population: a systematic review the impact of changes in population health and mortality on future prevalence of alzheimer's disease and other dementias in the united states crimmins, steve austad, and edward calabrese. key: cord-320208-uih4jf8w authors: li, diya; chaudhary, harshita; zhang, zhe title: modeling spatiotemporal pattern of depressive symptoms caused by covid-19 using social media data mining date: 2020-07-10 journal: int j environ res public health doi: 10.3390/ijerph17144988 sha: doc_id: 320208 cord_uid: uih4jf8w by 29 may 2020, the coronavirus disease (covid-19) caused by sars-cov-2 had spread to 188 countries, infecting more than 5.9 million people, and causing 361,249 deaths. governments issued travel restrictions, gatherings of institutions were cancelled, and citizens were ordered to socially distance themselves in an effort to limit the spread of the virus. fear of being infected by the virus and panic over job losses and missed education opportunities have increased people’s stress levels. psychological studies using traditional surveys are time-consuming and contain cognitive and sampling biases, and therefore cannot be used to build large datasets for a real-time depression analysis. in this article, we propose a corexq9 algorithm that integrates a correlation explanation (corex) learning algorithm and clinical patient health questionnaire (phq) lexicon to detect covid-19 related stress symptoms at a spatiotemporal scale in the united states. the proposed algorithm overcomes the common limitations of traditional topic detection models and minimizes the ambiguity that is caused by human interventions in social media data mining. the results show a strong correlation between stress symptoms and the number of increased covid-19 cases for major u.s. cities such as chicago, san francisco, seattle, new york, and miami. the results also show that people’s risk perception is sensitive to the release of covid-19 related public news and media messages. between january and march, fear of infection and unpredictability of the virus caused widespread panic and people began stockpiling supplies, but later in april, concerns shifted as financial worries in western and eastern coastal areas of the u.s. left people uncertain of the long-term effects of covid-19 on their lives. in december 2019, an outbreak of pneumonia caused by a novel coronavirus (covid-19) occurred in wuhan and spread rapidly throughout the globe [1] . the covid-19 outbreak has forced people to change their regular routine lives and practice social distancing. such a sudden change can drastically increase people's stress level and lead to other mental health issues. the difficulties caused by the covid-19 outbreak in different geographic regions can determine the cause and degree of stress in people, which corresponds to their risk of developing serious depression [2] . according to a poll [3] , nearly half (45%) of adults in the united states reported that their mental health has been negatively impacted due to worry and stress over the virus. as the pandemic continues, it is likely that the mental health burden will increase as people's sense of normalcy continues to be disrupted by social distancing, business and school closures, and shelter-in-place orders. the preexisting stress, constant unpredictability, and lack of resources lead to even greater isolation and financial distress. traditional mental health studies rely on information primarily collected through personal contact with a healthcare professional or through survey-based methods (e.g., via phone or online questionnaire). for instance, the patient health questionnaire (phq) is a self-administered version of the primary care evaluation of mental disorders (prime-md) diagnostic instrument for common mental disorders [4] . however, these survey-based methods are time-consuming and suffer from cognitive and sampling biases, and therefore cannot be used to build large datasets for a real-time depression analysis [5] . furthermore, understanding of spatial epidemic trends and geographic distribution patterns of covid-19 provides timely information on people's risk perception of epidemics. however, these important spatial and environmental leading factors are difficult to include in a survey-based method to model covid-19 related mental stress. geographic information system (gis) and social media data mining have become essential tools with which to examine the spatial distribution of infectious diseases [6] [7] [8] , and can be used to investigate the spatiotemporal pattern of mental stress caused by the pandemic. for instance, social media data (e.g., twitter data) provide a unique opportunity to learn about the users' moods, feelings, and behaviors that reflect their mental health as they experience daily struggles [8] [9] [10] . many articles focused on using feature-based approaches to perform sentiment and emotional analysis using twitter data [11] [12] [13] [14] . for instance, go and colleagues [11] investigated the usage of unigrams, bigrams, and their combination in training the classifiers for sentiment analysis of tweets. various supervised classifiers were trained, including maximum entropy, naïve bayes [15] , and support vector machine (svm) classifiers and their performance on the n-grams was compared. however, some methods previously used [11] have become outdated; for instance, they took emoticons into account for their sentiment index, but nowadays lots of twitter users use emojis more frequently [16] . barbosa and feng [17] showed that n-grams are not useful in classifying tweets, as unused words in tweets can cause problems during classifier training. pak and paroubek [18] proposed the usage of microblogging features like hashtags, emoticons, re-tweets, and comments to train an svm classifier and showed that it resulted in higher accuracy than training using n-grams. several articles address the effect of using part-of-search (pos) tag features in text classifiers [18, 19] . abadi and colleagues [19] investigated pos, lexicon, and microblogging features. the results showed that the most relevant features are those that combine prior polarity with the pos tags of the words. however, there have been mixed results reported on the usage of pos tags. go and colleagues [11] showed that the pos tags caused reduced performance, although pos tags can be strong indicators of emotions in text and serve as a helpful feature in opinion or sentiment analysis [18] . moreover, bootstrapping approaches, which rely on a seed list of opinion or emotion words to find other such words in a large corpus, are becoming more popular and have proven effective [20] [21] [22] [23] . mihalcea, banea, and wiebe [23] described two types of methods for bootstrapping the subjectivity lexicons into dictionary-based and corpus-based. their research began with a small seed set of hand-picked subjective words, and with the help of an online dictionary produced a larger lexicon of potential candidate words. a similar bootstrapping model was effectively used to build a sentiment analysis system for extracting user-generated health review about drugs and medication [20] . however, all the aforementioned methods only detect the general emotion of tweets and lack the ability to model depression levels in detail. latent dirichlet allocation (lda) is one of the most commonly used unsupervised topical methods, where a topic is a distribution of co-occurring words [24] . however, the topics learned by lda are not specific enough to correspond to depressive symptoms and human judgments [25] . the unsupervised method can work with unclassified text, but it often causes topics overlap [26] . later, the lda method was extended by using terms strongly related to phq-9 depression symptoms as seeds of the topical clusters and guided the model to aggregate semantically-related terms into the same cluster [27] . however, this approach only detects the presence, duration, and frequency of stress symptoms, ignoring the spatial context or environmental factors that are important in modeling the covid-19 related mental stress. to identify phq related text and unrelated text, a sentiment analysis index generated by python textblob was used [27] , which only calculates the average polarity and subjectivity over each word in a given text using a constant dictionary [28, 29] . work based on the lda probabilistic generative model was found to have limitations related to interpreting high dimensional human input factors which makes it difficult to generalize generative models without detailed and realistic assumptions for the data generation process [30] [31] [32] . in this article, we propose a corexq9 algorithm that integrates correlation explanation (corex) learning algorithm and clinical phq lexicon to detect covid-19 related stress symptoms at a spatiotemporal scale in the united states. we aim to investigate people's stress symptoms in different geographic regions caused by the development of the covid-19 spread. since twitter data are high-dimensional human input data with diverse terms used to express emotions, we used the corex algorithm, a method intended to bypass the limitations of lda implementation and minimize human intervention [33] . after that, we developed a fuzzy accuracy assessment model to visualize the uncertainty of the analytical results on the map. the rest of the article is organized as follows: section 2 introduces the material and methods used in the research work including the introduction of data collection and processing methods, basilisk and machine learning classifier, and the proposed corexq9 algorithm. the results and discussion are presented in sections 3 and 4, respectively. section 5 draws conclusions. twitter data used in this article were collected through the twitter api from january to april 2020 for the continental united states. the collected data contained 80 million tweets (~70 gb), which posed significant computationally intensive challenges for the traditional gis computing environment. to address this challenge, we used a jupyter computing environment deployed on the texas a&m high performance computer. we filtered the collected twitter data using coronavirus related entities (e.g., hashtag, trends, and news). then, we removed irrelevant information (e.g., non-english language tweets, punctuation, missing data, messy code, url, username, hashtags, numbers, and query terms) from the filtered tweets. some adjustments and normalizations (e.g., uniform lower case, nonmaize vectorized tweets, standardize time sliced tweets) were also made in order to fulfill the common requirements of machine learning models. however, the stop words were removed later when applying the proposed algorithm to match the tweet phrase with lexicon. after that, the tweets were tokenized using the natural language toolkit's (nltk) tweettokenizer [33] . we also replaced repeated character sequences by using the length value of three for any sequences of length three or greater (3+), since most users often extend words or add redundant characters to express strong feelings. tweets with an exact geospatial tag and timestamp were mapped to the corresponding county using reverse geocoding method [34, 35] . other tweets (e.g., without geotags but containing user-defined location information in the user's profile) were geocoded to their corresponding county using a fuzzy set search method and city alias dataset [36] . we excluded tweets that did not have geotags nor user-defined location information. one of the key innovations in our research was to map the covid-19 caused stress symptoms at a temporal scale. in this case, we set the temporal scale to biweekly starting from 26 january 2020, so the number of tweets collected in each county could be sufficient for accurate and reliable analysis. we used the basilisk bootstrapping algorithm to find semantic lexicons that could be used to divide the tweets into two categories: stressed and non-stressed. the bootstrapping approach to semantic lexicon induction using semantic knowledge, also known as the basilisk algorithm, was developed by thelen and riloff in 2002 [37] . this approach can extend to divide the tweets into multiple categories across different areas [22] . it employs a bootstrapping method to determine high-quality semantic lexicons of nouns. the algorithm takes a huge unannotated corpus from where it finds new related words and assigns them to the different semantic categories (e.g., stressed and non-stressed in our case). it is a form of categorization that is based on the seed words manually provided to the algorithm. these seed words are bootstrapped to identify new words that fall within the two categories. basilisk must be seeded with carefully selected terms for it to be effective. the two categories of seeds used for this task consisted of 20 words each (table 1) [38]. the first category contained words describing stress and were used to bootstrap other words semantically related to stress or carrying a similar context. the second category contains words that describe non-stressed or a relaxing behavior. these two categories can be thought of as words that fall at the opposite ends of a stress level spectrum. before the bootstrapping process, the patterns were extracted on the unannotated corpus. this is used to extract all the noun phrases that were either the subject, direct object or prepositional phrase. the noun phrases were extracted from the corpus using the stanford dependency parser [39] . it is a natural language parsing program used to find grammatical structure in sentences and can be used to find relationships or dependencies between nouns and the actions or words that form a group and go together. the dependency parser was run on all the sentences in the corpus and dependency relations were extracted for each word in the text (in the conll-u format [40]). for each tweet, the following dependency information was extracted. the conll-u format of the extracted dependency pattern consists of the index, text, lemma, xpos, feats, governor, and dependency relations ( table 2 ). these extracted dependency relations were used to extract patterns that were used by the basilisk algorithm to generate seeds. these extraction patterns were created for each dependency relation obtained in the previous step. the extraction patterns consisted of noun phrases and the dependency of them with other related words in the sentence. this acted as the input to the bootstrapping method. after the input was generated, the next step was to generate the seeds using basilisk. the seed words from the initial pattern pool enlarge with every bootstrapping step. the extraction patterns were scored using rlogf metric [41] , which is commonly used for extraction pattern learning [41] . the score for each pattern was computed as: rlogf(pattern (i) ) = f i n i * log 2 f i , where f i represents the number of category members extracted by pattern (i) and n i is the total number of nouns extracted by pattern i . this formula was used to score the patterns with a high precision or moderate precision but a high recall. the high scoring patterns were then placed in the pattern pool. after this process, all head nouns co-occurring with patterns in pattern pool were added to the candidate word pool. at the end of each bootstrapping cycle, the best candidates were added to the lexicon thus enlarging the lexicon set. the process used related to basilisk, as proposed by thelen and riloff, can be described using the algorithm shown on table 3 (for notation description see appendix a). this performs the categorization task of assigning nouns in an unannotated corpus to their corresponding semantic categories. using the words generated by the basilisk algorithm, we counted the total number of occurrences of any of the keywords in both categories. after the total count of stress and non-stress words in each tweet was obtained, we determined whether the tweet was in the category of stressed or non-stressed or neutral. this was done by finding the maximum of the stress and non-stress word counts in three conditions: (1) if there were more stress words than non-stress words, we annotated the tweet as expressing stress. (2) if the number of non-stress words is greater than the number of stress words, we annotated the tweet to express relaxed behavior. (3) if the count was zero for both stress and non-stress words, we did not annotate the data. thus, tweets and their corresponding labels generated using this process were the initial training set, which was used to train a classifier to classify the other unannotated tweets. table 3 . illustration of the basilisk algorithm [41] . procedure: lexicon = {seed words} for i := 0 1. score all extraction patterns with rlogf 2. pattern pool = top ranked 20 + i patterns 3. candidate word pool = extractions of patterns in pattern pool 4. score candidate words in candidate word pool 5. add top five candidate words to lexicon 6. i := i + 1 7. go to step 1. the universal sentence encoder [42] was used to generate word embeddings. these text embeddings convert tweets into a numerical vector, encoding tweet texts into high dimensional vectors that are required to find semantic similarity and perform the classification task. it takes a variable length english text as input and outputs a 512-dimensional vector embedding. the encoder model was trained with a deep averaging network (dan) encoder [15] . after the word embeddings were obtained for each stressed and non-stressed category tweet, a technique was used to make the two classes equalized. to do this, we selected the category with fewer samples and made the other category a similar size by removing samples. this ensured that the training process was not biased towards a particular class. before training the classifier, the data were split into training and validation sets. the data were randomly shuffled and put into the two datasets, with 80% used as the training dataset. to obtain the best performance, multiple classifiers were used, and performance was compared using accuracy metrics. the classifiers used in the training process were svm [42] , logistic regression [43] , naïve bayes classifier [44] , and a simple neural network. svm handles nonlinear input spaces and separates data points using a hyperplane using the largest amount of margin. as a discriminative classifier, svm found an optimal hyperplane for our data, which helped with classifying new unannotated data points. we used different kernels to train the svm. the hyperparameters were tuned and the optimal value of regularization and gamma were also recorded. the logistic regression classification algorithm can be used to predict the probability of a categorical dependent variable. the dependent variable is a binary variable that contains data coded as 1 (stressed) or 0 (non-stressed). the logistic regression model predicts p(y = 1) as a function of x. prior to training, it shuffles the data. it uses a logistic function to estimate probabilities to calculate the relationship between independent variable(s) and the categorical dependent variable [45] . naïve bayes is another probabilistic classifier which makes classifications using the bayes rule. this classifier is simple and effective for text classification. a simple neural network consisting of three dense layers were used to train our datasets. the loss function and optimizer used in the training is binary cross entropy and rmsprop, respectively. training was done for 40 epochs with a batch size of 512. table 4 illustrates the performance evaluation of these classifiers. after the model was trained, the model was run on the unannotated tweets to label them. to label the sentence embeddings for the tweets, the same procedure was used as for the training set. the universal sentence encoder extracts 512 features and created vectors that were used to classify the tweets based on the model. the svm classifier with linear kernel was used to predict the probabilities of the tweets because it had the best trained models (see table 4 ). here, a threshold of 0.75 was set to determine if the tweet belonged to a particular category or not. if the probability of the tweet was above 0.7 for that category, the tweet was classified with the corresponding label. the tweets and labels generated using the above process were then used to train another classifier to generate the final model for classification of the entire unannotated corpus. here, a logistic regression model was used to train tweets and their corresponding labels generated using the above process to ensure that the model was robust and was not overfitted on the initial set of tweets that were filtered out using the basilisk generated keywords. the trained model had an accuracy of 90.2% on the validation data. in this article, we propose a novel corexq9 algorithm to detect spatiotemporal patterns of covid-19 related stress. table 5 illustrates the general structure of the corexq9 algorithm. the input of the algorithm was the stressed-related tweets derived by using the trained models (see sections 2.2.3 and 2.2.4) to all the processed covid-19 related tweets. we assessed the level of stress expressed in covid-19 related tweets by integrating a lexicon-based method derived from established clinical assessment questionnaire phq-9 [46] . table 6 illustrates the phq-9 lexicon examples and their corresponding mental stress symptoms. procedure: 1. shallow parsing each tweet into tweet_pharse using spacy 2. for each word_set in phq_lexicon do 3. calculate average vector of word_set and tweet_pharse using glove 4. match word_set with tweet_pharse set using cosine similarity measure 5. append each matched tweet_pharse to word_set 6. calculate tf-idf vector for all the tweets and transform the calculated value to a sparse matrix x 7. iteratively run corex function with initial random variables v random 8. estimate marginals; calculate total correlation; update v random 9. for each word_set in phq_lexicon 10. compare v random and word_set with bottleneck function 11. until convergence the phq-9 lexicon contains about 1700 clinical words, which is difficult to understand and match with the spoken language that is often used on twitter. therefore, we used the following methods to transform phq-9 lexicon to human understandable language by appending matched tweets to their best match phq-9 categories. in the first step, each tweet was placed into a set of phrase sets using natural language processing toolkit spacy [44] (see table 4 , procedure 1). after that, the tweets and phq-9 lexicon were vectorized using global vectors for word representation (glove), wikipedia, and gigaword 5 model (with 300 dimensional word vectors and four million unique tokens) [45] . glove provides a quantitative way to distinguish the nuance difference of two words (e.g., happy or unhappy), which is useful to match phrases set with the phq-9 lexicon. those pre-trained vectors were loaded to gensim [47] to perform average vector and cosine distance calculation (see equations (1) and (2)). we appended all phrases that have the similarity rate higher than 0.8 to their corresponding phq-9 lexicon (see table 4 , procedures 3-5). given any words in a phrase, the average vector was calculated using the sum of the vectors divided by the number of words in a phrase: given any two average vectors v a and v b of two phrases, the cosine similarity, cos θ, is represented by next, a sparse matrix (e.g., a vocabulary dense matrix) for stressed corpus was calculated by transforming those tokenized and vectorized tweets using frequency inverse document frequency (tfidf). the mathematical formula of tfidf is illustrated below: where t denotes the terms; d denotes each document; and d denotes the collection of documents. the first part of the formula t f (t, d) calculates the number of times each word in covid-19 corpus appeared in each document. the second part of id f (t, d) is made up with a numerator d = d 1 , d 2 , . . . d n and a denominator | {d ∈ d : t ∈ d}|. the numerator infers the document space, which is all documents in our covid-19 stress corpus. the denominator implies the total number of times in which term t appeas in all of our documents d. the id f (t, d) can be represented by we utilized scikit-learn tfidfvectorizer to transform preprocessed tweets to a sparse matrix [48] (see table 4 , procedure 6). after that, the sparse matrix and lexicon are used by the anchored corex model to perform anchored topic modeling [32] . the total correlation tc [49] (for notation description see appendix a) of each topic is calculated by anchoring the corex model with the document sparse matrix. the total correlation in our phq-9 lexicon detection can be expressed using kullback-leibler divergence as below [50] . where p(x g ) represents the probability distribution and tc(x g ) is non-negative or zero factorizes of p(x g ) (see appendix a for more detail). in the context of phq-9 detection, x g represents the group of word types among the covid-19 corpus. note that each vector in the tfidf matrix is based on the distance between two probability distributions, which is expressed as cross-entropy entropy(x) [51, 52] . when introducing a random variable y, the tc can explain the correlation reduction in x, which is a measure of the redundant information that the word types x carry about topic y [30] . the total correlation can be represented by: where i(x : y) = entropy(x) + entropy(y) − entropy(x, y) (for notation description see appendix a). thus, the algorithm starts with randomly initialized variables α i,j and p(y i |x i ), where α i,j are indicator variables of tc that are assigned to 1 if the topic is detected and p(x i ) represents the approximate empirical distribution (see table 4 , procedure 7). then, the correlation explanation updates both variables iteratively until the result achieves convergence. in each iteration, the estimate marginals p(y j |x i ) = x p(y i |x)p(x)δ x i and mutual information tc are calculated (notation description see appendix a). next, the update for a t i,j in each t step is calculated by where λ conduct a smooth optimization of the soft-max function [53, 54] . finally, the soft labeling of any x (for notation description see appendix a) can be computed by after the soft-max function α converges to the true solution at a particular step α k in the limit λ → ∞ , the mutual information terms can be ranked by the informative order in each factor. to perform semi-supervised anchoring strategies, gallagher and reing proposed the combination with bottleneck function and total correlation [32] . the bottleneck function can be represented by: the connection with corex and anchor words can be described by comparing equation (6) with equation (9). the same term i(x : y) in two equations represents the latent factor and the variable z corresponds to x i . it is worth noting that z is typically labeled in a supervised learning task [54] and β is a constant parameter to constrain supervising strength so that α = β can imply a word type x i correlated with topic y j . in this case, z was represented by each variable generated by the enriched phq-9 lexicon. to seed lexicon to detect topics, we can simply anchor the word type x i to topic y j , by constraining the β (see table 5 , procedures 8-11). the symptoms of covid-19 related stress were visualized at the county level biweekly from 26 january. here, we used the fuzzy accuracy assessment method to evaluate the uncertainty of final phq stress level for each county [55, 56] . we summarized the implementation of fuzzy accuracy assessment for a thematic map as presented by gopal and woodcock to explain our model evaluation for the phq map [55] . let x be a finite universe of discourse, which is the set of county polygons in the study area. let ζ denote the finite set of attribute membership function (mf) topics categories to the d in x; and let m be the number of categories |ζ| = m, (e.g., nine phq categories). for each x x, we define χ(x) as the mf classes assigned to x. the set: defines the data. the subset s ⊂ x of n data is used. a fuzzy set is associated with each class c ζ where µ c (x) is the characteristic of mf of c. the fuzzy set can be represented as: to implement a decision-making system for fuzzy accuracy, the model uses a boolean function σ that returns results of 0 or 1 based on whether x belongs to the class c with respect to the matrix a. that is, σ(x, c) = 1 if x "belongs" c, and σ(x, c) = 0 if x does not "belong" to c. then σ(x, c) is 1 if the numeric scale of the mf for x in category c(µ c (x)) is maximum among all map categories µ c (x), and we set the boolean function σ as max follows: according to the fuzzy set accuracy assessment, the final phq value for each county was selected based on the max function, meaning each county was colored based on the majority tweet phq value derived from the proposed corexq9 algorithm. since the accuracy assessment was based on a comparison of the phq label assigned to each county with the evaluation given by the expert (e.g., in each county, the majority tweet phq label). the rating system can thus be expressed as linguistic variables that describe the uncertainty associated with the evaluation of the class label. here, the linguistic variables are described below: 1. score 1: understandable: the answer is understandable but may contain high levels of uncertainty; 2. score 2: reasonable: maybe not the best possible answer but acceptable; 3. score 3: good: would be happy to find this answer given on the map; 4. score 4: absolutely right: no doubt about the match. it is a perfect prediction. figure 1 illustrates the fuzzy mf created for the fuzzy accuracy assessment analysis. the x-axis represents the percentage of the tweets that belong to the assigned final phq category. the y-axis represents the value of the degree of the membership function corresponding to the linguistic score. for instance, if a county was assigned to a phq category 3, and 80% (e.g., x = 0.8 in figure 1 ) of the tweets within this county polygon were labeled as phq-3 using the corexq9 algorithm, the corresponding mf should be absolutely right with membership value equal to 1. the accuracy assessment score was further visualized on the phq stress map to show the spatial uncertainty of the analysis results. the numeric scale of the mf for in category ( ( )) is maximum among all map categories ′ ( ), and we set the boolean function as follows: according to the fuzzy set accuracy assessment, the final phq value for each county was selected based on the max function, meaning each county was colored based on the majority tweet phq value derived from the proposed corexq9 algorithm. since the accuracy assessment was based on a comparison of the phq label assigned to each county with the evaluation given by the expert (e.g., in each county, the majority tweet phq label). the rating system can thus be expressed as linguistic variables that describe the uncertainty associated with the evaluation of the class label. here, the linguistic variables are described below: 1. score 1: understandable: the answer is understandable but may contain high levels of uncertainty; 2. score 2: reasonable: maybe not the best possible answer but acceptable; 3. score 3: good: would be happy to find this answer given on the map; 4. score 4: absolutely right: no doubt about the match. it is a perfect prediction. figure 1 illustrates the fuzzy mf created for the fuzzy accuracy assessment analysis. the x-axis represents the percentage of the tweets that belong to the assigned final phq category. the y-axis represents the value of the degree of the membership function corresponding to the linguistic score. for instance, if a county was assigned to a phq category 3, and 80% (e.g., x = 0.8 in figure 1 ) of the tweets within this county polygon were labeled as phq-3 using the corexq9 algorithm, the corresponding mf should be absolutely right with membership value equal to 1. the accuracy assessment score was further visualized on the phq stress map to show the spatial uncertainty of the analysis results. since corexq9 represents topic and potential symptoms as a lexicon-based topic modeling, traditional measures such as regression correlation and log-likelihood are unnecessary for the semantic topics. therefore, to evaluate the baseline performance of the corexq9 model, we first involved the semantic topic quality coherence measure methods with other common topic models. we compared corexq9 with lda and non-negative matrix factorization (nmf) [57, 58] . in addition, we used frobenius normalized nmf (nmf-f) and generalized kullback-leibler divergence nmf (nmf-lk) for a closer comparison with traditional topic modeling. all models were trained with a since corexq9 represents topic and potential symptoms as a lexicon-based topic modeling, traditional measures such as regression correlation and log-likelihood are unnecessary for the semantic topics. therefore, to evaluate the baseline performance of the corexq9 model, we first involved the semantic topic quality coherence measure methods with other common topic models. we compared corexq9 with lda and non-negative matrix factorization (nmf) [57, 58] . in addition, we used frobenius normalized nmf (nmf-f) and generalized kullback-leibler divergence nmf (nmf-lk) for a closer comparison with traditional topic modeling. all models were trained with a randomly selected covid-19 twitter dataset. the topics generated by those models were scored by topic coherence measures to identify the degree of semantic similarity between high-scoring words in the topic. a common coherence measure is umass which calculates and scores the word co-occurrence in all documents [59] : where d(w i , w j ) represents the number of documents containing both w i and w j words and d(w i ) counts the ones containing w i , and c represents a smoothing factor. the intrinsic umass [59] coherence measure calculates these probabilities over the same training corpus. additionally, the extrinsic uci measure [58] introduced by david newman uses a pairwise score function, which is based on pointwise mutual information (pmi). it can be represented by: where p(w i ) represents the probability of seeing w i in a random document, and p(w i , w j ) is the probability of seeing both w i and w j co-occurring in a random document. those probabilities are empirically estimated from an external dataset such as wikipedia. the higher the topic coherence measure score, the higher the quality of the topics. in our baseline evaluation, we calculated the coherence scores by setting the range of topic numbers from 10 to 30. the abnormal and low-quality topics were cleared and the average coherence scores (table 7) were calculated by the sum of all coherence scores divided by the number of topics. on average, the corexq9 algorithm has a better umass score than lda and nmf. even though the uci score was slightly lower than two types of nmf algorithms, we can take the external estimation dataset as an uncertainty factor of this coherence model because the result of the comparison was still meaningfully coherent and it has the competitive functionality of the semi-supervised feature, which exceeded the usable range of nmf. in our research, the methods described above were combined to generate the final thematic map. to summarize processes for each detailed procedure, the workflow for the research is shown in figure 2 . first, starting from data collection, we prepared a twitter dataset, basilisk lexicon, and phq-9 lexicon. then, we cleaned each tweet and extracted its location information using the method mentioned in section 2.1. to engage time series analysis, the whole twitter dataset was formatted and sorted by unix timestamp before being sliced into two-week intervals. third, two lexicons were separately assigned to corexq9 and basilisk algorithm (mentioned in section 2.2) with the prepared twitter dataset. in the end, we decomposed the result generated by anchored corex model into spare matrix in order to group by all tweets in county level for visualization. note that each row of the results from the corex algorithm represents the correlations index within an individual tweet explained by nine phq levels so that we can reverse convert the result to its original tweets. the selected top symptoms and topics are present in table 8 . the fuzzy accuracy assessment results of the study are illustrated in figure 3 . on each map, the individual county is colored according to the assigned phq index using the proposed algorithm and fuzzy assessment accuracy assessment method. the numbers on the map represent the spatial uncertainty indices derived from the fuzzy accuracy assessment. each number represents the assessment score calculated from section 2.2.6. for most of the hot spots areas in figure 3 , the values are greater than two, which indicates middle to high accuracy results have been reached for those regions. higher scores for an area indicate a larger percentage of the topics being present in this area at specific time region. the fuzzy accuracy assessment results of the study are illustrated in figure 3 . on each map, the individual county is colored according to the assigned phq index using the proposed algorithm and fuzzy assessment accuracy assessment method. the numbers on the map represent the spatial uncertainty indices derived from the fuzzy accuracy assessment. each number represents the assessment score calculated from section 2.2.6. for most of the hot spots areas in figure 3 , the values are greater than two, which indicates middle to high accuracy results have been reached for those regions. higher scores for an area indicate a larger percentage of the topics being present in this area at specific time region. the results also present the spatiotemporal patterns from january to april (shown in figure 3a -g. table 8 shows the detected stress symptoms and topics generated from corexq9. each map represents the spatial distribution of stress symptoms over a biweekly period. it indicates that most of the regions have low to medium phq values (topic 0-3) during january and february, since information about the u.s. covid-19 outbreak was not publicly available in the u.s. during that time. most counties that have a low phq level contain general covid-19 related topics that are tied to the cases in asia and general symptoms of covid-19 (e.g., "wenliang li" (a chinese doctor) [60] , "south korea confirms", "coughing", "sneezing"). from the end of january, a few hotspots appear in some major u.s. cities such as san francisco, denver, los angeles, and seattle with topics related to "mistakenly released", "vaccine", "pandemic bus", and "china death" (see table 8 , figure 3b ,c). for instance, the keyword "mistakenly released" reflects news story in february about the first u.s. evacuee from china known to be infected with the coronavirus being mistakenly released from a san diego hospital and returned to quarantine [61] . people who living in california reacted strongly to this news (figure 3d) . later, on 8 march (figure 3c,d) , the phq level started to increase rapidly due to the covid-19 test stations available, increased number of covid-19 death cases, and a shelter-in-place order in many states (see table 8 , march). an interesting pattern was found that the number of counties with a high phq value kept growing until 5 april and started to decrease after the second week of april [62] . figure 4 illustrates the number of increased cases in the u.s. from january to may 2020. results show that the phq stress level in our results matches well with the number of increased cases illustrated in the johns hopkins coronavirus resource centers' statistical analysis results [61] . this means the number of new cases reduced due to the social distancing practice, and at the same time, the level of people's major concerns in many geographic regions reduced as well. the results also present the spatiotemporal patterns from january to april (shown in figure 3a g. table 8 shows the detected stress symptoms and topics generated from corexq9. each map represents the spatial distribution of stress symptoms over a biweekly period. it indicates that most of the regions have low to medium phq values (topic 0-3) during january and february, since information about the u.s. covid-19 outbreak was not publicly available in the u.s. during that time. most counties that have a low phq level contain general covid-19 related topics that are tied to the cases in asia and general symptoms of covid-19 (e.g., "wenliang li" (a chinese doctor) [60] , "south korea confirms", "coughing", "sneezing"). from the end of january, a few hotspots appear in some major u.s. cities such as san francisco, denver, los angeles, and seattle with topics related to "mistakenly released", "vaccine", "pandemic bus", and "china death" (see table 8 , figure 3b,c) . for instance, the keyword "mistakenly released" reflects news story in february about the first u.s. evacuee from china known to be infected with the coronavirus being mistakenly released from a san diego hospital and returned to quarantine [61] . people who living in california reacted strongly to this news (figure 3d) . later, on 8 march (figure 3c,d) , the phq level started to increase rapidly due to the covid-19 test stations available, increased number of covid-19 death cases, and a shelter-in-place order in many states (see table 8 , march). an interesting pattern was found that the number of counties with a high phq value kept growing until 5 april and started to decrease after the second week of april [62] . figure 4 illustrates the number of increased cases in the u.s. from january to may 2020. results show that the phq stress level in our results matches well with the number of increased cases illustrated in the johns hopkins coronavirus resource centers' statistical analysis results [61] . this means the number of new cases reduced due to the social distancing practice, and at the same time, the level of people's major concerns in many geographic regions reduced as well. our results also show a meaningful explanation of the spatial pattern caused by people's risk perception to various media messages and news during the pandemic. in march 2020, people in the united states had mild concerns about the uk prime minister boris johnson's talk of "herd immunity" [65] and social distancing (see table 8 , phq0, march). on the other hand, the major stress came from topics such as cases of deaths (e.g., in washington state), lack of food and covid-19 protection equipment (e.g., panic buy), and the increasing number of confirmed and death cases in our results also show a meaningful explanation of the spatial pattern caused by people's risk perception to various media messages and news during the pandemic. in march 2020, people in the united states had mild concerns about the uk prime minister boris johnson's talk of "herd immunity" [65] and social distancing (see table 8 , phq0, march). on the other hand, the major stress came from topics such as cases of deaths (e.g., in washington state), lack of food and covid-19 protection equipment (e.g., panic buy), and the increasing number of confirmed and death cases in the united states. figure 3d ,e shows that most of the hotspots were located in washington, california, and new york, and florida matched with to the march covid-19 increased cases map (see [61] . in april, keywords such as "death camps", "living expenses", "white house", and "economy shrinks" (see table 8 ) appeared most often in the high phq value categories, which indicated that people's major concerns shifted to financial worries due to businesses shutting down and the economic depression [66] . our study was conducted to perform a spatiotemporal stress analysis of twitter users during covid-19 pandemic by the corexq9 algorithm. according to the model evaluation results, the proposed corexq9 had the best baseline performance among other similar algorithms such as lda, nmf-lk, and nmf-f models. in addition to the corexq9 algorithm, we applied a fuzzy accuracy assessment method to the corexq9 analysis results to visualize the spatial uncertainty of the analysis results. this enables expert knowledge (e.g., phq rating of tweets) to be integrated in the social media data mining process. the result of our observed pattern reasonably matched the relevant events and epidemic trends. ideally, the analytic result of our collected twitter dataset is expected to support the research of mental health for the entire u.s. population as a sample case. in our cleaned twitter dataset, those tweets were posted by 1,410,651 users, which represent over 0.4% of the u.s. population. however, a previous investigation found that the 22% of american adults who use twitter are not uniformly distributed across age [66, 67] . another study found that twitter users are getting younger [68] , but the actual age, gender, and race of twitter users from those investigations have been controversial [55] . to generalize the psychology health analysis to the whole u.s. population, further work related to the user demographic is required to reduce the influence of the sample bias. the thematic maps we created for phq topics distribution were assessed based on fuzzy sets. the purpose of this commonly used method for categorical maps is to allow explicit accounts for the possible ambiguity regarding the appropriate map label [55, [69] [70] [71] [72] . a wide variety of statistical techniques have been proposed for the accuracy assessment of thematic maps [73] . in the future, we can use the standard deviation approach to estimate the quantity derived from the distribution of the tweets as a count on specific category if the assessment is focused on how the number of labeled phq tweets were distributed in each category. even though our datasets were preprocessed and selected with entities on covid-19 related topic, some of the tweets might be outside of the topic or are influenced by other objective factors. our future focus of uncertainty assessment of the thematic maps could be to extend to spatial uncertainty [74] , temporal uncertainty [75] semantic uncertainty [76] , etc. our assessment task can be considered a criterion referenced task that can focus on a selected phq level and can represent the majority level in any location. the fuzzy area estimation methods were extended based on previous research [72] . category assessment based on fuzzy sets can estimate the accuracy of classes as a function of levels of class membership [77] . here, we used biweekly data as a temporal scale for the analysis. our research group continues collecting twitter data for this project, so analysis could be applied to more fine-grained temporal scales in the future. since coivd-19 is a global pandemic, this project could be extended to a global scale to compare the results across different countries. in the future, the model could be applied to other cases to detect the related stress symptoms and provide real-time spatial decision support for addressing the problem. an end-to-end spatiotemporal analysis system could be built if all of the modules were integrated; this would increase the efficiency of determining the potential symptoms and causes of public mental health problems. in this article, we proposed the corexq9 algorithm to analyze the covid-19 related stress symptoms at a spatiotemporal scale. the corex algorithm combined with clinical stress measure index (phq-9) helped to minimize human interventions and human language ambiguity in social media data mining for stress detection and provided accurate stress symptom measures of twitter users related to the covid-19 pandemic. there was a strong correlation between stress symptoms and the number of increased new covid-19 cases for some major u.s. cities such as chicago, san francisco, seattle, new york, and miami. people's risk perceptions were sensitive to the release of covid-19 related public news and media messages. many frequently appearing keywords in the high phq value categories represent the popular media and news publications at that time. before march, most regions had mild stress symptoms due to the low number of reported cases caused by the unavailability of test stations, creating a false sense of security among the public in the united states. the number of cases increased suddenly in march due to governmental confirmation of the seriousness of the pandemic in the united states and shelter-in-place orders in many states. from january to march, a major concern for people was being infected by the disease and there was panic-buying behavior, but this shifted to financial distress later in april along coastal eastern and western united states. our main contributions are as follows: first, we introduced a specialized stress tweets classifier, which narrows down the theoretical algorithms to practical usage on the public health area and demonstrates more effectiveness than traditional sentiment index classifiers. second, we framed corexpq9 as a topic detection model in our research. we explored the latent connection between the social media activity and phq-9 depression symptoms and topics in united states. finally, as a supplement methodology for the existing questionnaire-driven mental health research, our integrated system was used to glean depression topics in an unobtrusive way. the proposed algorithm provides an innovative way to analyze social media data to measure stress symptoms under covid-19 pandemic at a spatiotemporal scale. by doing this, we were able to observe spatiotemporal patterns of stress symptoms and answer the questions of what the major concerns related to the pandemic in different geographic regions at different time scales were. in the future, this model could be applied to other cases to detect related stress symptoms and provide real-time spatial decision support for addressing arising issues. the authors declare no conflict of interest. table a1 . notation table. pattern pool a subset of the extraction patterns that tend to extract the seed words candidate word pool the candidate nouns extracted by pattern pool are placed in candidate word pool tc total correlation, also called multi-information, it quantifies the redundancy or dependency among a set of n random variables. kullback-leibler divergence, also called relative entropy, is a measure of how probability distribution is different from a second, reference probability distribution [50] . p(x g ) probability densities of x g i(x : y) the mutual information between two random variables p(y|x) y's dependence on x can be written in terms of a linear number of parameters which are just the estimate marginals δ the kronecker delta, a function of two variables. the function is 1 if the variables are equal, and 0 otherwise. a constant used to ensure the normalization of p(y|x) for each x. it can be calculated by summing |y| = k, 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area estimation this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-280605-2i4gk7et authors: bachmann, maría consuelo; bellalta, sofía; basoalto, roque; gómez-valenzuela, fernán; jalil, yorschua; lépez, macarena; matamoros, anibal; von bernhardi, rommy title: the challenge by multiple environmental and biological factors induce inflammation in aging: their role in the promotion of chronic disease date: 2020-10-14 journal: front immunol doi: 10.3389/fimmu.2020.570083 sha: doc_id: 280605 cord_uid: 2i4gk7et the aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. at any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to 5–8% of the population of industrialized countries. several environmental factors can play an important role for modifying the inflammatory state. genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in 50% to 75% of patients. several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. the interaction between inflammation and the environment offers important insights on aging and health. these conditions, often depending on the individual’s sex, appear to lead to decreased longevity and physical and cognitive decline. in addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. poor psychological environments and other sources of stress also result in increased inflammation. however, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging. the inflammatory response is different in men and women. adult females develop stronger innate and adaptive immune responses than males. these sex-related differences can determine the ability of immune cells to generate an effective inflammatory response, which translates into epidemiological differences on the prevalence of various pathologies, including allergies (22), asthma (23, 24), autoimmune diseases (25), anaphylaxis (26), neonatal sepsis (27), and cancer (28), among several pathologies. the immune response of women is polarized towards an increased production of th2 cells, t regulatory cells (treg), m2 macrophages, il4, il10, and gata-3 cytokines, and decreased th1, th17, tbet, and rorgt lymphocytes (29-31). on the contrary, men show an immune response that depends on th1 lymphocytes (32, 33), high il33 production (34) and low levels of reactive mast cells (35) . men have also an increased response of microglia in the central nervous system (cns) and an increased presence of tnfa and prostaglandins in response to inflammatory stimuli (36). differences in inflammatory response between men and women vary among specific tissues. in the cns inflammation, women show greater levels of b-cell (cd19+, cd5+, cd1d hi b10) migration from the spleen to the site of injury than men, followed by an increase of macrophages/microglia (cd11b+, cd206), which appears to generate a lower neuroinflammatory response in female compared with male mice (37). in addition, women develop an increased immunoreactivity due to high numbers of ifn-producing dendritic cells (38, 39). female mice tend to have m2 phenotype and activated eosinophils and mast cells show a higher reactivity than in male mice (35, 40) . however, in response to an acute inflammatory stimulus, males produce higher amounts of inflammatory cytokines, cd8a+ neutrophil and t cells infiltration of the injury site (41). conversely, the inflammatory microenvironment in female mice is characterized by an increased production of antibodies (42, 43) and a differential pattern migration of antibody-secreting cells (42). the immune system responds differently in men and women not only because of the influence of sex hormones, but also differences in the patterns of autosomal methylation and x chromosome methylation, which determine distinctive profiles of gene expression (43, 44). sex hormones exert antagonist effects on the immune system: both estradiol and testosterone have a suppressive effect on the immune response (45). estrogen is the sex hormone with the greatest impact on the immune response, being described as one of the non-modifiable regulators of the immune system, due to its immunoregulatory and protective effects in many inflammatory models (46). however, this is contradictory with the fact that women have a higher prevalence of autoimmune diseases than men, although estrogens should be a protective condition (47). the sex-dependent difference in the immune response is time-, and estrogen dose-dependent (29). variations on the estrogen concentration during the ovulatory cycle, puberty or menopause, can promote the development of immune-related diseases (48). mice exposed to chronic estrogen-treatment generate hormone resistance, decreasing the clonal expansion of treg lymphocytes in autoimmune diseases (49, 50) . estrogen regulates immune response primarily through aand b-estrogen receptors (era/b), mitogen-activated protein kinase (mapk) pathways, estrogen-dependent 3′-5′-cyclic adenosine monophosphate (camp) response element-binding (creb), and modifications in the production of camp in immune cells (51). in addition to estrogen receptors, the presence of il receptors influences the type of immune response; female macrophages express greater amounts of il4 receptors than males. il4 receptors favor the m2 phenotype when stimulated by estrogen. in agreement with that, estrogen induces an increased expression of il4 on naive cd4+ t cells (40, 52) . for a better general view of estrogen´s mechanisms and effect on the innate immune system cells we recommend reviews that have extensively covered those topics (53-56). sex regulates gene expression in multiple human tissues, in fact, one third of the autosomal genes that are expressed in a sexbiased manner exhibit androgen or estrogen hormonal response elements (57, 58). sex hormones play a strong role in sexually dimorphic gene networks (59), inducing aberrant expression in immune response genes via differential methylation ccl18 cxcl5 il5 (60) . there are changes in the methylation pattern of sex-dependent immune response genes during embryonic development, which are reinforced in puberty by the estrogenmediated induction of active forms of chromatins that are maintained during adulthood (61) . immune response-related genes located in chromosomes 3 and x are differently expressed in b lymphocytes depending on the sex of the individual (62). among the differentially expressed genes that are relevant for the immune/inflammatory response, can be mentioned the toll-like signaling, cytokine receptors, jak-stat pathway and genes related to the activation of t-cell receptors (63) . phenotypically, the different pattern of gene expression may explain the greater female t-cell expandable capacity when exposed to an antigen (64) . female t cells present higher activation and division capacities than their male counterparts. however, male t cells can develop greater infiltration potential and a lower self-reactive phenotype than female ones (65, 66) . these differences could be due to the high expression of peroxisome proliferator-activated receptors (ppars) (64) , prostaglandins, and cyclooxygenase-2 (cox-2) in males (67) . the influence of sex on the immune response is observed throughout life and is accentuated with aging. in the neonatal stage, women have a lower concentration of regulatory t lymphocytes than men (68) . during childhood, men develop a more intense immune response and are more likely to develop infections by various pathogens compared with women (69, 70) . with increasing age, the dynamics and proportion of lymphocytes and myeloid cells differ depending on the sex due to the differential expression of 144 genes of the immune response in men and women (71) . also, in aged individuals, epigenomic changes generate a more robust innate and pro-inflammatory response in men and an increased activity in the adaptive immune response in women (72, 73) . in recent times, during the covid-19 pandemic, it has been observed that the infection by sars-cov-2 in older adults shows conspicuous differences; men have elevated plasma levels of il8 and il18 and a high amount of monocytes whereas women develop a robust activation of t lymphocytes (74) . this differences in the immune response could explain the higher mortality of covid19 in men than in women (75, 76) . to recapitulate, sex hormones and genetic expression patterns in men and women can generate distinct immune and inflammatory responses that determine singularities in the epidemiological distribution of immune diseases. research protocols in immune response and inflammation must be redefined to avoid results biased by sex. furthermore, research in women is urgently needed to define the efficacy for women of several therapies that were originally tested in men. the increase in noncommunicable diseases (ncds), such as obesity, hypertension and cancer as well as the low-grade chronic inflammation that characterizes most ncds (77) can be affected by environmental factors that change the immune response. lifestyle factors like nutrition can modulate the immune system. it has been reported in mice that western diet-induced systemic inflammation and reprogramming of myeloid cell precursors is mediated by the activation of the nlrp3 inflammasome, which is a key sensor of the innate immune system for metabolic danger signals, such as uric acid and cholesterol (78) . metabolic regulation appears to be very robust and long lasting, being reported that proper nutrition during pregnancy can reduce the risk for ncds in the offspring even at adult age (79, 80) . the impact of the diet on the immune response and inflammation some diet types can result in metabolic and epigenetic changes that affect immune function (81) , as reported in populations that consume a high-fat and low-fiber western diet, who show a prevalence of ncds higher than populations that consume a mediterranean diet or a diet based on bioactive compounds, like the hydroxytyrosol in olive oil (82) (83) (84) . there is evidence supporting the anti-inflammatory activity of phenolic extracts from olive oil, such as their ability to reduce lipopolysaccharide (lps)-stimulated nitric oxide (no) production by the raw-264.7 macrophage cell line. the hydroxytyrosol stearate and the hydroxytyrosol oleate decrease no production in a concentration-dependent manner (85) . in addition, olive oil extracts increase total plasma glutathione concentration (86) , increasing the antioxidative response of the individual. nordic diet has many similarities with the mediterranean diet, but its effects on low-grade chronic inflammation are less known. both diets include abundant fruits, vegetables, whole grain products, fish and vegetable oil, but restrict saturated fat and red and processed meats (87, 88) . observational (89, 90) and interventional (91, 92) studies report an inverse association between the adherence to nordic diet and the concentration of high sensitivity c-reactive protein (hscrp). single intervention studies reported beneficial effects, reducing il1 receptor a (il1ra) (87) and cathepsin s (93) , and downregulation of inflammatory mediators in the adipose tissue (94) and peripheral blood mononuclear cells (pbmcs) (95) . a key nutrient in fish are the n3 polyunsaturated fatty acids (pufas) (88) . the greenland inuit population, which has a high dietary intake of n3-pufas, have a lower incidence of myocardial infarction than the danish population (96) . numerous studies associate the cardioprotective effects of n-3 pufas to their effect on immunomodulation (97) (98) (99) , and control of inflammation, including neuroinflammation during aging (100) . the mechanism of the anti-inflammatory effects of n3-pufas n3-pufas can regulate the transcription and expression of inflammatory mediators such as cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, and monocyte-macrophages (101) (102) (103) (104) . anti-inflammatory effect of eicosapentaenoic acid (epa), docosahexaenoic acid (dha) and their biologically active metabolites (d and e resolvinsmediators derived from omega-3 fatty acids, primarily epa and dha that block the production of proinflammatory mediators and regulate leukocyte trafficking to inflammatory sites) can be mediated through one of the mechanisms capable of reducing inflammation of raw-264.7 cells and of primary intraperitoneal macrophages (105) . one of the mechanisms is the activation of g-protein coupled receptors (gpr), ea. gpr120 inhibition of toll-like receptor 4 (tlr4)-mediated inflammatory response, which blocks nfkb activation. the other is mediated by nuclear receptors, particularly ppars-a/g. dha binds to ppars with high affinity resulting in the activation of anti-inflammatory cascades (106) , which appears to be responsible for the beneficial health effects (97) . the inhibition of nfkb-mediated pro-inflammatory activity (107) is the common mechanism of immunomodulation by n3-pufas, being dha more effective than epa in reducing lps-n3-pufas induced inflammatory cytokine production by macrophages (108) . n3-pufas are incorporated into phospholipid bilayers and in human atherosclerotic plaques. their incorporation is associated with a reduction in the number of foam-and t cells, and a decrease in inflammation (109) . the increased incorporation of n3-pufas in membranes affects both the innate and adaptive immune responses, impairing the maturation of dendritic cells and the function of macrophages, as well as the polarization and activation of t and b cells (110) (111) (112) . it is well known that n3pufas compete with n6-pufas for being incorporated into cell membranes and for the active sites of cox-2 and lipoxygenase, resulting in the production of less potent pro-inflammatory or even anti-inflammatory mediators, such as the 3-series of prostaglandin and thromboxane (113) . resolvins reduce also neutrophil-derived ros production, favoring neutrophil apoptosis and clearance by macrophages, and inhibit chemokine signaling (114) . the partial agonist/antagonist activity of resolvin e1 (rve1) on the leukotriene b4 receptor on polymorphonuclear cells (pmns), inhibits nfkb activation, reduces release of pro-inflammatory cytokines and reduces infiltration by pmn (115) . moreover, rve1 reduces tnfa and ifng presence in the aortic wall, decreases the levels of the inflammatory marker crp and reduces macrophage infiltration of the intima. thus, rve1 attenuates atherosclerosis and atherosclerotic plaque formation (116) . aging is associated with the activation of inflammatory signaling pathways (117, 118) , which can be targeted by specific nutrients with anti-inflammatory effects, such as n3-pufas (119, 120) . in the brain, the main n3-pufa is dha, representing 12-14% of total fatty acids (121) . aging and neurological disorders are associated with decreased levels and turn-over rate of brain n3-pufas (122) (123) (124) (125) . in aged mice, n3-pufa supplementation and diets enriched in dha have been reported to revert age-induced spatial memory deficits and impairment on learning and memory (126) (127) (128) . in older adults, a low consumption of n3-pufas and decreased erythrocyte dha levels are associated with cognitive impairment (129, 130) . dietary supplementation with dha is positively correlated with an improvement in declarative memory test performance, improved cognitive function (131, 132) and a lower risk of developing neurological disorders (133) . the probable mechanisms by which n3-pufas mediate their effects in the resolution of age-related neuroinflammation are the increased synthesis of n3-pufa-derived rvd1 and decreased n6-pufaderived oxylipins, displaying an anti-inflammatory profile (134, 135) . to recapitulate, the evidence indicates that n3-pufas and their bioactive metabolites have immunomodulatory and antiinflammatory properties. potential cardioprotective lipid mediators, through multiple mechanisms, including changes in cell membranes composition, and modification of both cell signaling and gene expression, shift the pattern of lipid metabolites toward a more anti-inflammatory metabolite profile. dietary habits may be essential regulators of the inflammatory profile and promote healthy aging, reinforcing the recommendation of a n3-pufa rich diet. the long term chronic psychological stress is increasing among the world's population (136) . its circuit arises at high cortical centers through the limbic system to the hypothalamus, where corticotropin-releasing factor (crf) is produced, which is responsible for inducing the pituitary gland to liberate adrenocorticotropic hormone (acth) that signals the adrenal cortex to synthesize and secrete glucocorticoids (gcs) (137) . stress also activates the sympathetic nervous system (sns), particularly the adrenal medulla, activating chromaffin cells to produce epinephrine (epi), a main stress hormone along with gcs. the latter plays a key regulation feature inhibiting the hypothalamic-pituitary-adrenal (hpa) axis through negative feedback at the pituitary gland, hypothalamus, and medial prefrontal cortex, reducing crf secretion [rewieved in (138) ]. the interplay of social and environmental stressors induces inflammation through multiple biological mechanisms, including epigenetic factors (139) . studies in rats show that the methylation patterns of genes involved in the stress response, such as the glucocorticoid receptor (nr3c1) and crf, can be modified by psychosocial factors from early childhood (140) . similarly, early life adversity induces acute and long-lasting epigenetic modifications in nr3c1 genes, regulating hpa axis and cytokine production, reinforcing the importance of the activation inputs during critical periods of development (137, 141) . acute short-term emotional stress, such as speaking in public, leads to a transient increase in circulating inflammatory biomarkers and natural killer (nk) cells by the sns catecholaminergic activity (142) . on the contrary, chronic stress results in a reduction of cytotoxic nk activity, determining a poorer response to cytokines (143) . therefore, stress appears to have short term beneficial immune effects, whereas chronic stress in the absence of immune challenge has the opposite effect (138, 144) , activating constantly the hpa axis with the consequent persistent elevation of systemic gcs and reduction of nk cell responsiveness to cytokines (143) , affecting the balance of the t helper cell type 1/type 2 (th1/th2) cytokine networks, predisposing to a wide range of diseases (145) . the stress magnitude has been associated with il1b mrna overexpression in peripheral pbmcs, providing a molecular mechanism by which psychological stress is translated into an immune system response (146, 147) . when stress becomes chronic, such as in depression, there is a maintained overproduction of inflammatory cytokines, which have been associated with gcs resistance. immune cells become less sensitive to their anti-inflammatory effects because of their persistent secretion, leading to chronic low-grade inflammation (147, 148) . activation of the innate and adaptive immune system by chronic mild stressors increases inflammatory cytokines gene expression, maturation and trafficking of dendritic cells (dc), increased macrophage number and t cells recruitment and activation. social stressors can induce an increase in inflammatory responses and a state of gcs resistance at different levels (144, 149) . the acute repeated social defeat stress (rsds) and chronic restraint stress (crs) models induce an inflammatory response that results in neuroinflammation and depressive behavior (150) . stress activates the hpa axis and the sympatho-adrenomedullar (sam) axis causing neuroinflammation by circulating cytokines that crossed the blood-brain barrier (bbb) at the circumventricular organs and by cytokine bbb transporters. an inflammatory response that promotes bbb permeability, allowing more inflammatory factors entering the brain, including crf, metalloproteinase-9, il6, and tnfa (150) . additionally, microglia produce chemokines that attract monocytes into the brain (150) . activation of sns and hpa axis through continuous psychological stress dysregulate cytokine production, and together with the stress hormones corticosteroids and catecholamines, can affect endothelial adhesion molecules, causing endothelial damage (138) . corticosteroids could facilitate the infiltration of monocytes by increasing the expression of il1 and il6 receptors on endothelial cells. these monocytes and lymphocytes, after attaching to such sites, would commence the process of infiltration into the wall vessels, leading to foam cell formation and thrombotic events (138, 151) . chronic unpredictable mild stress (cums) decreases body mass and impairs the metabolism of carbohydrates and lipids. a model for cums showed an increased liver and pancreas protein-lipid peroxidation and protein oxidation (152) . high ros production in both organs could be a result of a response mechanism to stress at the cellular level. in the liver, protein oxidation can be due to the regulation of metabolic impairments by gcs and epi (152). the antioxidant system of the liver is in general more efficient than the pancreas. however, it is insufficient to clear the reactive species increased as consequence of chronic stress, which could be due to alterations in the antioxidant enzymatic activity (138) . altogether, stress appears to have short term beneficial effects on the immune function, whereas chronic stress (138, 144) activates persistently the hpa, elevating systemic gcs, and impairing the cytokine balance. the overproduction of inflammatory cytokines lead to gcs resistance driven by immune cells that lose their sensitivity to gcs, leading to a state of chronic low-grade inflammation (138, 145) . this gcs imbalance, shares common features with aging, mediating an enhanced neuroinflammatory priming (153) . the presence of psychological stress potentiates the defective immune response observed in aging, which at the same time conditionate an exaggerated sickness response to immune challenges (such as chronic stress). thus, chronic stress contributes to the phenomenon of inflammaging, which promotes the development of several age-related pathologies, including atherosclerosis and diabetes among others [reviewed in (154) ]. additionally, there is an impairment of the antioxidant defense system to manage ros production after chronic stress, resulting in the damage of various tissues (138) . in addition, people exposed to chronic stress age rapidly, showing a faster telomere shortening in their cells (155) (156) (157) . on the other hand, epigenetic changes acquired during critical developmental stages could shape chronic stress-response along the lifespan, either promoting or reducing pathological aging (139, 140) . substance abuse, such as alcohol and drugs, are important triggers of chronic inflammatory processes (158, 159) . the effects of alcohol on human health are complex and depend on multiple factors. however, many of those factors are associated with the generation of immunosuppression and increased morbimortality in heavy users. those effects, which have been previously reviewed by goral et al. (160) will not be discussed in this review. here, we will describe the effect of cocaine and methamphetamine abuse. both drugs are potent psychostimulants that, when repeatedly consumed, significantly disrupt the functioning of the cns, and modify the regulation of the immune response, leading to a chronic neuroinflammatory state (161) . in general, it is known that drug abuse, among other factors, increases nfkb transcription of multiple proinflammatory genes that spread across brain cell types further amplifying of nfkb transcription, as has been reviewed by crews et al. (162) . cocaine (benzoylmethylecgonine according to the international common denomination) is a strong stimulant tropane alkaloid that acts by modulating the catecholaminergic neurotransmitter dopamine. studies of the striatum of mice after the administration of various drugs showed that 1 h after administration of 25 mg/kg cocaine, there is a significant increase in gene arrays for hypoxia-inducible factor 1 (hif-1), transcription factors, and cytokine receptors (il6r, tnfa). two hours after cocaine administration, there is an increased gene expression for various tnf receptors, inducible no synthase (inos) and adhesion molecules (163) . in the nucleus accumbens of mice stimulated with cocaine, there is a significant increase in matrix metalloproteinase 28 (mmp28), macrophage colony stimulating factor (mcsf) and major histocompatibility complex ii (mhc-ii) (164) . the brain of human subjects consuming cocaine shows an increased density of macrophages and activated microglia (165) . cocaine induces the activation of microglia through the endoplasmic reticulum stress and autophagy pathways (166) . studies of human and rodent immune cell populations after cocaine administration show decreased numbers of t lymphocytes, modulation of nk activity and cytokine production (167) . among brain glial cells, astrocytes are the most abundant, and perform critical functions, being involved in neurogenesis, promotion of neuronal survival, elimination of free radicals, and the production of no to maintain neuronal homeostasis (166) . nevertheless, astrocytes can also be activated by toxic stimuli, leading to a new phenotype called "reactive astrocytes", similar with the changes observed after inflammatory activation. this phenomenon has been described in various neuropsychiatric disorders, such as alzheimer's and parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis (166) . the reactivity of astrocytes to toxic stimuli, such as cocaine, infection or disease, potentiates the neuroinflammatory process (168) . methamphetamine (desoxyephedrine; meth) is a synthetic adrenergic agonist with psychostimulatory effects, structurally related to the ephedrine alkaloid and adrenaline. studies on the effect of meth are limited. however, it has been determined that its abuse affects the immune response. animals exposed to both acute and chronic meth use show alkalization of normally acidic organelles in immune cells, inhibition of antigen presentation, and impairment of phagocytosis (169) . meth also generates mitochondrial oxidative damage, dysfunction of t lymphocytes and decreased production of antibodies and cytokines (159) . meth has effects in various tissues (170) . in the lungs, the number of t lymphocytes decreases compared with that of untreated animals, indicating a reduction in circulating cd3+ cells, and levels of il6 and il10 increases. in the spleen, recruitment of pmn and the number of ly-6g+ and f4/80+ are increased, whereas cd3+ cells are significantly reduced. in addition, levels of tnfa, ifng, il6, and il12 are higher than those of control mice. in the liver, there is an increase of t lymphocytes and macrophages, hepatocellular atrophy, and increased levels of ifng, tnfa, il1b, -4, -6, -10, and -12 in the group exposed to meth compared with control animals (170) . in the cns, meth can induce the activation of calpains and caspases; the production of ros with the subsequent induction of oxidative stress, and the release of high amounts of glutamate, causing excitotoxicity (171) . recently, raineri et al. reported that meth induces activation of astrocytes and microglia, increasing the levels of il6 and tnfa mrna and its receptor (tnfr1) in the mouse striatum and hippocampus (172, 173) . medical advances have resulted in the increment of the average life expectancy in developed countries. the aging of the population is associated with an increase in the number of older people using drugs of abuse. from 2000 to 2012, the number of cocaine users aged 55 or older that required treatment for drug addiction in the us increased by 63% (174, 175) . aging is associated with low-grade basal inflammation that can be compounded by substance use. as cocaine exposure is associated with elevated inflammation and altered immune functioning, the presence of cocaine use disorder might exacerbate inflammatory processes in aging adults (176) . a recent report by soder et al, compared the levels of inflammation (through the neutrophil to lymphocyte ratio) in older adults with cocaine use disorder (cud) and in healthy older adults, finding that the group with cud had a significantly higher baseline level of inflammation (176) . the use of illegal drugs such as cocaine or methamphetamine has not been shown to affect cognitive function in older adults at the clinical level. however, the evaluation of the cognitive function of young drug users reveals a decreased performance compared with healthy young people. in fact, the cognitive function of young drug users is similar to that of adults older than 60 years of age (174, 177, 178) . in summary, both cocaine and meth can directly impair the immune response, induce the activation of glial cells and stimulate the release of pro-inflammatory mediators in the cns. all those effects cause relevant changes in glial cell regulation and inflammatory activation, triggering chronic neuroinflammation and potentiating pathological aging. air pollution has become an important threat to public health. air pollutants consider a mixture of gases such as nitrogen oxides (nox), sulphur oxides (sox), tropospheric ozone (o3), volatile organic compounds (vocs), and particulate matter (pm) (179) . pm can enter the respiratory tract leading to severe in situ damage as well as inducing additional systemic effects (180) . the world health organization (who) suggests a maximum annual exposure of 10 μg/m³ of pm2.5, however, the exposure of 90% of the world's population exceeds the proposed limit (181) . exposure to air pollutants is associated with increased morbimortality associated with respiratory, cardiovascular, metabolic, neurological, carcinogenic and autoimmune diseases (17, (182) (183) (184) . inflammation is the main pathophysiological mechanism induced by air pollutants. in terms of the molecular and cellular mechanism induced by pollutants, pm and sox can generate ros, inducing oxidative stress, together with mitochondrial dysfunction and the consequent energy deprivation (185) (186) (187) . as a direct consequence, nfkb and mapk inflammatory pathways are activated, triggering an innate immune activation (188, 189) . despite the attempts to resolve the inflammatory event, the outcome appears to be an imbalance in lymphocyte homeostasis and immune system dysregulation, with inhibition of th1 and treg lymphocytes (190) . there is also an increase of th2 lymphocytes and recruitment of eosinophils, resulting in respiratory disorders such as asthma (186, 191, 192) . in parallel, pm deactivates the nuclear factor erythroid 2 pathway (nrf2), involved in antioxidant regulation and prevention of oxidative stress, a necessary process for the resolution of inflammation. therefore, to maintain oxidation-reduction reactions becomes impossible, becoming a breaking point towards increased ros production and the non-resolution of the inflammatory event (193) . another mechanism of action of pollutants is the activation of the aryl hydrocarbon receptor (ahr) by toxic agents. the binding of pm to ahr increases circulating th17 and decreases treg lymphocytes. increase in th17 associates to the release of il17, promoting an abrupt increase of th2 lymphocyte response. these changes promote the dysregulation of the immune response associated with the development of autoimmune processes (193) . aberrant increases in th17 may result in increased inflammation, with consequences such as asthma and acute respiratory failure syndrome (ards), due to neutrophil infiltration and tissue damage (194) . studies suggest the existence of a decline in treg levels and, therefore, an inability to suppress th1, th2 and phagocyte responses (195, 196) . in addition, exposure to pm has been associated with fibrotic events, where il17 increases synthesis and secretion of collagen in the lung parenchyma (197, 198) . in addition, it has been described that pm also induces the expression of tgfb, directly promoting fibroblast differentiation, which could also induce collagen deposition followed by a lower antifibrotic process in the liver (199) . pollutants may promote direct dna damage through oxidation of nitrogenous bases. hu and yu described in a 2019 paper different mechanisms and changes in mirna expression that comprise specific targets of dna methyltransferases, which can impair the methylation of tumor suppressor genes (200) . furthermore, urban populations show increased levels of mitochondrial methylation genes due to pm exposure (201) . there is evidence of the existence of methylation, acetylation and phosphorylation of histones h3 and h4, markers found in genes involved in the activation of immune cells and cardiovascular diseases (200, (202) (203) (204) (205) . altogether, air pollutants can generate dna adducts promoting carcinogenesis and deteriorate telomerase activity, as reviewed by martens and nawrot (2016) , and contributing to continuous dna damage and premature aging (206, 207) . in vivo studies suggest that the inflammatory activation is doseand time-dependent. mice exposed to pm show that both variables are determinant for the outcome. however, inflammatory effects and major genetic changes appear to be especially dependent on the exposure to high concentrations of pm. one possible explanation is that a prolonged exposure could induce an adaptive response of the inflammatory activation (208) , which may be mediated by the inactivation of the nrf2 pathway, generating a loss of antioxidant capacity and deregulation of the immune system (193) . the resolution appears to depend on the exposure context. acute exposure would result in high levels of ros and damage, whereas prolonged stimulation, even a low-grade one, generates a constant production of ros and chronic low-grade inflammation (187) , consequent with the potentiation of disease risk and an epigenetic age acceleration (206) , promoting pathological aging. direct causes of the deregulation of the inflammatory resolution process resulting from inhaled contaminants are still unknown, however, the burden of associated chronic diseases is expected to increase. it is mandatory to intensify environmental policies specifically in lower-middle-income countries in prevention of the development of inflammatory conditions and the subsequent chronic diseases. aging, characterized by a progressive loss of cellular functions, is an inevitable physiologic process inherent to all living beings (209) . the number of older adults is increasing. during the next 30 years, up to 22% of the world population will be older than 60 years (210) . this demographic change is accompanied by a higher incidence of ncds accumulated in the aging population (211) . therefore, various strategies have been proposed to improve the health and quality of life of older adults (212), along with recommendations for the development of public policies that support the fiscal expenditure resulting from ncds (213) . one of the most studied events of aging is the impairment of the immune system, characterized by an aberrant-increased activation of the innate immunity (214, 215) , and high levels of circulatory inflammatory mediators that establish an inflammatory environment, and a decrease of the adaptive immune response (216, 217) and a decrease of the adaptive immune response (214) due to this low-grade chronic inflammation (214, 218) , which together would promote the inflammaging phenomenon (219) . interestingly, it is proposed that age would not be the cause per se of these diseases associated with aging (214) . thus, there is a deterioration of the immune system's response to external stimuli, which depends on the individual's history (218) . also, several epigenetic mechanisms can modulate the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events (220) . on the other hand, it is described that epigenetic clocks would be useful to analyze mechanisms associated with this environmental influence (221) . finally, they would be capable of modulating the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events (220). multiple age-dependent changes play important roles in the promotion of ncds, with increased oxidative stress standing out as one of the main mechanisms. over the last two decades, evidence has revealed that increased oxidative stress and inflammation are involved in various ncds such as alzheimer's disease (219) , rheumatoid arthritis (222) , cardiovascular diseases (223, 224) , and cancer (225), among others. also, recent studies propose that the activation of nfkb signaling pathways could be the main driver of these associations (226) (227) (228) (229) . interestingly, de almeida et al. showed different sources of low-grade chronic inflammation that promote cardiovascular disease (226) . in the cns, high levels of ros lead to the activation of astrocytes and microglia, further increasing the overproduction of ros and proinflammatory cytokines that promote the development of neurodegenerative changes (217, 230, 231) . in fact, several systemic biomarkers appear to be associated with neuroinflammation and the development of cns diseases associated with aging (230) . these modifications trigger the phenotype of senescent or aged cells characterized as sasp (216, 232) extensively studied in the context of the deleterious effects of aging. however, sasp is also essential for remodeling and promoting wound healing, which requires a strict control of the inflammatory response, thus avoiding the induction of cell aging phenotypes that contribute to the development of chronic inflammatory diseases (233) . the immune imbalance in aging occurs due to various alterations in cellular behavior and phenotype, which cause functional deficiencies in immune cells (3) . for example, this context induces polarization of macrophages towards an inflammatory phenotype characterized by strong activation of the inflammasome (234) . thus, these events could induce il1b and tnfa release, changes in the chemoattraction of neutrophils mediated by the reduction of the intercellular adhesion molecule 1 (icam-1) expression, and the aberrant activation of the phosphoinositide lipid kinase-3 (pi3k) (235) . also, there is a decrease in the expression of pattern recognition receptors (prr), which leads to the activation of proinflammatory signaling promoting tissue damage (215, 216) . finally, the reduced level of certain hormones due to the impaired hypothalamic function causes the loss of muscle mass and an increase in adipose tissue, further contributing to the release of inflammatory cytokines and changes in metabolism (236) . despite the remarkable effort being made to understand the basis of the processes underlying the inflammatory imbalance during aging, it is not fully understood. in aging, there are cumulative epigenetic changes that promote low-grade inflammation (220, 237) , including a decrease in the global genome methylation, with increased methylation in specific regions, as those with repressive histone marks of cd8+ and cd4+ t cells (238) and bivalent chromatin domains (239) and histone acetylation and methylation. however, the influence of genomic methylation during aging remains undetermined (237) . several studies correlate the methylation of multiple sites on cpg islands with the increase of the low-grade inflammation marker, crp (220, 232, 240) . nonetheless, stevenson et al. propose that the dna methylation could be better associated with the low-grade chronic inflammation than crp (237) . in addition, the age-related mitochondrial dysfunction, with the resulting oxidative stress and decreased atp production (241) , affect the expression and activity of dna methyltransferases, which are responsible for maintaining the methylation pattern of dna (242) . the reduced methylation results in the demethylation of the tnfa promoter in leukocytes and macrophages (243) and the adhesion of immune cells to the endothelium (244) . also, many epigenetic events contribute to the differentiation of proinflammatory t cells, th17 (220) , which can compromise immunocompetence, associated with repression of differentiation of immune cells, loss of treg function (240) and the alteration of the hematopoietic stem cells differentiation (245) . thus, epigenetic mechanisms appear to have a major role in the inflammatory imbalance, which are associated with the accumulation of damage in time that ultimately leads to the perpetuation of a constant inflammatory response. according to the who, 60% of the world population is sedentary, lacking the benefits of physical exercise (246) . conditions such as sedentarism, unhealthy diet, overweight, obesity and aging induce chronic low-grade inflammation. physical exercise increases the anti-inflammatory potential and reduces the pro-inflammatory effect (247) . this equilibrium is partly modulated through tlrs (248) , which are fundamental for the recognition of prrs, including the damage-associated molecular patterns (damps) and the induction of an inflammatory response in the absence of pathogens. there is evidence that in young people, physical exercise decreases tlrs expression, co-stimulatory molecules cd80/ cd86, and mhcii (248, 249) in cd14+ monocytes. physical exercise also affects the adipose tissue. exercising reduces tlr4 mrna expression and tnfa production in adipocytes (250, 251) in obese mice. chronic physical exercise decreases tnfa and tlr4 gene expression in the skeletal muscle (252) . the evidence suggests that obesity-or cerebral ischemiainduced neuroinflammation, which are associated with the overexpression of tlr2 and tlr4, may be reduced by physical exercise through the reduction of tlrs expression as well as their downstream signaling molecules (tnfa, il1b, myd88, traf6, 552 tak1, and nfkb), together with the reduced microglial activation (253, 254) . there is evidence that cigarette smoking induces inflammatory status [reviewed in (255) ]. however, exercise training reduces smoke-induced inflammation. in that sense, training for 30 min with endurance exercise for 5 days in smoke-exposed mice demonstrated that therapeutic exercise training significantly reduces the expression of il1b and tnfa mrna in rectus femoris (256) . physical exercise has been used as a therapeutic tool in chronic pathological conditions. in that sense, obese older adults (body mass index 38 ± 2 kg/m2; 69 ± 1 years) undergoing an exercise program consisting in physical therapy, endurance, and resistance for 90 min, 3 days per week, show a reduced expression of tlr4, il6, and tnfa mrna in skeletal muscle (257) . in older adults, 8-week physical exercise reduces the expression of tlr4 and tlr2, as well as tlrs downstream mediators, such as myd88, p65, pp38, trif, ikki/ikkϵ, irf3, and pirf7 in pbmcs (258) . similarly, dendritic cells from multiple sclerosis patients undergoing an exercise (endurance and resistance) program for 12 weeks reduce tnfa and mmp9 secretion when stimulated with a tlr4 ligand (lps in combination with ifng, or a tlr7 ligand) (259) , suggesting that long-term physical exercise decrease tlr responsiveness. on the other hand, high-intensity physical exercise in untrained individuals induces inflammation, resulting in the increased expression of tlr4, ap1, nfkb, and p65 in mice myocardium and in adipose tissue (260) (261) (262) . physical exercise associated with eccentric contractions causes expression of tlr and nfkb in skeletal muscle and liver in rats (263, 264) . furthermore, this phenomenon induces muscle damage, which can increase chemotaxis, attracting nk, cd8+ 559 t cells, macrophages and neutrophils to the site of injury, promoting the production of cox560 2, inos, monocyte chemotactic protein-1 (mcp-1), tnfa, il6, and il1b, in addition to the production of ros and the activation of nfkb (265, 266) . in healthy young males, one session of intense endurance exercise (1 h intense cycling immediately followed by 1 h intense running), increases plasmatic concentrations of il6 and il10, in addition to increased gene expression of proinflammatory il1 receptor (il1r) and tlr signaling pathways. moreover, plasma myoglobin changes in correlation with neutrophil tlr4 gene expression (r= 0.74), suggesting that their transcriptional activity was particularly induced by damps (267) . therefore, inflammation and muscle damage are mainly associated with the type and intensity of the exercise, with loads that exceed individual physical abilities. chronic physical exercise generates epigenetic modifications. the physical exercise associated with an energy expenditure >500 kilocalories per week, results in hypomethylation of the il10 gene and hypermethylation of the tnfa gene (268) , with an inverse correlation between tnfa methylation and tnfa mrna expression (269) . the methylation of the caspase recruitment domain (asc) of the apoptosis-associated specklike protein gene, the main regulator of inflammasome and promoter of the activation of il1b and il18, decreases with aging. however, older adults who maintain physical exercises regularly express higher levels of asc methylation than subjects not exercising, which would imply a decreased release of inflammatory cytokines (270) (271) (272) (273) . similarly, in review a 6-month walk training can induce hypermethylation of the nfkb-2 gene, suppressing inflammation through the inhibition of the nfkb pathway (274) . as life expectancy increases, age-related diseases thrive. aging is a complex multifactorial process of molecular and cellular decline that renders individuals susceptible to disease and death. maintenance of cell integrity, cell metabolism and host-defense mechanisms are tightly regulated by the surrounding microenvironment. a growing body of evidence in different biological models has contributed towards identifying biological mechanisms that ward off structural and functional deterioration. these data offer us insights into healthy aging. molecular integrity of the genome, telomere length, epigenetic stability, and protein homeostasis are all features linked to more youthful stages (regardless of the age), associated with mitochondrial fitness, metabolic regulation, efficient intercellular communication, stem cell renewal, and regenerative capacity in tissues. a good understanding of the environmental and endogenous mechanisms that underlie agerelated normal and deleterious changes, and how these pathways interconnect, remains a major challenge for slowing pathological aging while extending older adults' healthy lifespan. the study of the environmental influence on the development of complex-chronic diseases shows that in addition to genetic predisposition, the pathogenesis is promoted by changes in metabolism and behavior, cellular environment, and epigenetic regulation patterns. the type of nutrient, or environmental cytokine milieu dramatically affects not only the homoeostasis of tissues but also of complete organs and even of the whole individual. thus, tissue stress, malfunction, and damage may induce inflammation alarm responses, which result either in resolution of tissue damage, restoration of normal cell function or development of chronic disease ( figure 1 ). older adults often present inflammaging, characterized by increased levels of proinflammatory cytokines il1, il6, il8, tnfa/crp (275) . however, the cellular sources of these cytokines are partially unknown. the increased inflammatory cytokines have been proposed to be a driver of unsuccessful aging (increased morbidity, degenerative processes, or frailty) and shortened health-span. the inflammatory scenario is complex and occurs in response to various internal and environmental stimuli ( figure 1 ) mediated mainly by a high level of proinflammatory cytokines. indeed, in healthy aging, increased production of the anti-inflammatory cytokines tgfb and il10, can regulate the pro-inflammatory state (276, 277) . research into the impact of environmental factors on inflammaging is at an early stage and the involved mechanisms are not completely understood. several hypotheses have been developed to explain the chronic inflammation: aging-related increase of stress (278) and oxidative stress (279) , dna damage in senescent cells [reviewed in (280) ], and stem cell aging (281) . the proposed mechanisms are likely interdependent, resulting in the generation of ros causing oxidative damage and amplification of the cytokines secretion, thus perpetuating a vicious circle of systemic inflammation where tissue injury and healing mechanisms proceed in parallel while damage slowly accumulates over the lifespan of the individuals. endocrine and metabolic alterations are linked to the shift towards a pro-inflammatory profile, which could explain some age-related pathologies, such as alzheimer's and parkinson's disease, osteoporosis, diabetes, cancer, and frailty (282, 283) . regarding stress-induced immune modifications, new evidence suggests that cross talk signals between the cns, endocrine and immune system are required for optimal response to stress [discussed in (284) ]. various stressors can affect the activity and regulation of immune cells via direct regulation by the autonomic and peptidergic system or through the release of neuroendocrine mediators. moreover, neuronal catecholamines modulate immune cell functions. these interactions are bidirectional, cytokines produced by immune cells, such as il1, can modulate the production of corticotropin-releasing hormone (crh) by the hypothalamus. chronic diseases are favored by some modern living conditions, such as the intake of high-caloric foods and the low level of physical activity, or endogenous signals produced by the chronic stress of modern life. there are many challenges in conducting research on biosocial processes, which will define novel disease-trigger factors. tailor-made approaches will depend on genetics, epigenetics and a constellation of factors depending on the historical as well as the present exposure to the environment. although environmental factors also express themselves as epigenetic changes, the combinatorial effect of the multiple factors generates complex patterns of epigenetic regulation, and the concomitant exposure to environmental factors can further modify the individual response. all authors contributed equally on the conception of the work, the analysis of literature and preparing the content of the review. rbe drafted and organized the manuscript. all authors contributed to the article and approved the submitted version. endogenous and environmental factors can be mostly beneficial (in green) and deleterious (in red) or can have both beneficial and deleterious effects depending on the specific context. the interplay of lifespan endogenous and environmental factors regulates the aging phenotype depending on dna damage, epigenetic changes, and inflammation. these drivers can induce functional aging hallmarks: changes in endocrine and metabolic regulation, and defective immune regulation that will further determine the response of the individual. in yellow we show processes that can participate in both protection and damage. exposure to various alarm signals induce an acute inflammation that, when associated with deleterious environmental and biological factors, potentiates chronic inflammation, which can be further promoted by excess ros production and oxidative stress that results from mitochondrial dysfunction or nox2 activity, leading to inflammaging and eventually to age-related disease. on the contrary, in the presence of protective environmental and biological factors, the initial inflammatory activation will be resolved and lead to a healthy aging process. ros, reactive oxygen species. sarcopenic obesity and inflammation in the inchianti study physiological aging: links among adipose tissue dysfunction, diabetes, and frailty immunosenescence: the potential role of myeloid − derived suppressor cells ( mdsc ) in age − related immune deficiency the hallmarks of aging circulating c1q complement/tnf-related protein (ctrp) 1, ctrp9, ctrp12 and ctrp13 concentrations in type 2 diabetes mellitus: in vivo regulation by glucose aging, inflammation and the environment both genetic and dietary factors underlie individual differences in dna damage 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models and paradigms for stem cell ageing from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation aging and parkinson's disease: inflammaging, neuroinflammation and biological remodeling as key factors in pathogenesis impact of stress on aged immune system compartments: overview from fundamental to clinical data 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.copyright © 2020 bachmann, bellalta, basoalto, goḿez-valenzuela, jalil, lépez, matamoros and von bernhardi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-022506-fkddo12n authors: griffin, brenda title: population wellness: keeping cats physically and behaviorally healthy date: 2011-12-05 journal: the cat doi: 10.1016/b978-1-4377-0660-4.00046-6 sha: doc_id: 22506 cord_uid: fkddo12n nan o u t l i n e whereas feline practitioners are usually well versed in the creation of wellness programs tailored to individual cats, optimizing the health of a population of cats requires additional knowledge and poses unique challenges. these challenges will vary depending on many factors, including the nature and purpose of the population itself. indeed, veterinarians may be tasked with developing health care programs for cat populations in a wide spectrum of settings-from facilities housing laboratory animals, to animal shelters, home-based rescue and foster providers, care-for-life cat sanctuaries, breeding catteries, or large multicat households. regardless of the setting, a systematic approach to the health of the clowder is crucial for success. merriam-webster's dictionary defines wellness as "the quality or state of being in good health especially as an actively sought goal." 80 ensuring population health requires careful planning and active implementation of comprehensive wellness protocols that address both animal health and environmental conditions ( figure 46 -1). 41 addressing physical health alone is not sufficient to ensure wellness. for example, a cat may be in proper physical condition and free from infectious or other physical disease, yet suffering from severe stress and anxiety. in this case, the patient cannot be assessed as healthy, because its behavioral (emotional) state is compromising its health and well-being. thus physical health and behavioral health are both essential components of wellness, and preventive health care must actively address each of these. addressing the environment of the population is also critically important when considering wellness. even the best-designed facilities cannot favor good health in a multicat environment without thoughtful implementation of environmental wellness protocols. in small animal practice, environmental wellness is frequently not emphasized simply because many owners are accustomed to providing a reasonably healthy environment for their pets. in contrast, a structured program to address environmental wellness is essential in the more specific goals will vary depending upon the given population and its purpose. for example, in an animal shelter, specific goals of the wellness program might include decreasing the incidence and prevalence of infectious diseases in the shelter and following adoption, decreasing the incidence of problem behaviors in the shelter, decreasing the rate of return of cats to the shelter for problem behaviors, increasing the adoption rate, and so forth. in the context of a breeding colony, the goals might include increasing kitten birth weights, decreasing neonatal mortality, or improving socialization of kittens. by identifying and tracking measurable factors (often called performance targets in large animal medicine), it is possible to measure progress toward these goals. once baseline data (such as disease rates) are established, it is possible to measure the impact of protocol changes on population health by evaluating these performance targets. both medical records and a system for regular surveillance and reporting are required to accurately track and access trends in animal health. 41 early recognition is crucial for effective control of infectious disease and problem behavior in a group. therefore a regular system of health surveillance must be in place to monitor every individual. in a population setting, daily "walk-through rounds" represent the foundation of an effective animal health care program. rounds should be conducted at least once daily (preferably twice a day or more often, depending upon the needs of individual cats) for the purpose of monitoring and evaluating both physical and behavioral health. medically trained caregivers should visually observe every animal and its environment, taking note of food and water consumption, urination, defecation, attitude, behavior, ambulation, and signs of illness, pain or other problems. monitoring should take place before cleaning so that food intake and the condition of the enclosure, including the presence of feces, urine, or vomit can be noted. alternatively, observation logs can be completed by caregivers at the time of cleaning and reviewed during walk-though rounds. any cat that is observed to be experiencing a problem, whether it be signs of respiratory infection, diarrhea, anxiety, or obvious pain, suffering, or distress must be assessed and treated in a timely manner. regardless of length of stay, regular daily assessment is imperative to identify new problems (medical or behavioral) that may develop so that they can be identified and addressed in a timely fashion to ensure the welfare of the individual animal as well as that of the population. context of a population, regardless of the actual physical facility. proactive measures to maintain clean, sanitary environments that are not overcrowded-where cats are segregated by age and health status and provided with regular daily schedules of care by well-trained dedicated caregivers-are essential. 41 simply stated, the overarching goals of a population wellness program are to optimize both the physical and behavioral health of the cats as well as preventing transmission of zoonotic diseases. in other words, a population wellness program should be designed to keep animals "healthy and happy" while keeping human caregivers safe. 41 it is not difficult to identify a healthy population of cats: when wellness protocols are successful, cats "look healthy" and "act like normal cats." in other words, they appear in good physical condition and display a wide variety of normal feline behaviors, including eating, stretching, grooming, scratching, playing, rubbing, resting, and if allowed, courtship and breeding. just as changes in a cat's physical appearance should alert the clinician to potential problems, so should the absence of such normal feline activities and behaviors by members of the group. wellness goals must include maintaining the health of individual animals as well as that of the population as a whole. in the context of the population, the individuals that are physically or behaviorally ill serve as indicators or "barometers" of the health care and conditions of the population. when individuals are ill, their health and well-being is always a priority; however, it should also immediately trigger the clinician to ask, "why is this individual sick? what is the cause of its illness, and how can i prevent this from affecting others?" to optimize feline health, wellness programs must be carefully structured to address both the physical and behavioral health of the animals, which are intimately linked to their environment, making it crucial to systematically address environmental conditions as well. behavioral health 1. freedom from thirst, hunger, and malnutrition by providing ready access to fresh water and a diet that maintains full health and vigor 2. freedom from discomfort by providing a suitable environment, including shelter and a comfortable resting area 3. freedom from pain, injury, and disease by prevention or rapid diagnosis and treatment 4. freedom to express normal behavior by providing sufficient space, proper facilities, and company of the animals' own kind 5. freedom from fear and distress by ensuring conditions that avoid mental suffering in addition to early recognition of health problems, timely action is crucial to effectively limit their morbidity. ideally, all facilities that house multiple cats should have written policies and protocols in place that detail how medical and behavioral problems will be handled. 41, 51, 54 a committee or team of individuals composed of medical staff, managers, and caregivers can establish and oversee these policies and protocols. such protocols serve as guidelines for systematic triage and care of animals and help to prevent delays in care that may otherwise arise if such plans were not in place. policies and protocols should be based on medical facts, taking into account the entity's purpose or mission and the availability of resources for care. they should include a definition or description of the disease or condition in question, a description of the methods that will be used for diagnosis, and a general policy regarding the handling and disposition of affected cats. in addition, protocols should include details on notification, housing, decontamination, treatment, and documentation (box 46-1). just as quality-of-life assessment is the responsibility of every veterinarian as they guide the medical care of individual animals, quality-of-life assessment is also a critical part of population health care and monitoring. the factors that affect physical and mental well-being are broad, complex, and often vary substantially among individuals. 76 exacting criteria are lacking for the objective measurement of quality of life of cats. however, subjective assessments can and should be made by medical and behavioral personnel at regular intervals (weekly or even daily, as indicated) considering the most information possible. 76, 109 the "five freedoms," which were originally described by the farm animal welfare council in the 1970s, represent a benchmark for ensuring quality of life or animal welfare 35 (box 46-2). these principles provide a useful framework that is applicable across varying situations and species and have been widely accepted and endorsed by animal care experts. many agencies have used the five freedoms as the basis of recommendations for minimum standards of care for many species, including cats housed in catteries, shelters, and research facilities. 7, 13, 54, 87 the tenets of the five freedoms define essential outcomes and imply criteria for assessment but do not prescribe the methods by which to achieve those outcomes. regardless of the setting, population wellness programs should ensure the five freedoms for all cats. wellness always starts with prevention: it is far more time and cost efficient than treatment, and it is kinder to the animals and their caregivers. with this in mind, population wellness programs should provide broad-based, holistic approaches to preventive care, rather than being based on the control of a single disease or problem, regardless of the setting. maintenance of good health or wellness is especially challenging in populations with high turnover and interchange of cats of varying ages and susceptibilities, such as animal shelters. infectious diseases can become endemic in facilities where populations of animals are housed. even in closed populations, certain pathogens can be difficult to exclude or to eliminate once introduced. notably, upper respiratory viruses, dermatophytes, and coccidia are among the most difficult pathogenic agents to control because of their persistence in the environment through carrier states and/or resistance to environmental disinfection. in particular, upper respiratory disease is the most common endemic disease in cat populations and is impossible to completely prevent in an open population. feline herpes virus type 1 (fhv-1) and feline calicivirus (fcv) have been implicated as the causes of most infections: both viruses induce persistent carrier states and are widespread in the cat population. 36 cats that recover from fhv-1 remain latently infected and shed virus intermittently, especially following periods of stress. fcv carriers shed continuously for months to years following infection. a variety of other viral and bacterial pathogens may also contribute to feline upper respiratory disease, and bordetella, chlamydophila, and mycoplasma are problematic in some populations. feline infectious peritonitis (fip) is another disease that is nearly impossible to eradicate from a multicat environment, and sporadic cases can be expected to occur, especially in young cats. 93 fortunately, proper wellness programs can greatly limit the incidence and severity of diseases, even for pathogens that are difficult to control. the multicat environment also presents enormous opportunities for inducing stress. because of their unique biology, cats are particularly prone to experiencing acute stress and fear in novel environments. anything unfamiliar to a cat can trigger apprehension, activating the stress response. confinement in a novel environment can result in a wide variety of behavioral indicators of stress including hypervigilance, feigned sleep, constant hiding, activity depression, and loss of appetite, among others. in the long term, if cats are unable to acclimate or cope in their environments, chronic stress, fear, frustration, or learned helplessness may result. in group settings, signs of social stress may also manifest with medical decisions must be weighed in the context of the health of the population as well that of the individual, while considering animal welfare and the availability of resources for care. when large numbers of animals are involved, situations may arise in which animal health and welfare cannot be managed in the case of every individual animal. this may be due to physical or behavioral illness, or environmental conditions that negatively impact animal health, such as crowding. regardless of the cause, it may be necessary to euthanize affected individuals if no other remedies exist to relieve animal suffering or to protect population health. these decisions can be difficult and emotionally challenging, especially in instances where the individual could easily be treated or otherwise accommodated if adequate resources were available. however, such decisions may be crucial for disease control, animal welfare, and population health. that being said, euthanasia should never be used as a substitute for providing proper husbandry and care. indeed, a critical need for a comprehensive wellness program exists in every multicat setting. it is unacceptable to house animals under conditions likely to induce illness and poor welfare, and such conditions can be expected when wellness programs are not in place and carefully monitored. 41 when facilities elect to house cats with medical or behavioral problems, appropriate veterinary care must be provided. it is imperative that a humane plan for diagnosis, treatment/management, monitoring, and housing be implemented in a timely fashion. when determining if cats with special needs can be humanely cared for in a population setting, the following goals and considerations should be addressed: what measures must be implemented to prevent transmission of disease to other cats or people? can appropriate care realistically be delivered? will the care provided result in a cure or adequate management of the disease or problem behavior? can the facility afford the cost and time for care? how will it impact resources available for other cats? in the case of animal shelters, additional considerations should include will the cat be adoptable? what steps can be taken to minimize the holding time required for treatment? if the cat is not adopted, do humane long-term care options exist in the shelter? what welfare assessment will be used to measure quality of life in the shelter? 41 disease control efforts when disease is present. however, the best method of disease control is always prevention. when creating preventive medicine programs for a population, consideration must be given to all components of wellness: physical, behavioral, and environmental health. 41 with regard to promoting physical health, wellness programs should address the following essential elements: implementing population wellness protocols and ensuring quality and timely care require reliable systems for medical record keeping and animal identification. regardless of the system used, medical record keeping procedures should comply with state and local practice acts, guidelines provided by state and national veterinary medical associations, and, in the case of laboratory animals, regulations as prescribed by federal law, the increases in problem behaviors, including urine marking, spraying, or other inappropriate elimination; constant hiding; and/or aggression. 46 stress not only has the potential to negatively impact behavioral health but also physical health as well. the intimate link between stress and immunity has been well described. in fact, stress is a leading factor in the development of infectious disease and is particularly important in the pathogenesis of feline upper respiratory infections. 37, 47 wellness programs that reduce stress will also serve to minimize the morbidity of infectious disease. despite the fact that infectious agents can never be completely eliminated from the environment, it is still possible to maintain good health. this is because the development of disease is determined by a complex interaction of many factors surrounding the host, the infectious agent, and the environment. keeping these factors in mind provides a rational context for many of the recommendations in this chapter. some of the host factors that influence health and the development of disease include age, sex and reproductive status, immune status, body condition, stress, and genetics. 39 the amount and duration of exposure to an infectious agent (i.e., the "dose effect"), as well as its virulence and route of inoculation, also influence the likelihood and severity of disease. in addition, environmental conditions contribute to the development of infectious disease, including such factors as housing density, sanitation, and fluctuations in temperature or air quality. the fact that disease results from such a large combination of factors underscores the importance of a holistic and broad-based approach to population wellness. when infectious disease does occur in a population, general principles of infectious disease control should guide the response. these include . some facilities prefer to use safety collars that are designed to break away should the collar become caught on something. even for kittens, collars can be used and may be especially beneficial, because they will learn to wear them from an early age. microchips may also be used for identification and are safe and simple to implant (figure 46-4) . the procedure is well tolerated by the vast majority of cats without the need for sedation. unlike visual means of identification, a scanner is necessary for positive identification of a microchipped animal. for this reason, microchips are often used in conjunction with a visual means of identification and serve as important permanent means of backup identification. box 46-3 describes the proper technique for scanning for a microchip. during the last 2 decades, microchips of varying radiofrequencies (125, 128, and 134 khz) have been introduced in the united states. 4 the 125-khz chips have historically been the most common, whereas the accepted standard in the rest of the world is the 134-khz chip. because some scanners read only certain radiofrequencies, it is possible to miss detecting a microchip that is present, depending on the scanner being used. currently, there are efforts to standardize microchipping in the united states, including widespread distribution of universal (global) scanners to ensure that all implanted microchips can be reliably identified. once global scanners are widely available, the american veterinary medical association (avma) recommends adoption of the 134-khz (iso) microchip as the american standard, because this frequency is recognized as the international standard for microchips institute for laboratory animal research and institutional animal care and use committees. computerized records are preferred; however, written records may also be used. computerized records offer the advantage of mechanized reporting, which facilitates detection and monitoring of health trends in the population. a medical record should be prepared for each cat and should include the cat's entry date, identification (id) number, date of birth, gender, breed, and physical description, as well as historical and physical/behavioral examination findings. in addition, it should contain the dosages of all drugs administered and their routes of administration, including vaccines, parasite control products, other treatments, and anesthetic agents; the results of any diagnostic tests performed; any surgical procedure(s) performed; and other pertinent information regarding the animal's condition. standardized examination and operative reports may be used, but should allow for additions when necessary. identification of cats in the form of a neckband, collar and tag, tattoo, earband, and/or a microchip is also essential for preventive health care and ongoing surveillance of individuals. 45 whenever possible, some form of identification should be physically affixed to every individual cat. in addition, enclosures should be labeled with the cats' unique identification number and/or name. contrary to popular belief, most cats can reliably wear collars safely and comfortably. 65 many facilities use disposable collars, including commercially available plastic or paper neckbands made for animals or hospital-type wristbands made for human patients (figure 46 -2). commercially available cat collars with an id tag affixed in the rest of the world. efforts have also focused on improving, updating, and centralizing microchip registries. this is extremely important in the context of animal shelters. box 46-4 contains information on the use of collars and microchips as tools for improving cat-owner reunification. in laboratory settings, tattoos may be used as a means of permanent identification of cats ( figure 46 -5). 45 tattoos are most commonly applied to the inner pinna of the ear using a tattoo machine with multiple needles. care must be taken to properly disinfect the needles between patients. a significant disadvantage of tattooing is that tattoos can sometimes be difficult to read because of the presence of hair, fading, or distortion that may occur as the cat grows. in addition, their application requires anesthesia or heavy sedation. small stainless steel ear tags manufactured for wing banding of birds are especially useful for identifying newborn kittens in some settings and are highly economical (figure 46-6). 45 they can be placed without the need for anesthesia or sedation when kittens are less than 10 to 14 days old. placing earbands requires skill and experience. they must be positioned in such a way as to provide adequate space for growth of the ear, while seating them deeply enough in the ear margin to ensure a secure piercing far enough away from the edge. if placed too close to the ear margin, the ear flap may tear, resulting in loss of the band. other complications include local inflammation or infection at the site of the piercing. ear tags are a practical method for identifying individual kittens in institutional or commercial breeding colonies, because when applied skillfully, they are seldom lost and provide reliable, long-lasting visual identification. in contrast, private breeding catteries and animal shelters generally prefer to use methods that will not alter the cat's cosmetic appearance long term. colored ribbon, nail polish, or clipping of hair in various areas of the body can all be useful means of temporary kitten identification in the neonatal stage, especially when coat color or patterns do not easily allow individuals to be distinguished. every cat, including those surrendered by their owners, should be systematically scanned for the presence of a microchip at the time of intake, as well as prior to being made available for adoption or being euthanized. proper technique and scanning more than once are crucial to avoid missing microchips. 67, 68 a universal (global) scanner (e.g., one that will read all microchip frequencies that are currently in use) should be used to ensure that all microchip frequencies are detected. at this time, the only universal scanners available in the united states are the new home again global world scanner (schering plough, whitehouse station, ny) and the imax black label resq scanner (bayer animal health, shawnee mission, kans.). one of the most common causes of scanner failure is weak batteries; therefore it is imperative that batteries be checked and replaced regularly. to ensure a thorough scan and avoid missing chips, cats must be removed from carriers or cages prior to scanning. metal and fluorescent lighting may interfere with chip detection. metal exam surfaces should be covered with a towel or other material prior to scanning to minimize interference. the entire animal should be scanned using a consistent speed, scanner orientation, scanning pattern, and distance. â�¢ scanner orientation: the scanner should be held parallel to the animal. rocking the scanner slightly from side to side will maximize the potential for optimal chip orientation and successful detection. the button on the scanner should be depressed continuously during the entire scanning procedure. â�¢ scanning distance: the scanner should be held in contact with the animal during scanning such that it is lightly touching the hair coat. â�¢ scanner speed: the scanner should not be advanced any faster than 0.15 m/second (0.5 ft/second). scanning slowly is crucial, because universal scanners must cycle through various modes to read all possible chip frequencies. â�¢ areas of animal to scan: the standard implant site is midway between the shoulder blades, and scanning should begin over this area. if a microchip is not detected here, scanning should proceed systematically down the back, on the sides, neck, and shoulders-all the way to the elbows in the front and the hindquarters in the rear. â�¢ scanning pattern: the scanner should be moved over the scanning areas in an "s"-shaped pattern in a transverse direction (from side to side). if no microchip is detected, the scanner should be rotated 90 degrees, and then the "s"-shaped pattern should be repeated in a longitudinal direction (e.g., the long way) on both sides of the animal. this pattern of scanning will maximize the ability of the scanner to detect the microchip, regardless of its orientation. â�¢ less than 2% of cats are reunited with their owners, compared to as many as 15% to 19% of lost dogs. 86 â�¢ the use of collars and tags as visually obvious forms of identification is extremely valuable, although overlooked by many cat owners. 69 â�¢ cats wearing collars are more likely to be identified as owned and not mistaken for strays. â�¢ even indoor cats require identification in case they escape, and studies clearly demonstrate that visual identification improves the odds of pet-owner reunification. 69 â�¢ the provision of permanent identification in the form of a microchip represents an important backup, further improving the odds of pet-owner reunification because collars and tags can be lost. 66 â�¢ because owners and shelter staff often describe cat coat color and patterns differently, photographs that can be posted online are a useful method of improving lost-pet matching and enabling owners to look for their pet, even if they are physically unable to come to the shelter. â�¢ adopted animals should be sent home with id collars and microchips. â�¢ shelter staff should always register microchips before the cat leaves the shelter, because many owners will neglect to do so following adoption, making the microchip an ineffective means of identification. 66 â�¢ web-based search engines for pet microchip identification numbers (http://www.checkthechip.com and http://www.petmicrochiplookup.org) have been established in an effort to functionally centralize microchip registries by linking existing national databases. facility that houses cats establish a formal relationship with one or more veterinarians who have direct knowledge of their animal population. this is essential to ensure that medical protocols are established with the proper professional oversight, and helps to ensure compliance with local veterinary practice acts that restrict the practice of veterinary medicine to licensed veterinarians. in facilities such as animal shelters, trained shelter staff can carry out preventive health care under the instructions of a veterinarian. 6 the success or failure of a population wellness program hinges in large part on its implementation and oversight. a knowledgeable, cohesive, and dedicated team, where accountability, responsibility, and lines of authority are well defined, is crucial for management success. as a part of the management structure and plan, veterinarians must be involved in the oversight of all aspects of animal care and must be given direct authority for the oversight of medical decisions. this requires that every physical examination is the clinician's single most important tool for evaluating health. following a standardized physical examination form will ensure a complete and systematic review of all body systems. a veterinarian should carefully examine any new cat entering a closed population prior to admittance. in the context of animal shelters, every cat that is safe to handle should receive a physical examination at or as close to the time of admission to the shelter as possible. in many shelters, a veterinarian may not be available to examine incoming animals. however, staff can and should be trained to perform basic evaluations including sexing, aging, body condition scoring, and looking for evidence of fleas, ear mites, dental disease, overgrown claws, advanced pregnancy, or other obvious physical conditions. of particular importance in the shelter physical examination are an accurate physical description of the animal and careful inspection for the presence of identification, both of which may aid in pet-owner reunification. 41 the gold standard for maintaining the health of a population is through exclusion of pathogens in combination with implementation of comprehensive wellness protocols. this requires that members of a population be free from specific pathogens when the group is established and that the colony be closed to any new individuals that do not meet the health standards of the group. 45 this is the foundation of disease control procedures in a laboratory animal setting, and these concepts should be applied to other population settings whenever possible. consideration should be given to testing for the following: feline leukemia virus (felv), feline immunodeficiency virus (fiv), dermatophytosis, intestinal parasites and infections (e.g., campylobacter, giardia, coccidia), as well as other endoparasites and ectoparasites. the setting and resources available, as well as the individual's history and physical examination findings, should guide the clinician's decisions regarding selection of testing for cats entering a specific population. when new stock is added to a closed colony, disease testing is imperative. the american association of feline practitioners (aafp) maintains detailed professional guidelines for the management of felv and fiv infections. identification and exclusion of infected cats is the most effective method of preventing new infections. cats and kittens should always be tested prior to entry to a closed population. those that test negative should be retested, because it the clinician should develop a program for physical health for the population that addresses all of the essential elements as noted. none of these should be considered as optional, but their implementation will depend on the setting, purposes, and resources of the group. the value of obtaining an accurate medical history on any cat entering a population is immeasurable, because it will often alert the clinician to the presence of potential problems. in a laboratory setting, obtaining cats from commercial purpose-bred colonies or institutional breeding colonies ensures that an accurate history will be available, maximizing the odds that only healthy cats will be added to the population. likewise, private breeding catteries should always strive to obtain an accurate medical history on any cat that may be accepted into the cattery. the introduction of cats from random sources to closed populations of cats risks the health of the population and should be avoided whenever possible. 45 in contrast, by their very nature, animal shelters must frequently receive cats from multiple random sources, and it will not always be possible to obtain accurate histories. in some cases, cats are brought in by animal control officers or good samaritans who have little if any information about them. furthermore, some shelters provide a location (e.g., drop-off cages) where cats can be relinquished after business hours. this practice should be discouraged; however, if facilities elect to do this, every effort must be made to obtain a history through questionnaires that can be completed when the cat is left. the presence of staff to directly accept cats and obtain a history at the time of relinquishment is greatly preferred. 41 even so, surrendering owners may or may not provide complete or accurate information, fearing that if they are honest about a pet's problems, the pet may be euthanized. nonetheless, when available, a history can be extremely valuable, saving time and money as well as preventing unnecessary stress for cats and staff alike. intake procedures should be in place to capture basic patient information, including both physical and behavioral data as well as the reason(s) for relinquishment. the importance of obtaining historical information cannot be overemphasized. in many cases, historical information may be used to expedite the disposition of the cat in the shelter. can be problematic. 2 in relation to population health, testing is of little value, because infected cats pose no risk to other cats. nonetheless, a clinician may elect testing as part of an initial database for individual cats, especially if they will be used for breeding. with heartworm tests readily available in combination with pointof-care felv/fiv tests, many animal shelters have been faced with determining whether or not to perform routine screening of cats in their care. to answer this question, it is helpful to consider the following: in consideration of these facts, the author does not recommend routine screening of cats for heartworm disease in shelters. monthly chemoprophylaxis, however, is a safe and effective option for cats sheltered in areas where heartworm infection is considered endemic. dermatophytosis or ringworm, the most common skin infection of cats, is a known zoonosis. it is caused by infection of the skin, hair, and nails with microscopic fungal organisms that cause varying degrees of hair loss and dermatitis. the dermatophyte that causes the majority of cases in felines is microsporum canis, which is responsible for greater than 96% of all cases. 82 if left untreated, most infections will spontaneously resolve within 12 to 14 weeks postinfection. however, during this time, the infected cat will infect the surrounding environment and other animals or humans in the area. not all cats infected with dermatophytosis develop lesions, and some may become chronic carriers. control of dermatophytosis is difficult, because the spores formed by m. canis can survive in the environment for up to18 months or longer and are extremely resistant to disinfectants and detergents. in addition, the presence of asymptomatic carriers makes it difficult to readily recognize all infected cats. for this reason, consideration should be given to culturing all cats prior to entry to a closed colony. in particular, persian cats may be predisposed to dermatophyte infection and can be particularly difficult to clear once infected. in closed colony settings, dermatophyte testing by culture is highly recommended unless the source of the cat excludes the possibility of infection (e.g., specific pathogen-free [spf] cats, purposebred laboratory cats). to screen cats using cultures, may take as long as 60 days following exposure for a cat to test positive. 62, 63 in the context of animal shelters, testing decisions are often influenced by the availability of resources. the aafp's guidelines include recommendations specifically for shelters. they state that all cats should ideally be tested at the time of entry and again in 60 days in case of recent exposure. when cats test positive on screening tests (e.g., point-of-care enzyme-linked immunosorbent assay [elisa] tests), the aafp recommends that the results be confirmed by additional testing, including testing over an interval of time, because false positives can occur. however, such confirmatory testing requires substantial time and monetary investment and may not be feasible in many shelters. in recognition of this, the association of shelter veterinarians established a policy statement on "management of cats who test positive for felv and fiv in an animal shelter," which states that the logistics and cost of holding and retesting unowned cats may be an ineffective use of resources. 5 in addition, it can be difficult to find homes for retroviruspositive cats, which in many instances translates into stressful, prolonged shelter stays. such long-term confinement may compromise quality of life and may compound the emotional stress of caregivers who may later be faced with euthanizing cats that have been held for long periods awaiting confirmatory testing or adoption opportunities. for all of these reasons, many shelters elect to euthanize cats that test positive on retrovirus screening tests. although it may be ideal for shelters to test cats on entry, it is not always feasible because of financial constraints. the next best practice might be to test cats prior to adoption as well as those that are housed in the shelter long term. in addition, cats should be tested prior to placement in group housing with unfamiliar cats and prior to investment, such as foster care, treatment, or spay/neuter surgery. however, given the limited resources of many shelters, the relatively low prevalence in healthy cats and the fact that transmission can be prevented by housing cats separately, it may not be cost effective for all shelters to screen every cat before selection for adoption. each shelter should evaluate its own resources and determine their best use. when testing is performed, samples must never be pooled, and the negative results of one cat (such as a mother cat) should not be extrapolated to other cats (such as her kittens). these practices are invalid and can falsely lead to misidentification of a cat's true infection status. 62, 63 if testing is not performed prior to adoption, adopters should be advised to have their new pet tested and to keep them separate from any other cats they may own prior to doing so. point-of-care heartworm tests for cats have recently become more widely available, but interpreting results vaccination protocols are typically applied uniformly to all of the individuals comprising the population. this simplifies their application and helps to afford the best possible protection for the group. detailed vaccination records should be maintained for each cat, including vaccine name, manufacturer and serial number, date, the initials of the person who administered it, and any adverse reactions. 96 proper vaccination can substantially reduce disease in cat populations, and serious adverse reactions are relatively rare. for this reason, vaccination against certain core diseases is recommended in all population settings. although exclusion of infectious disease is always a goal of health management, certain pathogens are so widespread that even with careful biosecurity in a closed population, an infection may be introduced to susceptible cats. only in the case of specific pathogen-free colonies, where there may be a compelling reason not to vaccinate as dictated by the purposes of the research, should vaccination be foregone. the aafp maintains published guidelines for vaccination of cats in a variety of settings and includes detailed recommendations for cats in animal shelters. 96 although many vaccines are commercially available for cats, only a few are recommended for routine use in populations. unnecessary use of vaccines should be avoided to minimize the incidence of adverse reactions and reduce cost. core vaccines involve diseases that represent significant morbidity and mortality and for which vaccination has been demonstrated to provide relatively good protection against disease. core vaccines for cats in a population setting include feline parvovirus (fpv or panleukopenia), fhv-1 (feline herpes virus type 1 or feline rhinotracheitis virus), and feline calicivirus (fcv). 96 these vaccines are usually given in a combination product commonly referred to as an fvrcp vaccine (feline viral rhinotracheitis, calicivirus, panleukopenia). in most cases, timely vaccination against panleukopenia will prevent the development of clinical disease. in contrast, vaccination against the respiratory viruses (fhv-1 and fcv) does not always prevent disease. in many instances, it affords only partial protection, lessening the severity of clinical signs but not preventing infection. to optimize response, modified live vaccines (mlv) should be used in most cases, because they evoke a more rapid and robust immune response and are better at overcoming maternal antibody interference than killed products. this is especially important in multicat environments in which the risk of infection is high, such as animal shelters, foster homes, as well as any population setting where upper respiratory disease is endemic. a samples should be collected using the mckenzie toothbrush method, where a new toothbrush is used to brush the cat's entire body, giving special attention to the face, ears, and limbs. in addition, if skin lesions are present, hair should be plucked around these areas for culture as well. campylobacter, salmonella, giardia, coccidia, tritrichomonas, and other gastrointestinal parasites and pathogens are common in some cattery situations and can be very difficult to eliminate once they are introduced. in fact, in some settings, these pathogens may become endemic and nearly impossible to eliminate. treatment of coccidia in shelter kittens is described in although clinical signs, such as diarrhea, may be associated with infection, some cats remain asymptomatic. these pathogens have the potential for high morbidity in a population (especially in young kittens), and some possess zoonotic potential. therefore routine fecal examinations, cultures, and/or empirical treatments should be considered prior to the introduction of new cats. it is well recognized that vaccination plays a vital role in the prevention and control of infectious diseases. protocols should be established in the context of the population's exposure risk, which will vary depending upon the setting. in the context of population medicine, ponazuril is a metabolite of toltrazuril that has proven activity against coccidia.* because there is no approved product for use in cats, the equine product marquis oral paste (15% w/w ponazuril; bayer healthcare) may be dosed at 50 mg/kg, po, once daily for 1 to 5 days. prophylactic treatment may be instituted in high-risk situations, such as young kittens in environments with documented infection. proper hygiene, including the use of disposable litter boxes and frequent removal of feces, is also necessary. oocysts survive in the environment and are not treated by routine disinfectants, such as bleach and quaternary ammonium compounds. with a history of upper respiratory infection) may benefit from vaccination prior to breeding to maximize passage of maternal antibody to their kittens. for pregnant cats in such environments, administration of mlv should be avoided, because the potential risk of injury to the developing kittens may outweigh the risk of infection in this case. vaccination of lactating queens should also be avoided in a low-risk environment. a series of vaccinations should be administered to kittens less than 4 months of age to minimize the window of susceptibility to infection and ensure that a vaccine is received as soon as possible after maternal antibodies have decreased sufficiently to allow vaccine response. 96 for kittens, vaccines should be administered every 2 to 4 weeks until they are 16 weeks (e.g., 4 months) of age or their permanent incisors have erupted. the minimum interval of 2 weeks is recommended in high-risk settings to narrow the window of susceptibility as maternal antibody wanes. a vaccination interval of less than 2 weeks is not recommended, because it may actually blunt the immune response from previous vaccination. 40 in the case of an outbreak of panleukopenia, extending vaccination to 5 months of age may be warranted to ensure than no animal remains susceptible. although the vast majority will respond by 4 months of age, a few may fail to respond, while others are provided with a boost to enhance the immune response. just as in owned pets, booster vaccines are generally not required until 1 year later for modified live vaccines but should ideally be administered once in 2 to 4 weeks whenever resources permit. this may be especially important for cats that were ill at the time of initial vaccination, as may be the case in an animal shelter. revaccination in long-term shelter facilities should follow the guidelines set forth for pets: boost at one year, then every 3 years for fvrcp. 96 vaccination against rabies virus is regarded as a core requirement for pet cats and is required by law in some jurisdictions. 96 thus vaccination against rabies is recommended in the context of private catteries. in contrast, rabies vaccination may be considered optional in most closed laboratory settings, because the risk of exposure should be absent and legal requirements may not apply. in animal shelters, vaccination against rabies is not generally recommended at the time of admission, simply because there is no benefit in terms of disease prevention or public health. 96 vaccination on admission will not provide protection against an infection acquired prior to entry, nor will it limit concern if a cat with an unknown health history bites someone soon after admission. rabies vaccination is recommended for cats prior to adoption when a veterinarian is available to administer it (or as otherwise legally prescribed by state laws). alternatively, rabies vaccination may be administered as single modified live fvrcp vaccine will usually afford protection to cats that are at least 4 months of age. in contrast, killed products require a booster in 2 to 3 weeks to confer immunity, making their use largely ineffective in such environments. 96 to ensure rapid protection against panleukopenia, injectable fvrcp vaccines are preferred, but intranasal vaccines may offer advantages for feline respiratory disease, because they have been shown to rapidly induce local immunity at the site of exposure. furthermore, intranasal vaccines may be better at overriding maternal antibody in young kittens. for this reason, they are often used to reduce the morbidity and severity of upper respiratory infection (uri) in preweaningage kittens. when intranasal vaccines are used in animal shelters, they should be used in combination with injectable fvrcp vaccines to ensure and optimize response against panleukopenia as well as the respiratory infections. 96 ideally, all cats should receive a mlv fvrcp vaccine at least 1 week prior to entering a population. in the context of an animal shelter setting, this is seldom feasible. vaccination immediately upon entry is the next best practice and can provide clinically significant protection for the majority of cats. if neither maternal antibody nor another cause of vaccine failure interferes, modified live vaccinations against panleukopenia will often confer protection against disease in only 5 days. 12 intranasal vaccines against respiratory infections, including fhv and fcv, typically provide partial protection within 2 to 4 days. 18, 30 in animal shelters, all incoming cats and kittens 4 weeks of age and older that can be safely handled should receive an injectable mlv fvrcp vaccine immediately upon entry. a delay of even a day or two significantly compromises the vaccine's ability to provide timely protection. even injured cats, those with medical conditions, and those that are pregnant or lactating should be vaccinated on entry, because vaccination will likely be effective and the small risk of adverse effects is outweighed by the high risk of disease exposure and infection in the shelter. 96 when vaccination of all cats on entry is not financially feasible, the next best practice is to vaccinate all those that are deemed adoptable at the time of entry or that are likely to be in the shelter long term. 41 whenever possible, vaccinated cats should be separated from those that will remain unvaccinated (e.g., those that will be euthanized following a brief holding period) as soon as that determination can be made. in contrast, in lower-risk settings, ensuring that cats are in good health prior to vaccination should be a priority. 96 vaccination of kittens with injectable fvrcp vaccinations may be delayed to 6 to 8 weeks of age. however, when respiratory disease is endemic, administration of intranasal vaccines beginning at 4 weeks of age may be beneficial. in breeding catteries, queens (especially those control and prevention of internal and external parasites represent another important component of a population wellness program. common products used for their management are described elsewhere in this book. of particular importance are roundworms and hookworms, common intestinal parasites with zoonotic potential (see chapter 23) . although uncommon, the risk of human infection from contaminated environments is real and can result in organ damage, blindness, and skin infections. for this reason, the centers for disease control and prevention and the companion animal parasite council strongly advise routine administration of broad-spectrum anthelminthics for their control. 17, 20 pyrantel pamoate is one of the most costeffective and efficacious drugs for treatment and control of roundworms and hookworms. in both shelter and cattery settings, the author recommends administration of pyrantel pamoate at a dosage of 10 mg/kg to all cats with re-treatment in 2 weeks and then at monthly intervals. 41 in shelters, if it is not possible to treat all cats at the time of entry, at a minimum, all cats that are deemed adoptable should be treated as soon as possible. in addition, kittens should be treated at 2-week intervals until 4 months of age. for cats with diarrhea, fecal examination (e.g., flotation or centrifugation, direct fecal smear and cytology) should be performed with treatment according to results. even if results are negative, the administration of broad-spectrum anthelminthics should be strongly considered. in animal shelters, ectoparasites, particularly ear mites and fleas, are also very common in cats and kittens. shelter staff should be trained to recognize infestation and protocols should be established for treatment. in terms of shelter treatment protocols, the author recommends treating ear mites with ivermectin, because it is highly efficacious and costs only pennies per dose. the recommended dosage is 0.3 mg/kg subcutaneously. for fleas, the author recommends topical treatment with fipronil (frontline, merial, duluth, ga.) as a spray or top spot. in particular, the spray is very cost effective. it is safe for use in cats of all ages, including pregnant and nursing mothers and neonatal kittens. in addition, fipronil also has activity against ear mites, cheyetiella, chewing lice, and ticks. 19, 100 spaying and neutering is another important consideration in the context of population wellness. 41 reproductive stress from estrous cycling in queens and sex drive in tomcats can decrease appetite, increase urine spraying/ marking and intermale fighting, and profoundly increase social and emotional stress in the group. for these reasons, spaying and neutering cats that will not be used soon as possible following adoption. 41 the latter may encourage new owners to establish a relationship with a private veterinarian. rabies vaccination is warranted when cats are housed long term in shelter facilities. in addition, if individual cats must be held for bite quarantines, they should be vaccinated against rabies in accordance with the current compendium of animal rabies prevention and control. 85 noncore vaccines include those that may offer protection against disease, but because the disease in question is not widespread or only poses a risk of exposure in certain circumstances, vaccination is only recommended based on the individual risk assessment of a population of animals. noncore vaccines include felv, fiv, chlamydophila, and bordetella. 96 vaccination against felv is not warranted in a closed population of cats in which there is no risk of exposure (e.g., most laboratory animal settings). in private catteries, a risk assessment should be done to determine if vaccination is warranted (e.g., cats permitted in outdoor enclosures, frequent introduction of cats from external sources, other opportunities for exposure). special consideration should be given to vaccinating kittens because of their high susceptibility to felv infection and the high likelihood that they will become persistently infected if exposed. in general, felv vaccination is not recommended in animal shelters when cats are housed short term. however, its use is warranted when cats are group housed when resources permit. 96 fiv vaccination is not generally recommended in population environments. a confounding feature of fiv vaccination is that vaccinated cats develop false-positive test results on most commercially available tests (see chapter 33) . if fiv vaccination is elected, vaccinated cats should be permanently identified (e.g., by use of a microchip) to help clarify their status. 96 chlamydophila felis (c. psittaci) and bordetella bronchiseptica vaccines may be of benefit when clinical signs of these diseases are present in the population and diagnosis is confirmed by laboratory evaluation. their efficacy is moderate, and reactions are more common than with most other feline vaccines; therefore ongoing use should be periodically reassessed. 96 some vaccines are not generally recommended for use because of undemonstrated efficacy, such as the feline infectious peritonitis (fip) vaccine. 96 wants whenever he or she chooses. dry food is used for this method of feeding, because canned products left at room temperature are prone to spoiling. the major advantage of free choice feeding is that it is quick and easy: caregivers simply need to ensure that fresh dry food is always available. major disadvantages include the fact that cats that are not eating may remain unrecognized for several days, especially when more than one animal is fed together, and some cats may choose to continually overeat and become obese. free choice feeding is an excellent method for cats that require frequent food consumption. these include kittens up to 5 to 6 months of age, queens in late gestation, and those that are nursing. unlike dogs, who are competitive eaters by nature, free choice feeding may benefit cats that are group housed, because it ensures that there will be ample time for all members to eat, provided that dominant members of the colony do not block the access of subordinate cats. 49 meal feeding using controlled portions of dry and/or canned food may be done as an alternative to or in conjunction with free choice feeding. when used alone, a minimum of two meals should be fed per day. 49 meal feeding is ideal for any cat that requires controlled food intake and facilitates monitoring of appetite. meal feeding also has the benefit of enhancing caregiver-cat bonding and provides a pleasant and predictable experience for cats when done on a regular daily schedule. using a combination of free choice plus once daily meal feeding takes advantage of the positive aspects of both methods and works well for most cats in a population setting. typically, dry food is available free choice, and a small meal of canned food is offered once daily. this combination approach accommodates the normal feeding behavior of cats by allowing them to eat several smaller meals throughout the day while allowing caregivers to monitor the cat's appetite at least for the canned food meal. as necessary for the individual cat, some may be fed additional meals of canned food to ensure adequate nutritional support. good body weight and condition and a healthy hair coat are evidence of an adequate nutritional plane and proper nutritional management. both appetite and stool quality should be monitored daily. normal stools should be well-formed and medium to dark brown. adult cats typically defecate once daily, although healthy adults may defecate anywhere between twice a day and twice a week. kittens tend to produce a larger volume of stool more frequently, which is often lighter in color and softer in form than that of adults. simple scales can be used for monitoring appetite (e.g., good, some, none), and fecal scoring charts are available. the author recommends the purina fecal scoring system chart available from nestlã© purina petcare company (figure 46-7) . for breeding is recommended. in animal shelters, spaying and neutering cats prior to adoption will ensure that they do not reproduce and contribute to the surplus of community cats. this will also serve to enhance husbandry, because the procedures rapidly decrease spraying, marking, and fighting; eliminate heat behavior and pregnancy; and greatly mitigate stress. in addition to reducing stress and odor, spaying and neutering sexually mature cats will facilitate group housing, which is often beneficial for cats, especially when housed longer term (see below). the medical benefits of spay/neuter have also been well described, including dramatic reductions in the risk of mammary carcinoma, elimination of cystic endometrial hyperplasia, pyometra and ovarian cancer in queens, and decreased risk of prostate disease in toms. 55 thus spaying and neutering favors both individual as well as population health. proper nutrition has a profound impact on wellness. not only is it essential for management of healthy body weight and condition, good nutrition is also known to support immune function. a regular diet of palatable commercial food consistent with life stage should be offered, and fresh water must always be available. although some cats tolerate changes in food without apparent problems, it is important to recognize that for others, changing from one diet to another can cause loss of appetite and/or gastrointestinal upset. for this reason, it is generally best to provide the most consistent diet possible. 49 whereas this may be relatively easy to do in a laboratory or cattery setting, it can be more challenging in a shelter environment. some pet food companies offer feeding programs for animal shelters, providing a consistent food for purchase at a special rate for shelters. however, some shelters rely heavily on donations of food. in this case, by requesting donation of certain brands of food, shelters are able to provide a consistent diet whenever possible. it is also feasible to mix donated foods with the shelter's usual diet to minimize problems caused by abrupt diet changes while taking advantage of other donated products. the wild ancestors of domestic cats hunted to eat, feeding up to 30 times in a 24-hour period. this style of feeding behavior is preferred by many domestic cats that would nibble throughout the day and night, consuming many small meals if left to their own devices. although this is true, most cats are capable of adapting to either free choice or meal feeding as their daily feeding pattern. 15, 49 there are advantages and disadvantages to each in a population setting. with free choice or ad libitum feeding, food is always available such that a cat can eat as much as he or she trends in body weight, because both weight loss and gain can compromise health and well-being. appropriate grooming is also essential to ensure wellness and must never be considered as optional or purely cosmetic. most cats require minimal grooming because of their fastidious nature. however, long-haired cats are notable exceptions, often experiencing matting of the hair coat without regular grooming sessions. matted hair coats are not only uncomfortable for the animal, but may lead to skin infection. overgrown nails can also be a problem for some cats, particularly those that are geriatric or polydactyl. the provision of appropriate surfaces for scratching will encourage cats to condition their own claws; and a system for regular inspection of the hair coat and nails should be established. in addition to ensuring proper coat and nail maintenance, regular grooming sessions provide an excellent opportunity to monitor body condition; and some cats enjoy the physical contact and attention. in high-risk settings, the use of stainless steel combs or undercoat rakes that can be readily disinfected are generally preferable to the use of in addition to appetite and stool quality, it is essential to monitor body weight and condition. body condition can be subjectively assessed by a process called body condition scoring, which involves assessing fat stores and, to a lesser extent, muscle mass. fat cover is evaluated over the ribs, down the top line, tail base, and along the ventral abdomen and inguinal (groin) areas. body condition score charts have been established on scales of 1 to 5 and 1 to 9. the author recommends use of the purina body condition score chart which is based on a scale of 1 to 9 with 1 being emaciated and 9 being severely obese (see figure 3 -3). cats should be weighed and their body condition scored at routine intervals. ideally, body weight should be recorded at entry to the population and then weekly during the initial month of care, after which it could be recorded once a month or more often as indicated based on the individual's condition. this is especially important for cats, because significant or even dramatic weight loss may be associated with stress or illness during the first few weeks of confinement in a new setting. on the other hand, in long-term-housed cats, excessive weight gain may occur in some individuals. therefore protocols must be in place to identify and manage unhealthy score 4 -very moist (soggy); distinct log shape visible; leaves residue and loses form when picked up. score 5 -very moist but has distinct shape; present in piles rather than as distinct logs; leaves residue and loses form when picked up. score 6 -has texture, but no defined shape; occurs as piles or as spots; leaves residue when picked up. score 7 -watery, no texture, flat; occurs as puddles. score 2 -firm, but not hard; should be pliable; segmented appearance; little or no residue left on ground when picked up. score 3 -log-like; little or no segmentation visible; moist surface; leaves residue, but holds form when picked up. fecal scoring system bristle brushes because the latter are impossible to disinfect and have the potential to spread common skin infections such as ringworm. dental health is another component of wellness. in the context of population wellness, it may not be the highest priority; however, it should always be a consideration in terms of individual health care and well-being. this is important because periodontal disease will occur unless it is actively prevented, and plaque and tartar buildup may contribute to serious health concerns, ranging from oral pain to chronic intermittent bacteremia and organ failure. feline tooth resorption and gingivostomatitis are also common conditions of the feline oral cavity that can lead to chronic pain, affecting the cat's appetite and ability to self-groom, and negatively impacting quality of life. when painful dental disease is present, a plan for timely treatment should be identified and implemented. preventive dental care may include tooth brushing, dental-friendly diets, and treats and chew toys in combination with periodic professional dental care. 33 these should be tailored to meet the needs of individuals in the population to optimize dental health. cats with stomatitis should be removed from breeding programs. 72 wellness protocols may also be dictated by the specific needs of certain breeds of cats. for example, persian, himalayan, and other brachycephalic cats are predisposed to respiratory disease and tend to be more severely affected than other cats because of their poor airway conformation. because of the high likelihood of exposure in a shelter setting, these cats should be housed in highly biosecure areas that are well ventilated and should be prioritized for immediate adoption or transfer to foster care or rescue. 41 in the author's experience, even intranasal vaccination of these breeds can result in severe clinical signs of respiratory disease and is best avoided. just as a physical wellness program must be tailored to the population in question, a behavioral wellness program, composed of all of the essential elements, should be created to meet its specific needs as well. even when animals will only be housed for short periods, considerations for behavioral care are essential to ensure humane care. short-term confinement can induce severe stress and anxiety, and when confined long term, cats may suffer from social isolation, inadequate mental stimulation, and lack of exercise. a behavioral wellness program should strive to decrease stress from the moment cats arrive at a facility until the moment that their stay ends. as previously described, a thorough behavioral history will provide an important baseline for action and follow-up. understanding the importance of minimizing stress in cats and possessing the ability to recognize and respond to it are essential to facilitate a cat's transition into a population. 42, 46 staff should be trained to evaluate cats beginning at intake and to recognize and respond to indicators of stress. active daily monitoring of cats for signs of stress or adjustment should be performed, and staff should record their findings daily, noting trends and making adjustments in the care of individual cats and the population as indicated. in animal shelter environments, proper behavioral care of cats also requires an understanding of the wide spectrum of feline lifestyles and an approach tailored to the individual needs of each group. domestic cat lifestyles and levels of tractability range from the most docile, sociable housecat, to free-roaming strays and truly unsocialized feral cats that will not allow handling. stray cats include those that may have been previously owned or are "loosely owned" neighborhood or barn cats. 79 because of their lack of socialization, capture, handling, and confinement are especially stressful for feral cats. however, fearful cats may resort to overt aggressive or may "teeter on the edge" of defensive aggression regardless of their socialization status. in fact, even the tamest house cats may exhibit the same behaviors as feral cats when they are highly stressed (figure 46-8) . 29, 42 these responses can compromise cat welfare and staff safety and hinder adaptation to a new environment. regardless of their demeanor, all cats and kittens should be provided with a hiding box in their enclosure at the time of entry, because the ability to hide has been shown to substantially reduce feline stress. 14 for those cats that are severely stressed or reactive, covering the cage front, in addition to providing a hiding box, and posting signage to allow the cat "chill out" time for several hours or even a few days can facilitate adaptation. 42 this is important because, once highly stressed or provoked, cats often remain reactive for a prolonged time and may become more reactive if they are stimulated again before they have been allowed a period of time to calm down. 9 soft bedding should be available for comfort and so that cats may establish a familiar scent, which aides in acclimation to a new environment. care should be taken during cleaning procedures to minimize stress and noise, behavioral evaluation may be useful, especially for cats that will be re-homed. several evaluations have been recommended, but none are scientifically validated for predicting future behavior with certainty. 3, 103, 107 nonetheless, this form of evaluation may be useful for determining behavioral needs while cats remain in a facility, as well as guiding appropriate placement. box 46-6 describes common components of a feline behavioral evaluation (figure 46-10) . housing design and operation can literally make or break the health of a population. 45 regardless of the species in question, housing should always include a comfortable resting area and allow animals to engage in species-typical behaviors while ensuring freedom from fear and distress. 54 it is not sufficient for the design to address only an animal's physical needs (e.g., shelter, warmth). it must meet their behavioral needs as well, and both the structural and social environment are essential considerations for housing arrangements. furthermore, the environment must provide opportunities for both physical and mental stimulation, which become increasingly important as length of stay increases. 54 a sense of control over conditions is well recognized as one of the most critical needs for behavioral health. 77 thus housing design must provide cats with a variety of satisfying behavioral options. specifically, housing arrangements must take into account the following feline behavioral needs 46 : â�¢ opportunities for social interactions with humans and/or other compatible cats and cats should be allowed to hide while their cage is quietly tidied and replenished around them as needed. commercially available "cat dens" are ideal for this purpose, because they can be secured from a safe distance such that the cat is closed inside a secure, familiar hiding place during cleaning procedures (figure 46-9) . cats should be returned to the same cage and only spot cleaning should be performed to preserve their scent, which is necessary for stress reduction. if it becomes necessary to house the cat in another location, the den and towel should accompany the cat to ease the transition. finally, the use of commercially available synthetic analogues of naturally occurring feline facial pheromones (feliway, veterinary product laboratories, phoenix, ariz.) have been shown to be useful for stress reduction in cats during acclimation to new environments and can be sprayed onto bedding and allowed to dry prior to use or dispersed in the room using plug-in diffusers. 48 the way in which cats are handled at intake has a profound impact on their behavior, health, and wellbeing and will impact the cat's ability to adapt to its new environment. when stress is successfully mitigated, cats are more likely to adapt and to "show their true colors" rather than reacting defensively. during a period of a few days, many cats that did not appear to be "friendly" at intake will become tractable and responsive to their human caregivers, facilitating care. 42 aside from informally "getting to know" cats during their initial acclimation period in a facility, a systematic â�¢ the ability to create different functional areas in the living environments for elimination, resting, and eating â�¢ the ability to hide in a secure place â�¢ the ability to rest/sleep without being disturbed â�¢ the ability to change locations within the environment, including using vertical space for perching â�¢ the ability to regulate body temperature by moving to warmer or cooler surfaces in the environment â�¢ the ability to scratch (which is necessary for claw health and stretching, as well as visual and scent marking) â�¢ the ability to play and exercise at will â�¢ the ability to acquire mental stimulation because these needs will vary depending upon such factors as life stage, personality, and prior socialization and experience, facilities should maintain a variety of housing styles in order to meet the individual needs of different cats in the population (figure 46-11) . 46 managing housing arrangements for a population of cats of varying ages, genders, personality types, social experiences, and stress levels requires knowledge of normal feline social behavior and communication. 46 during the past 2 decades, knowledge of feline social structure has evolved from the widespread belief that cats are generally an asocial and solitary species to the realization that they are social creatures. 24, 25 with the exception of solitary hunting, free-roaming cats perform responses are observed and recorded for each of the following: â�¢ the tester approaches cage, stands quietly for 20 seconds, then offers verbal encouragement. â�¢ if deemed safe to proceed, tester opens the cage door and calmly extends an open hand towards the cat, then attempts to gently touch the cat's head. â�¢ if the caregiver is unsure if this is safe to do, a plastic hand may be used to gauge the cat's receptiveness to touch (see figure 46 -9). â�¢ if the cat allows handling, the cat is gently lifted and carried to a secure, quiet room for further observation. â�¢ the tester sits quietly on a chair and/or the floor; the tester calls and solicits the cat's attention. â�¢ the tester pets the cat on the head. â�¢ the tester strokes the cat down the back several times. â�¢ the tester picks up the cat and hugs it for 2 seconds. â�¢ with the cat standing on the floor, the tester strokes the cat down the back and firmly but gently grasps the base of the tail and lifts the cat off of its hind feet for 1 second. the tester repeats this a second time. â�¢ the tester engages the cat in play with an interactive toy. in some instances, it is difficult to determine if a cat will accept handling. to prevent injury to staff, a plastic hand (assess-a-hands; great dog productions, accord, ny) is used to approach this cat. as the hand approaches, the cat appears tense (a) but begins to relax and accepts petting (b and c) . the absence of normal behaviors (e.g., grooming, eating, sleeping, eliminating, stretching, greeting people). defensive behavior may involve characteristic postural and/or vocal responses, and is usually motivated by fear. disruptive behavior involves destruction of cage contents and creation of a hiding place. stereotypic behaviors (e.g., repetitive pacing, pawing, and circling) may also develop as a result of stress but generally occur less commonly. as an illustration of these feline behaviors, consider the responses of a typical social domestic cat when caged in a novel environment (box 46-7 and figures 46-12 to 46-15) . behavioral signs of stress may be further classified as active communication signals or passive behaviors. 46, 90 signals of anxiety, fear, aggression, and submission may be subtle or obvious and include vocalization (growling, hissing), visual cues (facial expression, posturing of the body, ears, and tail), and scent marking (urine, feces, various glands of the skin). passive signs of stress include the inability to rest/ sleep, feigned sleep, poor appetite, constant hiding, absence of grooming, activity depression (decreased play and exploratory behavior), and social withdrawal. high-density housing exacerbates these signs. lowsocial-order cats in such an environment may exhibit decreased grooming, poor appetite, and silent estrus. 44 cats that are consistently fearful or anxious may hide, most of their activities within stable social groups where cooperative defense, cooperative care of young, and a variety of affiliative behaviors are practiced. affiliative behaviors are those that facilitate close proximity or contact. cats within groups commonly practice mutual grooming and allorubbing (e.g., rubbing heads and faces together). this may serve as a greeting or as an exchange of odor for recognition, familiarization, marking, or development of a communal scent. cats of both genders and all ages may exhibit affiliative behaviors, and bonded housemates often spend a large proportion of their time in close proximity to one another. 8 maternal behavior is the primary social pattern of the female cat, and cooperative nursing and kitten care are common. if allowed, queens form social groups along with their kittens and juvenile offspring. 24, 25 tomcats typically reside within one group or roam between a few established groups. within groups of cats, a social hierarchy or "pecking order" forms. 24, 90 once established, this hierarchy helps to support peaceful co-existence of cats within a stable group, minimizing agonistic behaviors between members. social hierarchy formation occurs within groups of cats that are sexually intact, as well as in those that are neutered. knowledge of behavioral signaling is critical for successful management of housing arrangements. manifestations of both normal and abnormal behavior indicate how successfully an animal is coping with its environment. common behavioral expressions of feline anxiety may manifest with inhibited or withdrawal behavior, defensive behavior or disruptive behavior. 46, 90 inhibited or withdrawal behavior refers to activity depression or â�¢ fear is typically the initial response, and if threatened by the proximity of unfamiliar caregivers, defensive aggression may be displayed. alternatively, the cat may freeze or appear catatonic. â�¢ if provided with a box for concealment, the cat will hide or otherwise slink against the back of the enclosure, behind the litter box, or disrupt the cage and hide under the paper. â�¢ given time, most cats become more active and engage in greeting behaviors, coming to the front of the cage and pawing or mewing as caregivers approach. â�¢ if the cat remains confined with time without adequate periods of exercise, mental stimulation, and social companionship, stress and frustration will manifest with activity depression and withdrawal (lying in the litter box, failure to groom, failure to greet caregivers, and, in some cases, displaying aggression towards caregivers). â�¢ displays of stereotypic behavior (such as pacing) may occur; however, inhibited or withdrawal behaviors are much more common (see unfamiliar or new cats entering the group. within an established group, however, most social conflicts are not characterized by overt aggression. instead, the main mode of conflict resolution is avoidance or deference (figure 46-16) . 9,46,90 deference behaviors include looking away, lowering the ears slightly, turning the head away, and leaning backward. large numbers of cats peacefully co-exist together, using such strategies for avoidance provided ample space and resources are available for all members of the group. 10 signs of social stress within groups of cats may manifest with overt aggression, increased spraying and turn their back, huddle, and avert their eyes from the gaze of other cats. hiding is a normal and important coping behavior; however, when hiding is occurring with increased frequency or in response to stimuli that did not previously cause hiding, it should be recognized as a sign of stress. 46, 90 in group settings, the complexity of the social structure cannot be overestimated. the internal structure of social groups rarely represents a straightforward linear hierarchy, except in very small groups of less than four to five animals. 25 in larger groups of cats, there are usually one or two top-ranking individuals and one or two obvious subordinates, while the remaining cats share the middle space. 9, 44 most cats within the group form affiliative or friendly relationships; however, some may fail to form such relationships and remain solitary. colony members commonly display aggression toward c marking, or constant hiding. 46, 90 lower-ranking cats may spend little time on the floor, remaining isolated on single perches or other locations where they may even eliminate, while higher-ranking cats remain more mobile, controlling access to food, water, and litter resources. 44 high-density housing conditions frequently result in such abnormal behaviors and are associated with increases in transmission of infectious diseases and reproductive failure as well. 50 cats are commonly housed in three basic arrangements: cage or condo units, multiple runs within a room, or free ranging in a room. 45 cage housing of cats should be avoided unless necessary for short periods for intake observation, legal holding periods in shelters as required by local ordinances, medical treatment or recovery, or to permit sample collection. 45 although space recommendations vary substantially in the literature, common sense dictates that a determination of necessary housing space should take into account the cat's length of stay. in the author's opinion, it is neither appropriate nor humane to house cats in traditional cage housing long term (e.g., more than 1 to 2 weeks). the design of short-term housing should include provisions for housing individual animals, litters, families, or bonded housemates for intake evaluation and triage. 41 housing must be easy to clean and sanitize, well ventilated, and safe for animals and caregivers. short-term housing should provide sufficient space to comfortably stand, stretch, and walk several steps; sit or lay at full body length; and separate elimination, feeding, and resting areas. litter boxes should be of appropriate size to comfortably accommodate the cats for which they are intended ( figure 46-17) . resting areas should include comfortable surfaces, soft bedding, and a secure hiding place to provide a safe refuge. 41 a hiding place is essential, because it reduces stress by allowing cats to "escape," facilitating adaptation to a new environment. the addition of a sturdy box to a cage will provide a hiding place as well as a perch (figures 46-18 and 46-19) . in addition, cages should be elevated off of the floor by at least 0.5 m (1.5 feet), because this serves to reduce stress as well. 73 in most instances, cage or condo style housing is used in most facilities for short-term holding at intake for observation, acclimation, and/or triage. runs or small rooms are also appropriate for intake housing, and offer cats the obvious benefit of additional space to meet their behavioral needs (figure 46-20) . regardless of their configuration, enclosures for short-term housing of cats should be large enough to allow them to stretch, groom, and move about while maintaining separate functional areas, at least 0.6 m (2 feet) apart, for sleeping, eating, and elimination. 41, 58, 98 laboratory guidelines in the united states call for a minimum floor area of 0.27 m 2 (3 ft 2 ) for cats weighing less than 4 kg and 0.36 m 2 (4 ft 2 ) for cats weighing 4 kg or more, with a minimum height 0.6 m (2 ft). 54 a resting upper respiratory infections. double-sided enclosures (e.g., cat condos) are ideal for meeting these specifications and have the benefit of easily allowing cats to remain securely in one side of the enclosure while the opposite side is cleaned (figure 46-22) . this helps to minimize stress, prevent exposure to infectious disease, perch is also required. current guidelines (european convention for the protection of vertebrate animals used for experimental and other scientific purposes, ets123) promulgated by the council of europe (http:// www.coe.int) for laboratory cats are similar, but proposed revisions call for substantially more floor space for cats, at 1.5 m 2 (16.7 ft 2 ) per adult cat with a height of at least 2 m (6.5 ft). 21,22 the revisions, which have not been approved to date, also call for the provision of shelves, a box-style bed, and a vertical scratching surface. animal shelter facilities in the united states have traditionally been equipped with small perchless cages (e.g., 0.5 to 0.75 m or 1.5 to 2.5 ft long) that are poorly designed for housing cats. the association of shelter veterinarians (http://www.sheltervet.org) recommends a minimum enclosure size of 1 m 2 (11 ft 2 ) for adult cats. 7 commercially available cages are typically approximately 0.75 m (2.5 ft) deep (e.g., an arm-length deep so that they can be readily accessed); therefore a cage with a length of 1.2 m (4 ft) is required to provide this approximate square footage, and it will also allow for adequate separation of food, water, and litter ( figure 46-21) . similarly, the cat fanciers' association (http:// www.cfa.org) recommends a minimum of 0.85 m 3 (30 ft 3 ) of space per cat for those weighing 2 kg or more. 16 cubic measurements take into account the use of vertical space in addition to floor space, which is crucial for improving the quality of the environment. for example, a 0.85-m 3 (30larger enclosures also allow for better air circulation, which is an important consideration for control of feline housing for a single cat. note the large 4-ft long cage, provision of a secure hiding place and perch with bed, separation of litter from resting and feeding areas, and appropriately sized litter box for this large cat. housing units are available for cats and serve to separate functional living areas and provide improved opportunities for exercise and exploration. this unit (tristar metals, boyd, tex.) is constructed of powder-coated stainless steel, which is highly durable and easy to disinfect but less noisy than uncoated stainless steel. note the elevation from the floor and the grills on both the front and back, which allows flow-through ventilation. is both mentally and physically stimulating for cats and preferably that which is esthetically pleasing to humans. 41 the latter is an important consideration to facilitate adoption in animal shelters. and, even in other types of facilities, it is important to create a pleasant environment not only for the animals, but also for their caregivers. studies indicate that employee satisfaction improves animal care and staff retention, both of which may positively impact population health and well-being. 95 for long-term housing of cats alternatives to traditional cage housing should be afforded. 41, 45, 46 at an absolute minimum, cats that are cage housed must be released each day and allowed an opportunity to exercise and explore in a secure enriched setting. for long-term housing, most cats will benefit from colony-style housing, provided there is sufficient space, easy access to feeding and elimination areas, an adequate number of comfortable hiding, and resting places and careful grouping and monitoring to ensure social compatibility among cats. not every cat, however, will thrive in a group setting, and certain individuals will require enriched single housing, depending on their unique physical or behavioral needs. these may include cats that bully other cats or are otherwise incompatible and those with special medical needs. it is important to recognize that such singly housed cats will require more regular contact with their human caregivers and higher levels of mental and physical stimulation in order to maintain behavioral health during long-term confinement. whenever possible, long-term housing of such individuals should be avoided. when cats are housed in amicable groups, it is easier to maintain proper behavioral welfare in the long term, because many of their social and emotional needs can be met by conspecifics. group housing affords cats with opportunities for healthy social contact with others, which, in turn, provides additional mental and physical stimulation. when properly managed, this housing arrangement enhances welfare.* insufficient space and crowding or poor compatibility matching of cats serves to increase stress and negates the benefits of the colony environment. group housing should never be used as a means of simply expanding the holding capacity of a facility. in animal shelters, the high turnover rate of cats contributes substantially to feline stress levels, especially in the context of groups of unfamiliar animals. because it may take days to weeks to acclimate to a group environment, enriched individual housing may be preferable when a brief stay is anticipated. however, the benefits of enriched social group housing become evident when stays extend beyond a few weeks. 41 and preserve staff safety, which are especially crucial for newly arrived cats. traditional cages can be modified into condo-style enclosures by creating portals to adjoin two or three smaller cages (figure 46-23) . regardless of the precise specifications of the enclosures, the importance of the overall quality of the living environment cannot be overemphasized. this includes a holistic approach to husbandry, with careful attention to the way in which cats are handled, noise levels, the provision of creature comforts, positive contact with caregivers, and strict avoidance of overcrowding, as well as good sanitation, medical protocols, and careful monitoring to ensure health and welfare. for long-term housing (e.g., greater than 2 weeks), consideration should also be given to providing space that a b *references 28, 45, 46, 57, 59, 70, 71, 74, 90, 98, 99. breeding age should be avoided whenever possible. at a minimum, mature tomcats should be neutered to prevent intermale aggression, urine spraying, and breeding. 41 reproductively intact females may be co-housed with other intact females or with neutered males. in contrast, in breeding colonies, harem-style housing may be used to facilitate breeding (e.g., a few queens with a tomcat). it is also advantageous to house compatible pregnant queens together before delivery, because they will usually share nursing and neonatal care ( figure 46-24) . 45 after delivery, pairing of queens becomes more difficult. when tomcats are not breeding, they can usually be co-housed with a spayed female, a neutered male, or a compatible juvenile for companionship. other recommended groupings in the context of a breeding colony include postweaning family groups, prepubertal juveniles, or compatible single-sex adults. personality type there are two basic feline personality types: cats that are outgoing, confident, and sociable and those that are relatively timid and shy. 105 cats with bold, friendly temperaments tend to cope and adapt more readily than shy, timid cats. a subset of the bold, friendly personality type is the "assertive" or "bully" cat. 90 bully cats constantly threaten other cats in a group setting in order to control access to food, litter, perches, or the attention of human caregivers. to maintain harmony, removing cats of this personality type from a colony is usually necessary. reassignment is possible, but may prove difficult, necessitating single housing. shy, timid cats sometimes have difficulty interacting successfully with more dominant members of a group or may fall victim to a bully, resulting in chronic stress and increased hiding. placement of shy cats in smaller groups or with calm juvenile cats, where they will not be intimidated or harassed, is generally rewarding and often helps them to "come out of their shells." 46, 90 similarly, dominant cats will often accept calm, younger cats, as opposed to other adults by whom they may feel threatened. and finally, in the case of some dominant males, the introduction of a female cat will be more likely to be successful. 46, 64, 90 the precise space requirements for long-term housing of cats will vary, because it is dependent on many factors (box 46-8). 46, 59 of paramount importance is that group size must be small enough to prevent negative interactions among cats and to permit daily monitoring of individuals. cats typically prevent social conflict through avoidance, and adequate space must be available so that cats can maintain social distance as needed. crowding can make it impossible for animals to maintain healthy behavioral distance, creating situations where individuals may not be able to freely access feeding, resting, or elimination space because of social conflicts over colony careful attention to groupings of cats is essential for success. family groups and previously bonded housemates are natural choices for co-housing, 11, 46 but unfamiliar cats may also be grouped using careful selection criteria. many cats do have preferences for housemates, necessitating conscientious compatibility matching combined with the provision of a high-quality environment. groupings of unfamiliar cats should always be given priority for the largest available enclosures. in addition, cats should always receive appropriate health clearances prior to admission to a group. these should be determined by the specific protocols of the facility; but in most cases, minimum requirements would include that cats be free of signs of contagious disease, tested for felv and fiv, vaccinated against fvrcp, and treated for parasites. in addition to prior relationships, selection criteria for groupings should include age, reproductive status, and personality. age age is an important consideration regarding housing arrangements. 46 to ensure proper social and emotional development, kittens should be housed with their mother at least until they are weaned. because it can be behaviorally beneficial, it is desirable for them to remain with her for a longer period of time when this is feasible. in fact, queens frequently do not fully wean their kittens until 12 to 14 weeks of age if left to their own devices. if older kittens are housed with their mother, it is important to provide a perch that allows her the option of periodically resting away from them if desired. most queens will accept the kittens of another cat; therefore young orphan or singleton kittens should be housed with other lactating queens and/or kittens of similar age/size. in a shelter setting where there is a high turnover of cats, it may be beneficial to house young kittens up to 4 to 5 months of age in large cages or condos for biosecurity purposes. juveniles and adults can be housed in colony rooms or runs but should be segregated by age (e.g., juveniles 5 to 12 months old, young adults, mature adults, geriatrics). well-socialized juveniles tend to adapt quickly in a group setting with other cats of similar ages and exhibit healthy activity and play behavior. in contrast, mature adults and geriatric cats often have little tolerance for the high energy and playful antics of many younger cats, which can cause them substantial stress. for this reason, adult cats should be kept separate from juvenile cats, and aging or geriatric cats separate from other age groups. in animal shelters, compatible cats that enter the shelter together should be housed together regardless of age, whenever possible. unless cats will be used for breeding, group housing of sexually intact cats of all of these reasons, housing cats in small groups is preferred. 41, 45, 46 in most instances, the author recommends housing cats in compatible pairs or small groups of not more than three to four individuals. housing cats in runs is ideal for this purpose (figure 46-25) . a well-equipped, 1.2-ã� 1.8-m (4-ã� 6-ft) run can comfortably house two to three adult cats depending on their familiarity and compatibility, or up to four juveniles (e.g., 6 to 12 months old). juveniles tend to accept a slightly higher housing density than adults. likewise, previously bonded housemates and families will generally peacefully co-exist at a higher density than will unfamiliar cats. when runs are used, they must have a top panel and should be at least 1.8 m (6 ft) high to allow caregivers easy access for cleaning and care. if chain-link is used, 2.5-cm (1-inch) mesh is ideal, but larger mesh can be used. existing dog kennel runs can be converted into areas for cat housing. this is an important and practical consideration in animal shelters, because many shelters have experienced a decrease in dog intake, while the need for improved cat housing is great. cats and dogs should never be co-housed in the same area; thus conversion should result in an exclusive cat housing area. for colony rooms, the author recommends a minimum enclosure size of approximately 3 to 3.5 m ã� 5 to 5.5 m (10 to 12 ft ã� 16 to 18 ft) for colonies of up to a maximum of eight adult cats, or in the case of juveniles, a few more. doubling the size of an enclosure does not necessarily allow a twofold increase in the number of cats that can be properly housed. another author recommends 1.7 m 2 (18 ft 2 ) per cat as a general guideline for group housing, resources. both crowding and constant introduction of new cats induce stress and must be avoided to ensure proper welfare. the addition of new cats always results in a period of stress for the group, and if there is constant turnover within the group, cats may remain stressed indefinitely. high turnover also increases the risk of infectious disease. if cat group numbers are small, disease exposure will be limited, facilitating control. for â�¢ length of stay â�¢ overall quality of the environment, including use of vertical space â�¢ overall quality of behavioral care â�¢ physical and behavioral characteristics of the cat (e.g., age, personality type, prior experience, and socialization) â�¢ individual relationships between cats (e.g., family groupings, previously bonded housemates, versus unfamiliar groupings and degree of social compatibility among cats) â�¢ turnover of cats (e.g., frequency of introduction of new members) â�¢ total room size â�¢ absolute number of cats â�¢ individual needs and levels of enrichment being used to meet these needs enabling caregivers to better monitor individual appetites and litter box results while allowing cats a period of rest away from one another. alternatively, individual enclosures may only be used for brief periods for meal feedings of canned food, with dry food available free choice in the colony. this sort of arrangement can also be used to facilitate introduction of new cats to the group and represents a desirable option. if design and biosecurity procedures permit, portable intake enclosures could even be transferred to group rooms to smooth the transition of new cats from intake to long-term housing areas. tremendous individual variation exists among cats in the context of social relations with other cats. although introduction of some previously unfamiliar cats will seem effortless and uneventful, introduction of others will result in considerable stress, not only for the new cat but for the entire group as well. 46 for this reason, introductions should always be done under supervision, and whenever possible, they should be gradual. to accomplish this, a new cat can be kept in a separate cage within or adjacent to the group enclosure equipped with food, water, litter, and a hiding box. usually, within a few days, it will be evident by the behaviors of the cats whether or not the new cat can be transferred into the group enclosure without risk of fighting. wellsocialized kittens and juvenile cats frequently adapt readily to group accommodations, and prolonged introductions may not be necessary unless they are shy or undersocialized. in established groups of cats, the introduction or removal of individuals will require a period of adjustment and may result in signs of social stress for members of the colony. these signs usually subside once a new social hierarchy and territorial limits (usually favored resting places) are established. in some cases, arrangement of incompatible cats, even within visible distance of one another, may create substantial anxiety, necessitating rearrangement (figure 46-26) . in the case of animal shelters, where population interchange is high, it is generally not feasible to maintain consistent groupings of cats. this underscores the absolute necessity of careful selection and compatibility matching, as well as maintaining a variety of housing styles. even in modestly populated, carefully introduced, environmentally enriched colonies, behavior problems may occur. for this reason, some facilities elect to use an "all in-all out" approach to avoid repeated introductions of new cats into stable groups. in animal shelters, bonded pairs and family groupings of cats frequently enter the shelter together and are usually perfect choices for co-housing. 46 because cats do have strong preferences for new roommates, caregivers must expect to find many that are incompatible as roommates. if only one or two cats are responsible for social destabilization of a group, they can acknowledging that many factors influence the spatial needs of cats, including the overall quality of the environment as well as the relationships of the individual animals. 59 in sanctuary and laboratory situations where cats are housed for months to years in stable colonies, larger groupings of cats may be feasible, provided ample space is available. 46 housing arrangements can also be created in which individual enclosures are maintained within a colony room. in this case, cats could be allowed to wander and interact freely in the colony room by day but be confined to their respective enclosures at night, for pair-housing of two adult cats. note the multiple separate areas for resting, perching, hiding, feeding, eliminating, scratching, and playing. b, cats enjoy the increased behavioral options provided by run-style housing. should exceed the number of cats and should be arranged in as many locations within the enclosure as possible. open single perches should be separated by at least 0.6 m (2 ft) or staggered at different heights to ensure adequate separation, while larger perches should be available for cats who choose to rest together in close proximity. many cats enjoy hammock-style perches or semienclosed box-style perches where they can hide. if there are not enough comfortable, desirable resting and hiding places, cats may choose to lie in litter boxes. comfortable bedding (that is either disposable or can be easily laundered) should be provided. not only do cats demonstrate preferences for resting on soft surfaces, they experience longer periods of normal deep sleep with soft bedding. 23 the environmental temperature should be kept comfortable and constant, and living quarters should be well ventilated, without drafts. by changing location within the colony (e.g., from the cooler surface of the floor to a sunny window), cats should be able to choose the environmental condition they prefer (figure 46-27) . 46 in colony rooms, installation of stairs, shelves, and walkways are ideal for increasing the use of vertical space (figure 46-28) . in larger rooms, installation of freestanding towers provides additional living and activity space and contributes to functionally reducing overcrowding (figure 46-29) . depending on the setting, it may not be desirable for cats to access areas above the level of an arm's reach so that cleaning is easy and cats can be easily retrieved from the highest perches if needed. colony room design should also ensure that cats cannot easily escape. in some cases, constructing a foyer at the entrance to the room will be necessary to minimize the risk of escape when the room is entered (figure 46-30) . in addition, ceilings should be constructed of solid surfaces, because cats can easily dislodge the usually be reassigned to another colony, because it is often the social grouping, not the individual, that is the problem. if a cat shows persistent incompatibility with other cats, he or she should be housed singly. studies indicate that cats that fight at the time of initial introduction are nearly 40 times more likely to continue fighting in the following weeks and months. 61 if overt fighting occurs, cats should be permanently separated. cohousing of incompatible cats or cats that fight is unacceptable. the success of group housing depends not only on selection of compatible cats and the size of the enclosure but also on the quality of the environment.* a variety of elevated resting perches and hiding boxes should be provided to increase the size and complexity of the enclosure and to separate it into different functional areas, allowing a variety of behavioral choices. the physical environment should include opportunities for hiding, playing, scratching, climbing, resting, feeding, and eliminating. whenever possible, a minimum of 1 litter box and 1 food and water bowl should be provided per 2 to 3 cats and arranged in different locations of the colony space, taking care to separate food and water from litter by at least 0.6 m (2 ft). in addition, placement should allow cats to access each resource from more than one side, whenever possible, without blocking access to doorways. litter boxes should not be covered, to allow easy access and to prevent entrapment or ambush by other cats. the number of resting boards and perches the importance of a cat-savvy staff that enjoys working with cats cannot be overemphasized. animal care staff must be willing to spend quality time interacting with cats to assure socialization and tractability. 45, 46 whenever possible, caregivers should be assigned to care for the same cats on a regular basis so that they become aware of the personality of each individual cat, which is necessary for detection of health problems, incompatibilities between cats, and, in the case of breeding colonies, estrous cycling. this is also important, because not all cats uniformly enjoy human companionship and will be more likely to be stressed by the presence of different caregivers, rather than becoming familiar and more at ease with one. in general, regular daily contact and socialization is essential to ensure that cats are docile, easy to work with, and have no fear of humans. caregivers should schedule time each day to interact with "their" cats aside from the activities of feeding and cleaning. some cats may prefer to be petted and handled, while others prefer to interact with caregivers by playing with toys (figure 46-32) . in particular, human contact is essential for proper socialization of young kittens. a sensitive period of socialization occurs during the development of all infant animals, during which social attachments to members of the same species and other species form easily and rapidly. in kittens, the sensitive period of socialization occurs between 2 and 7 weeks of age, and cats not properly socialized to humans during this time may never permit handling. 75, 105 beginning shortly after birth, kittens should be handled daily, talked to in a soothing panels typically used for dropped ceilings, and escape into the rafters (figure 46-31) . 46 in addition to contact with conspecifics, cats must be afforded time for pleasant daily contact with human caregivers. as previously discussed, daily social contact and exercise sessions with humans are especially important for individually caged cats. although social contact is usually highly desirable, it is not invariably pleasant for all cats. personality, socialization, previous experience, and familiarity contribute to whether or not social interactions are perceived as pleasurable, stressful, or somewhere in between. 46,77 a b voice, gently petted, and held. interactions should include play (stimulated with toys) as the kittens become ambulatory. for kittens housed in a shelter, socialization must always be balanced with infectious disease control, and caregivers should take precautions accordingly. other forms of stimulation, including those that engage the various senses, are important methods of enriching the living environment by promoting healthy mental and physical activity. for singly housed cats and longterm residents, appropriate levels of additional enrichment should be provided on a daily basis. the provision of birdfeeders, gardens, or other interesting stimuli in the external environment can enhance the internal environment of the colony. resting perches in view of windows or other pleasant areas of the facility are especially desirable. other novel and enriching visual stimuli include cat-proof aquariums with fish, water fountains, bubbles, perpetual motion devices, and videos especially designed for cats (figures 46-33 to . 32 a radio playing soft, low music in the room provides a welcome distraction and important source of play items that stimulate prey drive and physical activity, such as plastic balls, rings, hanging ropes, springmounted toys, plastic wands, and catnip toys, should also be provided but must be either sanitizable or disposable. empty cardboard boxes and paper bags are inexpensive, disposable, and stimulate exploration and play behavior as well as scratching. cats tend to be most stimulated by active toys, including wiggling ropes, wands with feathers, kitty fishing poles, and toys that can be slid or rolled to chase. 81 many cats enjoy chasing stimulation. in addition, it may help to habituate cats to human voices and prevent them from being startled by loud noises. most caregivers also enjoy listening to the radio, and happy caregivers create a relaxed environment. 45 the provision of scratching boards is especially important, and a variety of sturdy surfaces, both horizontal and vertical, should be provided for scratching. sisal rope, the backs of carpet squares, and corrugated cardboard are all useful (figure 46-36) . many cats like to smell and chew grass, and containers of cat grass or catnip can be introduced for brief periods to stimulate activity (figure 46-37) . providing novel sources of food is another important source of stimulation and can be easily accomplished by hiding food in commercially available food-puzzle toys or in cardboard boxes or similar items with holes such that the cat has to work to extract pieces of food ( figure 46-38) . 46, 102 positive reinforcement-based training obedience training using clickers with food rewards is an excellent form of enrichment, combining social contact with caregivers together with both mental and physical stimulation. positive reinforcement training using a target stick is a powerful tool for teaching shy cats to approach the front of an enclosure. teaching cats awaiting adoption to perform tricks is not only stimulating for them, but it often makes them more attractive to potential adopters (figure 46-39) . a cardboard tube, and a plastic container with holes). treats are hidden inside, and they will have to work to extract pieces of food. novel feeding is an excellent source of enrichment for cats that are housed long term. (e.g., feeding, cleaning, enrichment activities), and unpredictable caregiving has been shown to dramatically increase stress. 14 if events that are perceived as stressful (such as cleaning time) occur on a predictable schedule, cats learn that a predictable period of calm and comfort will always occur in between. cats also respond to positive experiences in their daily routines. for example, feeding and playtime may be greatly anticipated; thus scheduling positive daily events (e.g., a treat at 3:00 pm every day) should also be a priority. erratic periods of light and darkness are also known to be significant sources of stress for cats. animals possess natural circadian rhythms and irregular or continuous patterns of light or darkness are inherently stressful. lighting should be maintained on a regular the beams of laser pointers, small flashlights, or suspended rotating disco balls. commercially available electronic toys that stimulate play are especially useful in long-term settings (figure 46-40) . varied toys should be substituted regularly to ensure continued interest. 46 in some climates, cats may be housed comfortably in outdoor enclosures where fresh air, sunshine, and other stimuli can help to create a healthy environment ( figures 46-41 and 46-42) . 46, 60, 71 when indoor group enclosures connect to outdoor enclosures, it is important to have ample space for passage between them (e.g., more than one doorway) so that cats can pass freely. cats will also benefit greatly from consistent daily routines of care. they become entrained to schedules of care adoption, or euthanasia (when no other options exist) may be necessary to ensure cat welfare. achieving population wellness requires a healthy environment. thus the clinician's final task in creating a population wellness program is to develop tailored schedule, with lights on by day and off by night. whenever possible, full-spectrum and/or natural lighting is ideal. housed cats require active daily monitoring by staff trained to recognize signs of stress and social conflict. to the inexperienced observer, such signs may appear subtle (figure 46 -43) . it is often the absence of normal behaviors (such as engaging in grooming or exercise) or subtle social signals (such as covert guarding of resources or dominant staring) that signify problems. careful observers will note these behaviors and respond accordingly to ensure that stress or conflicts do not persist. when cats are well adjusted and housing arrangements meet their behavioral needs, they display a wide variety of normal behaviors, including a good appetite and activity level, sociability, grooming, appropriate play behavior, and restful sleeping (figure 46-44) . 44, 46 ultimately, the success of adaptation of cats to a new environment will depend on both the quality of the environment and the adaptive capacity of the individual. although most adapt to new environments with time, some never adjust and remain stressed indefinitely, ultimately resulting in decline of physical as well as emotional health. when cats fail to adjust to their environment and remain markedly stressed and fearful despite appropriate behavioral care, every effort must be made to prevent long-term stays. depending on the circumstances, transfer to another colony room, foster care, a shelter will result in saving more lives. to the contrary, euthanasia rates are highly correlated to intake rates, regardless of the number of animals that a facility houses. in many instances, keeping more animals in the shelter may actually reduce the organization's ability to help animals, because time and resources are tied up caring for a crowded, stressed population, rather than focusing on adoption or other positive outcomes. in shelter medicine, the term population management is used to refer to an active process of planning, ongoing daily evaluation, and responding to changing conditions as an organization cares for multiple animals. 7, 88 the major goal of population management is to minimize the amount of time any individual animal spends confined in the shelter, while maximizing the organization's lifesaving capacity. moving animals through the system efficiently is the foundation of effective population management. to move animals through the shelter more quickly, delays in decision making and the completion of procedures (e.g., intake processing, transfer from holding to adoption areas, spay/neuter surgery) must be eliminated or minimized whenever possible. in openadmission shelters, even delays of 1 to 2 days can have a dramatic effect on the shelter's daily census, particularly for shelters handling thousands of animals per year. this, in turn, affects the ability to provide adequate care. it is important to recognize that effective population management does not change the final disposition of an animal. it does mean that determinations are made as soon as possible, which serves both the individual animal as well as the population as a whole. for wellness programs to be effective, a clean and sanitary environment must be maintained. not only does this promote cat and human health, but it also promotes staff pride as well as public support. in addition to protocols for routine daily cleaning and disinfection procedures, protocols should be in place for periodic deep cleaning and disinfection as well as procedures to be used in the event of disease outbreaks. when crafting protocols, it is important to recognize that cleaning and disinfection are two separate processes. the cleaning process involves the removal of gross wastes and organic debris (including nonvisible films) through the use of detergents, degreasers, and physical action. although this process should result in a visibly clean surface, it does not necessarily remove all of the potentially harmful infectious agents that may be present. disinfection is the process that will destroy most of these agents, but it cannot be accomplished until surfaces have been adequately cleaned. 27 disinfection is usually accomplished through the application of chemical compounds or disinfectants. the most commonly used of these are reviewed in protocols focused on optimizing environmental conditions that favor cat health. once again, all essential elements as noted should be addressed. perhaps the most critical aspect of environmental management is to ensure a modest population density. high population density increases opportunities for introduction of infectious disease while increasing the contact rate among members of a group. both the number of asymptomatic carriers of disease, such as those with upper respiratory infection, as well as susceptible cats in a given group are likely to increase, enhancing the odds of disease transmission among group members through both direct contact as well as fomites. in addition, crowding also increases the magnitude of many environmental stressors (e.g., noise levels, air contaminants) and compromises animal husbandry, all of which induce unnecessary stress and further inflate the risk of disease in the population. indeed, crowding is one of the most potent stressors recognized in housed animals. 77 although adequate space for animals is essential, it is crucial to recognize that crowding is not solely dependent on the amount of available space. it is also a function of the organization's ability to provide proper care that maintains animal health and well-being. every organization has a limit to the number of animals for which it can provide proper care. when more animals are housed than can be properly cared for within the organization's capacity, caregivers become overwhelmed, and animal care is further compromised. 7, 54, 88 in animal shelters, crowding may also negatively impact adoption rates, because potential adopters often find crowded environments to be overwhelming and uninviting. if disease spread results as a consequence of the environmental conditions, animal adoptions may be further disrupted. although unexpected shelter intake may occasionally result in temporary crowding, a good wellness program dictates that protocols must be in place to alleviate crowding and maintain a modestly populated environment for the health and protection of the animals and staff. 41 regardless of the setting, facilities must limit the number of animals housed to the number for which they can provide proper space and care. there are three basic methods of reducing crowding: (1) limiting the admission (or births) of new animals into a population, (2) increasing release of animals from a population, and (3) euthanasia. in animal shelters, management practices that minimize each animal's length of stay and programs that speed or increase adoption, owner reunification, or transfer (e.g., to rescue or foster care) help to minimize crowding and maximize the number of animals that an organization can serve. it is a common misperception that housing more animals in â�¢ although commonly used, they must be applied to clean hands and allowed 20 seconds of contact time to be effective. â�¢ they are highly effective against bacteria, but have only moderate activity against viral agents, including feline calicivirus (fcv). â�¢ they should not be used as a substitute for hand washing or the use of gloves. â�¢ chlorhexidine is the most commonly used biguanide and is relatively expensive. its major use is as a surgical preparation agent. â�¢ although biguanide compounds have broad antibacterial activity, they have limited efficacy against viruses and are ineffective against nonenveloped viruses, such as panleukopenia and fcv. therefore they are not recommended as general-purpose environmental disinfectants. â�¢ household bleach (5.25% sodium hypochlorite) is the most commonly used chlorine compound and is an excellent, safe, and highly cost-effective disinfectant when used correctly. â�¢ at a dilution of 1 : 32, bleach is highly effective against bacteria and viruses, including nonenveloped viruses, such as panleukopenia and fcv. â�¢ solutions must be made fresh daily and stored in opaque containers, because bleach is highly unstable once mixed with water and degrades in the presence of ultraviolet light. â�¢ surfaces must be thoroughly cleaned with a detergent, rinsed, and dried prior to the application of bleach, because it is ineffective in the presence of detergents and organic material. â�¢ proper disinfection requires 10 minutes of contact time with a bleach solution. â�¢ although bleach is not effective when mixed with detergents, it can be safely and effectively mixed with quaternary ammonium compounds, which do provide some cleaning activity. 101 therefore this combination can be used for cleaning and disinfection, provided gross organic material is first removed and adequate contact time is allowed. the addition of bleach improves the disinfection properties of the solution, making it effective against nonenveloped viruses, including panleukopenia and fcv. 101 â�¢ concentrations stronger than a 1 : 32 dilution can result in respiratory irritation for both animals and people, as well as increased facility corrosion, and are therefore not recommended for routine use. â�¢ at a dilution of 1 : 10, bleach will destroy dermatophyte spores. however, cats must be removed from the environment prior to application of this concentration. â�¢ the use of calcium hypochlorite (wysiwash, st. cloud, fla.) is becoming more common and offers the potential advantages of reduced contact time and a neutral ph, which prevents corrosion. 108 oxidizing agents quarantine involves the holding of healthy-appearing animals. it is most useful when animals enter a closed population to ensure that they are not incubating disease when they are introduced into the general population. quarantine areas, with rigid biosecurity procedures in place, should be used to segregate healthy animals for observation. the use of such areas not only allows apparently healthy animals to be observed for developing signs of infectious disease, but it also allows time for response to vaccination in a highly biosecure environment where exposure risks are minimized. the use of quarantine is a mainstay of effective infectious disease control programs and is intended to prevent the introduction of disease into a population. it should be used whenever it is feasible to implement effectively, such as in a laboratory setting, a private cattery, or a low-volume, limited-admission sanctuary setting. however, quarantine practices are not effective in most animal shelters, because the high volume and turnover of animals precludes proper implementation of a true quarantine where an "all in-all out" system is used. 41 instead, incoming animals are usually added to the "quarantine group" on a daily basis, effectively defeating the purpose of true quarantine and simply prolonging the animal's stay. this is especially concerning given the fact that a cat's length of stay in a shelter is a major risk factor for development of upper respiratory infection. 26 for this reason, the use of quarantine is not recommended in most shelter settings. instead, high biosecurity areas are recommended for housing the most susceptible animals (e.g., kittens less than 4 to 5 months of age). on the other hand, quarantine is warranted when a serious disease is discovered in a shelter population. if healthyappearing animals are exposed during a serious outbreak, quarantine procedures should be used to stop the movement of animals and prevent further spread of disease. if possible, temporary closure to admittance is also recommended. quarantine may also be required in bite cases to ensure compliance with state rabies laws. 41 the particular population setting will guide the clinician's determination regarding implementation and length of quarantine, if any. a 14-day quarantine is sufficient to determine that cats are not incubating many common infectious diseases, including feline panleukopenia. however, other diseases, including feline leukemia virus and dermatophytosis, can have longer incubation periods and will therefore require a longer quarantine period. 63, 82 in breeding colonies, early weaning and quarantine have been advocated to prevent infection of kittens with feline coronavirus. 1 pregnant queens are isolated, and product selection should take into consideration the conditions present in a given environment (e.g., the type of surface and the presence of organic matter) and the compound's activity against the pathogens for which the animals are at greatest risk. the nonenveloped viruses, panleukopenia and feline calicivirus, are of particular concern. it is important to note that despite product label claims to the contrary, multiple independent studies have consistently shown that quaternary ammonium disinfectants do not reliably inactivate these important feline pathogens. 31, 34, 56 in addition to selecting effective agents, ensuring adequate contact time followed by thorough drying of surfaces is essential for achieving proper disinfection. protocols should include detailed methods for achieving both cleaning and disinfection. when performed properly and regularly, these practices decrease both the dose and duration of exposure to infectious agents. box 46-10 outlines essential considerations for the development of cleaning and disinfection protocols. segregation refers to the separation of animals from the main group or into subpopulations as necessary to promote health. segregation of cats by physical and behavioral health status is essential for infectious disease control, stress reduction, and safety. 41 in animal shelters, segregation may also be required to ensure compliance with animal control procedures as prescribed by state or local ordinances. depending on the setting, consideration should be given to separating cats by gender and reproductive status (e.g., intact, neutered, in heat, pregnant, nursing), physical and behavioral health status (e.g., apparently healthy, signs of contagious disease, reactive, feral), and life stage. a variety of separate areas will be necessary, depending on the needs of the given population and the context of the setting. the wellness program should define these areas in order to optimize cat health, while providing for the necessary functions of the facility. depending on the setting, consideration should be given to establishing areas for quarantine, isolation, general holding, adoption, and long-term housing, as well as to tailoring these by life stage. 106 for example, the very young and very old typically require more specialized care than healthy juveniles and adults. kittens less than 4 to 5 months of age are particularly susceptible to infectious disease, and extra care must be taken to heighten biosecurity and limit their exposure. in particular, geriatric cats require comfortable, quiet quarters with careful attention to stress reduction 91 (e.g., the provision of a secure hiding place and a dedicated caregiver to enhance bonding and comfort). if cats are used for breeding, functional areas will be required to facilitate mating, queening, and kitten care. 45 and, as previously â�¢ staff must wear personal protective equipment, as necessary, to prevent exposure to chemicals and/or pathogens. â�¢ thorough cleaning and disinfection of enclosures should occur between occupants and as part of periodic deep cleaning procedures. â�¢ cats must be removed from enclosures during these procedures. â�¢ "spot cleaning" is generally sufficient for apparently healthy cats that will continue to occupy the same enclosure. â�¢ the cat remains in the enclosure while it is cleaned and soiled material is removed. â�¢ this method is often less stressful for cats (see figure 46 -8). â�¢ separate staff should clean and care for animals in areas with highly susceptible or sick animals, whenever possible. â�¢ at minimum, attention should be given to the order of cleaning. â�¢ the least contaminated areas should be cleaned before those that are the most contaminated. kittens are weaned as early as possible (e.g., 4 to 5 weeks of age) and placed in strict quarantine. in this manner, as maternal antibody wanes and kittens become susceptible to the virus, exposure and infection are prevented. however, it is important to note that the level of biosecurity required for success is difficult to achieve. furthermore, eventual exposure and infection are highly likely because of the ubiquitous nature of coronaviruses. 93 in addition, because of the importance of the mother-kitten relationship to normal social and emotional development, this management practice may not always be desirable. healthy environmental conditions in isolation areas promote recovery, and their importance cannot be overemphasized. 43 crowding, noise, and stress must be avoided, and facilities must be easy to clean and disinfect. room temperature should be warm and comfortable with good air quality. windows are ideal, because natural sunlight is always beneficial to animal health and healing. strict biosecurity in quarantine and isolation areas, with attention to traffic patterns and the use of protective clothing, such as gowns and shoe covers, is essential. footbaths are insufficient to prevent transfer of infectious agents on shoes. this is because disinfectants typically require 10 minutes of contact time and may be poorly effective in the presence of organic debris. in fact, footbaths may even contribute to the spread of disease. dedicated boots or shoe covers should be used when entering contaminated areas. 84, 104 in addition, separate, designated staff should care for animals in high biosecurity areas whenever possible. by design, traffic patterns should move from the healthiest and most susceptible groupings to the least susceptible, and finally to isolation areas housing sick animals. observation windows and signage are useful to reduce traffic flow into high-risk areas. staff hygiene is extremely important, and the importance of diligent hand washing cannot be overemphasized. where space or facilities are not available, foster care may represent a viable and medically sound option for quarantine or isolation in some settings. for instance, in animal shelters, foster care is particularly useful for the care of preweaning-age kittens. foster homes must be monitored to ensure that cats receive proper care and that resident animals are protected from disease exposure. 41 in addition to ensuring proper population density, segregation, and sanitation procedures, there are several other essential aspects of facility operations that must be incorporated into a population wellness program. these include heating, ventilation, and air conditioning (hvac) considerations, noise and pest control, general facility maintenance, and staff training. 41 extremes or fluctuations in temperature and humidity, as well as poor ventilation and air quality, can compromise animal health. poor ventilation and high humidity favor the accumulation of infectious agents, while dust and fumes may be irritating to the respiratory tract. many cats are particularly sensitive to drafts and chilling, both of which can predispose to upper respiratory general holding, housing, adoption, and other areas in many settings, general holding areas are used for housing healthy juvenile and adult cats at intake. in animal shelters, it is important to consider that length of stay is associated with an increased risk of feline upper respiratory disease and that vaccination against core diseases often rapidly confers immunity. for these reasons, holding periods should be minimized whenever possible. in some cases, holding times will be influenced by legally required holding periods prescribed by state laws that allow owners a chance to reclaim lost pets. legal holding periods are usually not required for owner-relinquished pets and preweaning-age animals, but a brief medical hold (e.g., 1 to 2 days) for evaluation and triage is usually warranted. regardless, management practices that reduce length of stay are generally best for population health in a shelter setting. immunity is often strengthened with time through a combination of both active and passive immunity resulting from vaccination and exposure. upper respiratory disease is often endemic in cat populations, and in open populations, constant introduction of large numbers of carriers and susceptible cats make exposure likely. as length of stay increases, many cats develop and recover from respiratory disease. as animals acclimate to their environment and gain immunity, less stringent biosecurity requirements may be required for long-term housing areas, depending on the particular setting. in animal shelters, the public is usually allowed to interact with cats in adoption areas, which is another management consideration. isolation areas are used to segregate sick animals from the general population. immediate isolation of animals with signs of infectious disease is critical to effective control. isolation should be targeted by age and disease. for example, separate isolation areas should be available for cats and kittens with respiratory disease and those with gastrointestinal disease, whenever possible. in populations where upper respiratory infection is problematic, having two isolation areas for cats with respiratory infections is ideal: one area for those cats with moderate to severe signs that will require more intensive monitoring and treatment, and a separate area for those cats with only mild clinical signs and those that have been treated and are nearly recovered. 43 when mildly symptomatic cats can be housed separately from those that are very ill, staff compliance with isolation procedures are often improved. cats with non-infectious conditions should also be housed in separate areas for treatment, and, in some cases, housing in the general population is appropriate. prevents exchange of air among them and is recommended, because air pressure gradients that recirculate or cause exchange of air between rooms have been associated with the spread of disease by aerosols. when applying these standard recommendations to a particular setting, there are some practical considerations that should be taken into account. 41 first, even when ventilation systems provide 10 to 15 room air changes per hour, airflow may be restricted inside of cages or other enclosures within the room. in other words, the body of the room may be well ventilated, yet inside the cages, the air may remain relatively stagnant. in this case, ventilation may be improved by altering the housing design or arrangement; for example, the use of flow-through cages, runs, free-range rooms, or outdoor access may result in improved air quality. when considering the recommendations for 100% fresh, nonrecycled air with separate ventilation systems in various areas of the facility, consideration should be given to the fact that respiratory pathogens in cats are not aerosolized because of the cats' small tidal volume. 36 although droplet transmission is possible, droplet spray does not extend more than 4 feet, and most transmission of respiratory disease in cats is through direct contact with infected cats, carriers, or fomites. 94 although this recommendation seems prudent to consider, especially for isolation areas, it is very expensive to install and operate this type of ventilation system throughout a facility, especially in very cold or very hot climates. if air quality remains good and the facility maintains effective comprehensive wellness protocols, it may not be necessary for animal health. 41 more research is needed on the impact of such air exchange, but in the meantime, the author recommends consulting with an hvac specialist to establish effective and efficient settings to suit the specific needs of the given population. in addition to ensuring good ventilation, it is imperative to use other strategies for maintaining good air quality, including regular maintenance of filters, control of dust and dander through routine vacuuming and periodic deep cleaning, and the use of dust-free litter. 52 noise control is another important consideration. it is crucial to keep cats out of auditory range of dogs, because many are profoundly stressed by the sounds of barking. also, staff should be trained to reduce or avoid other sources of noise whenever possible. the installation of sound-proofing systems may be necessary for noise abatement and stress reduction. routine pest control may also be required, depending on the setting. it may be necessary to treat the environment for fleas, ticks, or other insects or ectoparasites. products used to treat the environment must be selected carefully, because cats are extremely sensitive to the toxic effects of many insecticides. 78 in many instances, it will infection. heating, ventilation, and air conditioning (hvac) specialists are uniquely qualified to help establish and maintain the environmental conditions required for animal health. when facilities are designed specifically for housing animals, these specialists should be consulted beforehand to ensure installation of the most effective and efficient systems possible. in reality, many facilities that house cats, including private catteries and shelters, among others, were not originally built for this purpose. retrofitting existing facilities with the ideal hvac system is often neither logistically nor financially feasible. regardless, consultation with hvac specialists is recommended in order to maximize the potential of the facility's existing system. the recommended temperature range for cats is between 18â° c and 29â° c (64â° f and 84â° f) with a temperature setting in the low-to mid-20s celsius (70s fahrenheit) being typical. 54 the temperature setting should be determined according to the specific animals' needs. for example, neonatal kittens are more susceptible to hypothermia and generally require warmer temperatures than healthy adult cats. the location of the cats may also be a consideration. for example, enclosures located closer to floor level are often a few degrees cooler than those at higher levels. the exact temperature setting may also vary somewhat based on the season of the year. for instance, power companies typically recommend keeping the temperature between 25â° c and 26â° c (78â° f and 79â° f) during hot weather to conserve electricity and reduce power bills. 41 laboratory guidelines recommend 30% to 70% humidity for cats. 54 higher humidity (e.g., 70%) may be advantageous in areas housing cats with respiratory disease because moist air may be beneficial to the respiratory passages, whereas lower humidity (e.g., 40% to 50%) may be beneficial in other areas in order to reduce survival of infectious agents in the environment. although the range considered acceptable is large, a given room should have a relatively constant humidity (i.e., it should not have large fluctuations). hosing or even mopping a room usually results in temporary spikes in humidity, but these will be short lived in a well-ventilated room. 41 adequate ventilation is crucial for good air quality. this is especially important for cats, because good air quality is essential for control of upper respiratory disease. ten to fifteen air changes per hour is the standard recommendation for an animal room, but more or less airflow may be acceptable or necessary depending on the housing density. 54 theoretically, the best case scenario, and what is typical in laboratory animal settings, is for the hvac system to allow for 100% fresh (e.g., nonrecycled) air in each room so that the air entering a given room is exhausted out of the building and not recirculated to another room. maintaining separate ventilation systems for various rooms or areas of a facility ensure both the physical and behavioral health of cats, as well as a healthy environment. a proactive, holistic approach coupled with compassion is required. when these are combined with careful attention to the unique needs and stress responses of cats, the result will be "healthy, happy cats." be necessary to remove cats during their application and only return them to the environment once it is thoroughly dried and ventilated. if rodent control is necessary, the use of rodenticide baits should be avoided, because cats can be exposed even if the bait is not within their reach. rodents that have ingested the poisonous bait may enter an animal enclosure and, if the animal ingests the rodent, the poison will affect that animal. humane live traps can be used to capture rodents for removal from a facility. food containers should be kept tightly sealed, and clutter should be minimized to discourage pests in the environment. general building maintenance procedures (e.g., regular inspection and servicing with repairs as needed) are also important considerations for the maintenance of a healthy environment. for example, periodic resealing of floors may be required as well as maintenance of plumbing fixtures to repair leaks or other problems. developing and following written standard operating procedures and daily, weekly, monthly, and quarterly checklists will ensure that systematic schedules of maintenance are carried out in a timely fashion. 41 regular staff training is essential for implementing effective population wellness programs. simply stated, staff caring for animals must be qualified to do so. to a large extent, their knowledge and skill will determine the success or failure of the wellness program. embracing a culture of training promotes high-quality animal care as well as human safety. both formal and on-thejob training should be provided to ensure that a staff has the knowledge and skills required to perform their assigned tasks. protocols should be established for all levels of training, and a system should be in place to ensure proficiency. staff training should be documented, and continuing education should be provided to maintain and improve skills. finally, training must include the provision of information about zoonoses and other occupational health and safety considerations. 54 regardless of the setting, maintaining population health is essential for animal welfare as well as to meet the goals of the particular population. population health depends on implementation of comprehensive wellness protocols, systematic surveillance, and excellent management. facilities must establish goals for animal health, and wellness protocols must be regularly evaluated and revised to ensure that these goals are met. the bulk of efforts must focus on preventive strategies to control of feline coronavirus in breeding catteries by serotesting, isolation, and early weaning guidelines for the diagnosis, treatment and prevention of heartworm (dirofilaria immitis) infection in cats american society for the prevention of cruelty to animals (aspca): mission possible, comfy cats. shelter temperament evaluations for cats microchipping of animals association of shelter veterinarians (asv): board position statement on cats who test positive for felv and fiv association of shelter veterinarians (asv): board position statement on veterinary supervision in animal shelters association of shelter veterinarians (asv): standards of care for animal shelters gender differences in the social behavior of the neutered indoor-only domestic cat beaver bv: fractious cats and feline aggression a game of cat and house: spatial patterns and behavior of 14 domestic cats (felis catus) in the home affiliative behaviours of related and unrelated pairs of cats in catteries: a preliminary report immunisation against panleukopenia: early development of immunity cvma): a code of practice for canadian cattery operations behavioral and physiological correlates of stress in laboratory cats the cat: its behavior, nutrition and health cfa cattery standard minimum requirements feline heartworm (dirofilaria immitis) infection: a statistical elaboration of the duration of the infection and life expectancy in asymptomatic cats environmental factors in infectious disease immunoprophylaxis and immunotherapy infectious disease management in animal shelters scaredy cat or feral cat? animal sheltering the lowdown on upper respiratory infections in cats in griffin b: a model of non-invasive monitoring of feline ovarian function in the domestic cat domestic cats as laboratory animals recognition and management of stress in housed cats stress and immunity: a unifying concept effects of a synthetic facial pheromone on behavior of cats small animal clinical nutrition housing design and husbandry management to minimize transmission of disease in multi-cat facilities implementing a population health plan in an animal shelter: goal setting, data collection and monitoring, and policy development feline infectious disease control in shelters commission on life sciences, national research council: guide for care and use of laboratory animals questions and answers on the effects of surgically neutering dogs and cats virucidal efficacy of the newer quaternary ammonium compounds stress and adaptation of cats (felis silvestris catus) housed singly, in pairs, and in groups in boarding catteries effects of density and cage size on stress in domestic cats (felis silvestris catus) housed in animal shelters and boarding catteries socialization and stress in cats (felis silvestris catus) housed singly and in groups in animal shelters centers for disease control: healthy pets, healthy people responses of cats to nasal vaccination with a live, modified feline herpesvirus type 1 use of fipronil to treat ear mites in cats counsel of europe: guidelines for accommodation and care of animals. proposed revision to appendix a to the european convention for the protection of vertebrate animals used for experimental and other scientific purposes. minimum cage floor area for cats soft surfaces: a factor in feline psychological well-being social organization in the cat: a modern understanding social behavior and aggressive problems of cats descriptive epidemiology of feline upper respiratory tract disease in an animal shelter sanitation and disinfection all together now: group-housing cats behavioral differences between owner surrender and stray domestic cats after entering an animal shelter a clinical trial of intranasal and subcutaneous vaccines to prevent upper respiratory infection in cats at an animal shelter virucidal efficacy of four new disinfectants influence of visual stimulation on the behaviour of cats housed in a rescue shelter feline dental prophylaxis and homecare disinfection efficacy against parvoviruses compared with reference viruses farm animal welfare council: five freedoms feline respiratory disease experimental induction of feline viral rhinotracheitis in fvr-recovered cats evaluation of the efficacy of disinfectant footbaths as used in veterinary hospitals national council on pet population study and policy: the shelter statistics survey animal welfare advisory committee: companion cats code of welfare five key population management factors affecting shelter animal health cat housing in rescue shelters: a welfare comparison between communal and discrete-unit housing recognizing and managing problem behavior in breeding catteries quality of life in long-term confinement maddie's infection control manual for animal shelters a review of feline infectious peritonitis virus observations on the epidemiology and control of viral respiratory disease in cats employee reactions and adjustment to euthanasia related work: identifying turning points through retrospective narratives the 2006 aafp feline vaccine advisory panel report welfare of cats in a quarantine cattery recommendations for the housing of cats in the home, in catteries and animal shelters, in laboratories and in veterinary surgeries comfortable environmentally enriched housing for domestic cats efficacy of fipronil in the treatment of feline cheyletiellosis virucidal disinfectants and feline viruses the influence of food presentation on the behavior of small cats in confined environments validation of a temperament test for domestic cats evaluation of the effects of footwear hygiene protocols on nonspecific bacterial contamination of floor surfaces in an equine hospital the human-cat relationship editor: cattery design: the essential guide to creating your perfect cattery intercat aggression in households following the introduction of a new cat feline leukemia virus and feline immunodeficiency virus american association of feline practitioners feline retrovirus management guidelines intercat aggression: a retrospective study examining types of aggression, sexes of fighting pairs and effectiveness of treatment evaluation of collars and microchips for visual and permanent identification of pet cats characterization of animal with microchips entering shelters in vitro sensitivity of commercial scanners to microchips of various frequencies sensitivity of commercial scanners to microchips of various frequencies implanted in dogs and cats search and identification methods that owners use to find a lost cat provision of environmentally enriched housing for cats environmentally enriched housing for cats when singly housed managing oral health in breeding catteries assessment of stress levels among cats in four shelters enriching the environment of the laboratory cat the impact of paternity and early socialisation on the development of cats' behaviour to people and novel objects quality of life in animals development of a mental wellness program for animals toxicology brief: the 10 most common toxicoses in cats the domestic cat. perspective on the nature and diversity of cats definition of wellness behavioral effects of cage enrichment in single-caged adult cats treatment of dermatophytosis in dogs and cats: review of published studies development of an in vitro, isolated, infected spore testing model for disinfectant testing of microsporum canis isolates quality-of-life assessment in pet dogs aafp-aaha. feline life stage guidelines meet your match and feline-ality adoption program key: cord-318363-1mv5j4w2 authors: zvolensky, michael j.; garey, lorra; rogers, andrew h.; schmidt, norman b.; vujanovic, anka a.; storch, eric a.; buckner, julia d.; paulus, daniel j.; alfano, candice; smits, jasper a.j.; o'cleirigh, conall title: psychological, addictive, and health behavior implications of the covid-19 pandemic date: 2020-08-27 journal: behav res ther doi: 10.1016/j.brat.2020.103715 sha: doc_id: 318363 cord_uid: 1mv5j4w2 • the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing. • an integrative covid-19 stress-based model could be used to guide research focused on the stress-related burden of the pandemic. • this work could provide a theoretical and empirical knowledge base for future pandemics. around some of the most clinically important psychological disorders, addictive behaviors, and health behaviors for well-being. in the first section, we describe the covid-19 implications for mental health focusing on (a) anxiety/stress and mood disturbance, (b) obsessive compulsive symptoms and disorders, and (c) posttraumatic stress. such mental health problems, although certainly not exhaustive of the scope of psychological disorders impacted by covid-19, are some of the most common mental health issues in the general population and are frequently comorbid with chronic illness. in the second section, we focus on addictive behaviors, including (d) tobacco (combustible and electronic), (e) alcohol use and misuse, and (e) cannabis. these forms of drug use represent the most prevalent types of substance use and are frequently associated with chronic illness and premature death. in the third section, we spotlight health behavior and chronic illness by discussing the role of (f) sleep health and behavior, (g) chronic illness using the example of hiv/aids as an illustrative model, and (h) physical activity. health behaviors represent vital targets for the mitigation of covid-related disease and may play a key role in psychological adjustment and recovery. in the final section, we highlight sociocultural factors (e.g., race/ethnicity, economic adversity), developmental considerations, and the role of individual difference factors for psychological, addictive, and health behavior and chronic illness. we conclude by offering an integrative covid-19 stress-based model that could be used to guide research focused on the stress-related burden of the pandemic. fear is an adaptive defense mechanism that is fundamental for survival and involves several psychological and biological processes of preparation for a response to potentially j o u r n a l p r e -p r o o f threatening events. covid-19 represents a true threat, with many unknowns. if you are infected, there is a chance you could die, regardless of your current age, sex, or health status. as such, fear is a natural and adaptive response to this pandemic. on the other hand, every year tens of thousands die from influenza as well as many other preventable or unexpected causes. this raises the key question regarding the degree to which we should be anxious and fearful of . how much anxiety is reasonable? since even basic knowledge about covid-19 is undeveloped, it will be difficult to clearly discriminate between normal, adaptive fear responses and less adaptive responses. that said, such an overarching true threat and the concomitant stressors such as social isolation, economic uncertainty and so forth could in fact recalibrate what is considered a normal level of anxiety in the general population. research has demonstrated that trait levels of anxiety have increased in the us in recent decades, though the cause of such increases is unknown (twenge, 2000) . the covid-19 pandemic is likely to contribute to these basic levels of trait anxiety, thus creating a "new normal" level of anxiety. if we consider the likely general increase in anxiety and stress in the context of diathesisstress conceptualizations of mental illness, we expect that such a salient and broad reaching stressor to increase the incidence of pathological anxiety. anxiety conditions are already highly prevalent (bandelow & michaelis, 2015) , and we may see an increased incidence of anxiety psychopathology if the pandemic serves to push vulnerable individuals toward the expression of maladaptive levels of anxiety. moreover, those with preexisting conditions are likely to have their symptoms intensify. one could further speculate that forms of pathological anxiety will increase. first responders and hospital personnel, particularly in affected areas are already showing troubling signs of stress and psychopathology (joob & wiwanitkit, 2020) . it is highly j o u r n a l p r e -p r o o f likely that we will see increased rates of generalized anxiety and posttraumatic stress related to the pandemic and its sequelae. beyond the somewhat vague notion of covid-19 acting as a stressor to increase both normal and pathological anxiety, it is interesting to consider the specific mechanisms that play a role in this process. there are several well-established parameters that relate to the genesis and maintenance of anxiety that seem highly relevant to the current situation. these processes include perceptions relating to predictability/certainty and controllability of threat (barlow, 2004) . coming across a shark while swimming is quite different from viewing the same shark in an aquarium since a potential threat in the wild is far less predictable or controllable than one in an enclosure. historically, epidemics and pandemics were considered divine punishments that were essentially uncontrollable. although medical understanding of pathogens has advanced, globalization now facilitates the spread of pathological agents, which diminishes the degree to which we can control them. similarly, naturally occurring mutations and adaptation of viruses ensure that novel pathogens like covid-19 will emerge and spread. these conditions leave us in a state of uncertainty, except that we can be certain that covid-19 and other infectious agents will persist. thus, covid-19 affects many of the core anxiety generating mechanisms since it leads to a sense of diminished predictability and controllability along with increased uncertainty relating to a true threat. ultimately, the covid-19 pandemic creates an ideal environment for the onset, maintenance, and exacerbation of anxiety symptoms and syndromes. the dsm-5 posttraumatic stress disorder (ptsd) criterion a (american psychiatric association [apa] , 2013, p.271) defines trauma as "exposure to actual or threatened death." individuals who are closer to that exposure --providing healthcare to those infected, witnessing j o u r n a l p r e -p r o o f the deleterious and perhaps deadly effects of the virus on patients or loved ones, enduring losses of patients, family, or friends --might experience the crisis as potentially traumatic. people on the frontlines of the pandemic, including healthcare personnel, first responders, grocery store clerks, and other essential workers, encounter the threat of possible exposure to the virus regularly and on an ongoing basis. similarly, incarcerated populations and those who might feel compelled, financially or otherwise, to work in close quarters without adequate personal protective equipment (e.g., factory workers) may be exposed to the covid-19 virus for extended periods without perceived or actual recourse and suffer negative mental health repercussions as a result. covid-19 survivors, particularly those who might have struggled through various medical procedures and prolonged hospitalizations, may emerge with unique or shared constellations of mental health reactions from risk to resilience. additional high-risk groups include healthcare professionals or first responders who may have experienced significant moral injuries (jinkerson, 2016; joannou, besemann, & kriellaars, 2017; williamson, stevelink, & greenberg, 2018 ) as a result of making unfathomable decisions on the job (e.g., providing admission or ventilator access to one patient at the sacrifice of another). yet, in addition to considering direct impacts of the novel covid-19 virus on our population, it is imperative to understand the secondary potentially traumatic effects of the pandemic on individuals and communities. the combination of prolonged stress, close quarters, and self-isolation guidelines has increased risk of domestic violence, child abuse, and substance use (abramson, 2020; national institute on drug abuse, 2020; santhanam, 2020; taub, 2020) . indeed, physical and sexual violence may escalate without the regular societal checks provided by employers, schools, and loved ones. furthermore, such violence may stem from and/or intensify more unbridled substance use (carter et al., 2020) emerging from a context where j o u r n a l p r e -p r o o f uncertainty and unpredictability are high, practical stressors (e.g., unemployment, financial stress, food insecurity) may be difficult to problem-solve, and social supports may be distant. furthermore, in this pandemic, issues of grief and loss are inevitably interwoven with those of potential trauma. spiritual and emotional grief processes to honor and emotionally mourn the losses of loved ones may be interrupted by this pandemic, potentially exacerbating or prolonging grief, traumatic bereavement, or ptsd reactions. to understand the effects of covid-19 on the mental health of those who experience it as potentially traumatic, we need to recognize first that the impacts of trauma may not be fully determined nor completely recognizable until after the traumatic stressor has concluded. the covid-19 crisis is going to have a long, yet undetermined course, and thus our ongoing reactions to it are dynamic but indicative of peri-traumatic rather than post-traumatic coping (bell, boden, horwood, & mulder, 2017; lapid pickman, greene, & gelkopf, 2017) . based upon decades of research, we can expect the majority of the population, regardless of level of proximity to or interaction with covid-19, to demonstrate resilience and to recover psychologically in the aftermath of the pandemic (alisic et al., 2014; kilpatrick et al., 2013) . a relative minority, the proportions of which are unknown, may emerge from the crisis with clinical or subclinical ptsd or with exacerbation in pre-existing ptsd symptoms and related mental health conditions (e.g., depression, substance use disorder). women are at heightened risk of ptsd following potentially traumatic events (gaffey et al., 2019; rattel et al., 2019) and racial/ethnic minority populations may be especially impacted due to socioeconomic inequities and health-related disparities with regard to financial security and access to healthcare and treatment (asnaani & hall-clark, 2017; cross et al., 2018; sibrava et al., 2019) . the intersections of trauma and the covid-19 pandemic are complex. many constellations of interweaving risk and protective factors, learning histories, and life circumstances can affect how trauma histories and potentially traumatic experiences during the covid-19 crisis can affect individual journeys of recovery. for example, more unbalanced, negative individual interpretations of the covid-19 crisis and related changes in beliefs about oneself, others, or the world may have lasting deleterious effects (e.g., "i am damaged"; "people cannot be trusted"; "the world is dangerous and unsafe"; beierl, böllinghaus, clark, glucksman, & ehlers, 2019; bernardi & jobson, 2019; köhler, goebel, & pedersen, 2019; losavio, dillon, & resick, 2017; scher, suvak, & resick, 2017) . similarly, avoidance of thoughts or emotions related to the covid-19 crisis may increase the risk of developing ptsd symptoms and/or exacerbating or maintaining pre-existing trauma-related symptoms (e.g., orcutt, reffi, & ellis, 2020) . additional risk factors for the development or exacerbation of ptsd symptoms include a prior history of trauma or mental health disturbances, depressed or anxious mood, significant concurrent life stressors (e.g., financial problems, job loss, relationship stress), low social connectedness or support, sleep disturbance, substance use, and emotional numbing or detachment (colvonen, straus, acheson, & gehrman, 2019; cusack et al., 2019; germain, mckeon, & campbell, 2017; hancock & bryant, 2018; shalev et al., 2019; steenkamp et al., 2017; vujanovic & back, 2019) . navigating the covid-19 crisis requires a tolerance of uncertainty that is challenging for all, but especially trauma survivors who may have endured, sometimes over months or years (e.g., combat, childhood abuse), unfathomable circumstances that were, by definition, unpredictable and uncontrollable (e.g., raines, oglesby, walton, true, & franklin, 2019; vujanovic & zegel, 2020) . undoubtedly, social connection and a sense of community and collectivism, hope, psychological awareness, and healthy coping will j o u r n a l p r e -p r o o f differentiate risk versus resilience trajectories during and after this crisis (bernardi & jobson, 2019; long & gallagher, 2018; thompson, fiorillo, rothbaum, ressler, & michopoulos, 2018) . learning who suffers long-term negative effects of the covid-19 pandemic, why, and under what circumstances will help us to understand how to intervene most effectively to psychologically support trauma survivors in the aftermath of this and future societal crises. indeed, reactions of trauma survivors to the covid-19 crisis are also likely to be as diverse as the traumas and individuals themselves with the possibility of emergent themes. theoretically, individuals with histories of being directly impacted by natural disasters, people recovering from severe medical conditions, and those with histories of imprisonment or captivity may feel especially emotionally reactive to the large community-level impact, the social distancing and quarantining aspects of weathering covid-19, and the continual perceived health threat inherent to the pandemic. individuals with interpersonal trauma histories may experience a solidification or exacerbation of maladaptive beliefs relevant to trust, safety, or power. others may feel increased social detachment or engage in increased harmful, self-injurious, or suicidal behaviors, particularly those with mood or substance use disorders. for some trauma survivors, following social distancing and self-quarantine guidelines may lead to less frequent exposure to trauma-related reminders in the outside world and/or a lower perceived interpersonal threat due to social-isolation, but increased trauma-related avoidance during the covid-19 crisis in turn may exacerbate ptsd symptoms in the long-term. a high-risk subset may emerge who are slow or reluctant to heed public health guidelines due to a reaction against efforts to control, an increased risk-taking propensity, all-or-none thinking, or helplessness resulting from a history punctuated by traumatic, uncontrollable events. this may lead to incessant attempts, by some, to attain perceived control via closely monitoring news, stockpiling food, or supplies, and maintaining constant vigilance. for those affected by trauma prior to and/or during the covid-19 crisis, the current, chronically stressful global atmosphere where uncertainty reigns may feel especially overwhelming. for others, this crisis may foster growth and resilience as they endure and overcome a crisis of epic and unimaginable proportions. obsessive-compulsive disorder (ocd) is a common (1-2% incidence; (nestadt, bienvenu, cai, samuels, & eaton, 1998; ruscio, stein, chiu, & kessler, 2010) , disabling mental health condition characterized by presence of obsessions and/or compulsions (american psychiatric association, 2013; markarian et al., 2010) . symptoms present in a heterogeneous fashion across a number of dimensions, including contamination/cleaning, taboo obsessions (i.e., sexual, aggressive content), symmetry/repeating/ordering, and checking (mckay et al., 2006) . childhood onset occurs in over 50% of cases and symptoms run a chronic course without adequate intervention (pinto, mancebo, eisen, pagano, & rasmussen, 2006) . clinical presentation is further characterized by frequent comorbidity (stein et al., 2019) and variable degrees of insight (hamblin, park, wu, & storch, 2017) . the covid-19 pandemic is likely to have a number of effects on those with ocd, as well as those at risk. this includes the potential for symptom exacerbation and increased incidence of ocd cases, as well as having implications for assessment and treatment post-covid-19. patients with ocd commonly present with contamination obsessions and associated cleaning compulsions (mataix-cols, do rosario-campos, & leckman, 2005; pinto et al., 2006) . some individuals with contamination related ocd have reported that their symptoms have worsened in light of public health recommendations for increased cleaning behaviors (e.g., washing, wearing masks) and other safety behaviors (e.g., social distancing, wearing masks), j o u r n a l p r e -p r o o f which may be difficult for some patients to maintain within recommended guidelines. covid-19 has become a feared outcome for many patients with contamination-related ocd similar to other what has been observed with other infectious diseases (e.g., hiv). outside of contamination-focused symptomology, other obsessive-compulsive symptoms may be affected such as harm obsessions whereby someone fears that they may have unintentionally spread covid-19. stress has an established relationship with worsened obsessive-compulsive symptoms (adams et al., 2018; brander, perez-vigil, larsson, & mataix-cols, 2016) , and availability of coping strategies is taxed for many; this may further impact ocd symptom presentation as well as comorbidity patterns. although systematic data have not been presented, clinical accounts support symptom worsening for some affected individuals while, on balance, many others have not experienced negative symptomatic change. beyond worsening of symptoms in those with ocd, there is the possibility that there will be increased cases in the near future. this may involve those with subclinical symptoms or other risk factors experiencing onset or worsening of symptoms. the behavioral cycle of ocd/anxiety highlights the role of negative reinforcement in which rituals/avoidance are reinforced by distress reduction and creating a cognitive sense of control (i.e., not getting covid-19 is due to compulsions; rector, wilde, & richter, 2017) . in this scenario, a person with or at risk for ocd may engage in rituals/safety behaviors in response to obsessional distress which in turn reduces anxiety and is perceived as reducing the risk. reduction in distress may motivate further safety behaviors which, for some at risk, could begin to exceed recommended guidelines. while ordinary levels of risk have risen requiring increased hygiene, it remains to be seen what happens when risk levels decline. that is, do cleaning behaviors likewise decline or remain at elevated states thereby impacting diagnosis rates? assessment approaches should continue to capture j o u r n a l p r e -p r o o f obsessive-compulsive symptoms that are impairing, distressing and excessive relative to current risk levels and not count symptoms that reflect behaviors consistent with accepted public health standards. there are also treatment implications. the gold standard psychological treatment for adult and childhood ocd is cognitive behavioral therapy with exposure and response prevention (erp; mcguire et al., 2015; olatunji, davis, powers, & smits, 2013) . this treatment involves gradual exposure to triggers that evoke obsessive-compulsive symptoms while refraining from completing rituals or other avoidance behaviors. a core element to this treatment is that exposure to triggers involves exposure to 'ordinary' levels of risk. covid-19 understandably has shaken what is perceived as ordinary; fortunately, adept therapists have shifted their practice to utilize exposures that reflect this new normal such as relying on imaginal exposures or exposures targeting rituals in excess of public health agency recommendations. at the same time, some clinicians have negative attitudes towards exposure (meyer, farrell, kemp, blakey, & deacon, 2014) which is related to reduced practice of this core therapeutic technique (farrell, deacon, kemp, dixon, & sy, 2013) . it will be critical to provide guidelines established by expert erp clinicians for how providers integrate realistic covid-19 concerns into their ongoing practice, as well as that in the future. a concerning possibility is that erp treatment post-covid-19 is diluted by virtue of therapists not practicing exposures to the actual level of risk. cigarette smoking remains the leading cause of preventable death and disability globally. smoking may confer worse covid-19 outcomes given extensive evidence for the negative impact of smoking on lung health and respiratory function (tonnesen, marott, nordestgaard, j o u r n a l p r e -p r o o f bojesen, & lange, 2019). indeed, emerging evidence has identified smoking as a possible risk factor for adverse covid-19 prognosis and disease progression (patanavanich & glantz, 2020; vardavas & nikitara, 2020) . in the largest study of covid-19 patients, 16.9% of severely affected patience were current smokers relative to 11.8% of non-severe patients (guan et al., 2020 ). an inverse pattern emerged with non-smokers such that a greater proportion of nonsevere patients identified as a non-smoker relative to severe patients. moreover, 25.5% of covid-19 patients who either needed mechanical ventilation, were admitted to an intensive care unit, or died from complications related to the disease were current smokers relative to 11.8% of those not experiencing these outcomes. similar disparities in covid-19 severity across smoking status have been observed in other samples (w. j. zhang et al., 2020) . thus, these data, albeit preliminary and limited by sample size, indicate that smoking is a risk factor for covid-19 progression (w. . taking a biological perspective to understand why smokers are more susceptible to severe covid-19 symptoms, recent research has proposed that smoking and covid-19 susceptibility and symptom severity may be related to an upregulation of the angiotensin-converting enzyme-2 (ace2) receptor (brake et al., 2020) . ace2, a membrane-bound aminopeptidase that plays a vital role in cardiovascular and immune systems, is highly expressed in the heart and the lungs (turner, hiscox, & hooper, 2004; wang, luo, chen, chen, & li, 2020) . studies have established that ace2 is a receptor for the covid-19 virus (j. , and greater ace2 gene expression has been observed in smokers compared to non-smokers (brake et al., 2020; cai, 2020; emami, javanmardi, pirbonyeh, & akbari, 2020; tian et al., 2020; wan, shang, graham, baric, & li, 2020; zhao et al., 2020; . the upregulation in ace2 creates an environment that allows greater potential for covid-19 to j o u r n a l p r e -p r o o f infect human cells among smokers through more opportunity to bind to this receptor (olds & kabbani, 2020; zuluaga, montoya-giraldo, & buendia, 2020) . in part, this biological mechanism may help explain observed sex differences in covid-19. specifically, covid-19 symptom severity and mortality rates in china indicate worse outcomes for men than for women, where 52.1% of men and 2.7% of women are current smokers (parascandola & xiao, 2019; sun et al., 2020) . it is possible that the elevated smoking rates among men in china, and therefore greater upregulation in ace2, contributed to significant gender difference in covid-19 incidence and severity (j. . in addition to combustible cigarette smoking, there also is growing concern for the impact of electronic cigarette (e-cigarette) use on covid-19 infection and disease progression (lewis, 2020) . although it is believed that the worldwide distribution and adoption of ecigarettes has the potential to increase population-level vulnerability to respiratory infecting diseases (olds & kabbani, 2020) , such as covid-19, no studies have assessed e-cigarette use among covid-19 patients (farsalinos, barbouni, & niaura, 2020) . given evidence for the impact of various e-cigarette formulations on lung health and functioning (viswam, trotter, burge, & walters, 2018) as well as the fact that most e-cigarette users are former or current combustible cigarette users (mirbolouk et al., 2018) , it is possible that product use will critically impact the course of covid-19 among users. additionally, similar to combustible cigarette use, it has been theorized that e-cigarette use may engage an upregulation in ace2 that parallels that of combustible cigarette use and increases the likelihood of covid-19 infection (brake et al., 2020) . further research on these products and their influence on covid-19 outcomes is urgently needed. a final point to consider is the effect that the covid-19 pandemic itself has on smoking. one of the leading reasons for smoking is stress management (baker, piper, mccarthy, majeskie, & fiore, 2004; garey et al., in press) . the psychological effect of the current global environment, characterized by feelings of fear, uncertainty, isolation, and stress (mertens, gerritsen, salemink, & engelhard, 2020) , coupled with limited availability of adaptive coping tools due to regulations and consequences of covid-19 (i.e., social distancing, financial hardship) likely increases the risk for smoking onset, increased intensity, and relapse (patwardhan, 2020; stubbs et al., 2017) . smoking initiation and severity, in turn, increase susceptibility for covid-19 and worse disease-related outcomes. behavioral scientists must engage in targeted efforts to support current smokers and former smokers in achieving and maintaining cessation during this particularly challenging time. there are promising initial findings from smoking cessation programs implemented in smokers managing other infectious disease that may help guide some of these initiatives . as more is learned about covid-19, it is imperative that health care providers assess smoking (and e-cigarette) use status as well as relapse potential among former users and provide appropriate education and intervention to help mitigate the potential risk of this health behavior on disease infection and course. the (mis)use of alcohol is a leading risk factor for global disease burden and preventable death (degenhardt et al., 2018; organization, 2019) . alarmingly, alcohol use, high-risk drinking, alcohol use disorder (aud), and alcohol-related deaths were increasing before the covid-19 pandemic (grant et al., 2017; white, castle, hingson, & powell, 2020) . despite the widespread belief that moderate alcohol consumption may confer health benefits (diaz et al., 2002; j o u r n a l p r e -p r o o f et al., 2007) , more recent work suggests that any alcohol consumption is associated with health risks (griswold et al., 2018) . in fact, given the immunosuppressing effects of alcohol both generally and in the respiratory system specifically (molina, happel, zhang, kolls, & nelson, 2010; szabo & mandrekar, 2009) , it is germane to consider the role that alcohol consumption, whether chronic or in acute response to the ongoing crisis, may have on contraction of the covid-19 virus. in addition to the direct physiological impact of alcohol consumption on the body, the disinhibiting properties of alcohol (kumar et al., 2009; oscar-berman & marinković, 2007) may put individuals at risk for other risky/poor decisions (george, rogers, & duka, 2005) . for example, those under the influence of alcohol may be more likely to violate social distance protocols, exhibit poor hand washing procedures, or refuse/forget to wear a face covering in public, leading to potential exposure to and/or spreading of the virus. importantly, impulsivity has reciprocal relationships with alcohol such that consumption increases impulsive behaviors and individuals with greater trait impulsivity (mis)use alcohol to a greater extent (dick et al., 2010) . moreover, the effects of impulsivity on alcohol (mis)use can be amplified by other factors, such as stress, to confer greater risk for alcohol (mis)use (fox, bergquist, gu, & sinha, 2010) . it is well-documented that stress, both acute and chronic, is a trigger for alcohol (mis)use (becker, lopez, & doremus-fitzwater, 2011; blaine & sinha, 2017) . the covid-19 pandemic has brought about both acute (e.g., work displacement, limited availability of cleaning supplies) and chronic stress (e.g., financial difficulty, isolation) that likely will contribute to alcohol (mis)use for coping. it also is reasonable to expect that alcohol (mis)use will worsen during the crisis in response to the stress and uncertainty. for example, during the 2008-2009 economic recession, although there was a decrease in prevalence of alcohol use overall (i.e., increase in j o u r n a l p r e -p r o o f abstainers), there was an increase in prevalence of binge drinking (bor, basu, coutts, mckee, & stuckler, 2013) . this suggests that there may be a realignment/concentration of problematic drinking such that a greater segment of those who do consume alcohol may be doing so in a maladaptive or harmful way. although sales to restaurants and events have reduced markedly during the pandemic, sales of online and to-go alcohol have skyrocketed (nielsen, 2020) . given shelter in place orders and limits on socializing, it is possible that greater amounts of alcohol are being consumed at home/solitarily relative to social contexts. solitary drinking can, in some circumstances, lead to greater alcohol consumption than social drinking (kuendig & kuntsche, 2012) and is associated with greater alcohol-related consequences overall (christiansen, vik, & jarchow, 2002) . for many, the covid-19 pandemic has led to significant social isolation with in-person socializing virtually eliminated and many working from home (if at all). these conditions may also exacerbate a common reason for alcohol-related relapse: boredom (levy, 2008) . without other adaptive ways to manage stress, socialize, or simply occupy one's mind, it is possible that craving for alcohol may intensify. finally, there are important treatment implications for alcohol (mis)use during covid-19. individuals already report numerous barriers to seeking drug/alcohol treatment (mcgovern, xie, segal, siembab, & drake, 2006) . in the wake of the pandemic additional barriers may arise such as the perception that one's treatment is not a priority during a 'life or death' pandemic or not worth the risk of leaving one's home. alternatively, for those seeking treatment, there may simply not be local resources available or treatment facilities may have waitlists. although the use of telehealth services are growing in general (dorsey & topol, 2016) , there is more work to be done, with specific considerations for low-income individuals (e.g. recently unemployed) who j o u r n a l p r e -p r o o f may be reluctant to spend money on treatment, perceive treatment to be a luxury, or not have technological resources or a private location to engage in telehealth. affordable computer-based treatments without the need for a provider that focus on stress and alcohol use (paulus, gallagher, neighbors, & zvolensky, 2020) could be particularly pertinent during this pandemic. administration center for behavioral health statistics and quality, 2019) presumably due at least in part to legalization of recreational and/or medical marijuana at the state level (johnston, o'malley, miech, bachman, & schulenberg, 2015) . notably, cannabis users report using more cannabis during times of heightened distress following national disasters such as the september 11, 2001 terrorist attacks, a pattern that was especially prominent among individuals who experienced post-traumatic stress disorder and depression (vlahov et al., 2002) . it therefore follows that cannabis use and associated problems may increase during the covid-19 pandemic. cannabis use increases during times of distress to manage negative affect. in support of this contention, cannabis users report relaxation and tension relief as one of the most common reasons for use (copeland, swift, & rees, 2001; hathaway, 2003; reilly, didcott, swift, & hall, j o u r n a l p r e -p r o o f 1998). data from experimental studies support these self-reports. to illustrate, current cannabis users were randomly assigned to an anxiety-induction or non-anxious control condition and cannabis craving increased from before to during the task among participants in the anxiety condition, but not among those in the control condition (buckner, ecker, & vinci, 2013) . these data indicate that cannabis users were especially vulnerable to wanting to use cannabis during an anxiety-provoking situation, which has direct implications for the covid-19 pandemic characterized by heightened stress. notably, this effect was specific to cannabis craving and was not observed for craving for alcohol or cigarettes in this sample of cannabis users. coping motives are the most common reasons cited for wanting to use during laboratory-induced anxiety (buckner, zvolensky, ecker, & jeffries, 2016) . prospective data collected via ecological momentary assessment also confirm that anxiety is positively, significantly related to cannabis craving at the momentary level, and is related to greater subsequent craving (buckner, crosby, silgado, wonderlich, & schmidt, 2012) . further, although positive and negative affect were greater immediately prior to cannabis use compared to non-use episode, negative affect increased at a significant rate prior to cannabis use, and decreased at a significant rate following cannabis use; changes in positive affect were not significantly related to use (buckner et al., 2015) . further, the stress associated with the covid-19 pandemic may serve as trigger for lapse and/or relapse among individuals undergoing a cannabis quit attempt. in a qualitative interview following cannabis quit attempts, situations involving negative affect and exposure to others smoking cannabis were among the most difficult situations individuals reported in which to abstain (hughes, peters, callas, budney, & livingstone, 2008) . among cannabis users undoing a self-guided quit attempt, data from ecological momentary analysis indicated that although positive and negative affect were significantly higher during cannabis lapse episodes compared j o u r n a l p r e -p r o o f to non-use episodes, when negative and positive affect were analyzed simultaneously, negative affect, but not positive affect, remained significantly related to lapse (buckner, zvolensky, & ecker, 2013) . again, the most common reason for use cited during lapse episodes was to cope with negative affect. not only could covid-19 increase cannabis use, but cannabis use may exacerbate covid-19 symptoms given that smoking cannabis damages the lungs. respiratory toxins (including carcinogens) in cannabis smoke are similar to that of tobacco smoke but notably the smoking topography for cannabis leads to higher per-puff exposures to inhaled tar and gases (tashkin & roth, 2019) . further, respiratory symptoms such as chronic cough, sputum, and airway mucosal inflammation are also similar between cannabis smokers and tobacco smokers. the impact on respiratory functioning of cannabis smoke has led for the consideration of cannabis use as a pre-exiting condition that could increase the likelihood of more severe complications should one contract covid-19 (national institute on drug abuse, 2020). sleep is a fundamentally restorative process, but it is also highly responsive to stress (irwin, 2015) . during times of increased stress, sleep, quite paradoxically, serves both as a major line of defense and as a source of heightened vulnerability. these relationships derive from the fact that sleep and immunological functioning are reciprocally related: sleep promotes healthy immune responses and healthy immune responses (e.g., to infectious agents) promote deeper, more restorative sleep (opp, 2005) . precise mechanisms are of course complex, but several specific links are noteworthy. immune-signaling proteins called cytokines, such as tumor necrosis factor (tnf) and interleukin-1 (il-1) directly target infection and inflammation but are j o u r n a l p r e -p r o o f also known to promote sleepiness and non-rapid eye movement (nrem) sleep (jewett & krueger, 2012) . the hormone melatonin, which provides an endogenous marker of circadian phase peaks during the nocturnal sleep period but also has important immunomodulatory effects. conversely, the hypothalamus-pituitary-adrenal (hpa) axis and the sympathetic nervous system (sns), two primary stress response systems, are down-regulated during sleep, decreasing immune-regulating cortisol levels (besedovsky, lange, & born, 2012) . however, when sleep is inadequate or disrupted, alteration in these systems is readily observable. experimental sleep research provides overwhelming evidence for the detrimental effects of chronic sleep disruption on immune responses including increases in multiple inflammatory markers such as c-reactive protein, diminished immune response to vaccination, and enhanced susceptibility to bacteria and toxins (besedovsky et al., 2012) . rather than representing enhanced immunity, elevated levels of inflammation are associated with a range of health risks including cardio-pulmonary disease (libby, 2006) . sleep's inextricable role in human immunological functioning clearly place it at the forefront of critical behaviors during a pandemic. unfortunately, multiple aspects of the covid-19 pandemic threaten healthy sleep patterns which in turn endanger both physical and mental health. widespread uncertainty, 24-hour media coverage (including misinformation), fear for one's own health and the health of loved ones, and potential loss of employment/wages are but a few of the significant sources of stress present during these unprecedented times. heighted psychological and physiological arousal elicited by such stress falls in direct odds with a calm, quiescent state necessary for sleep onset and maintenance. further, common behaviors aimed at managing increased stress and anxiety such as smoking, alcohol consumption, and decreased physical activity can give rise to or worsen sleep disruption via known negative effects on sleep j o u r n a l p r e -p r o o f duration and quality (irish, kline, gunn, buysse, & hall, 2015) . moreover, sleep deprivation can amplify inflammatory responses (bollinger, bollinger, oster, & solbach, 2010) , increasing the risk for poor outcomes in covid-19 as unrestrained inflammation is implicated in the pathophysiology of the disease (gamaldo, shaikh, & mcarthur, 2012) . although predisposing (e.g., genetics) and precipitating (e.g., trauma) factors play a role, stress is considered a primary cause of insomnia (morin, rodrigue, & ivers, 2003 ) and among insomniacs, perceived inability to sleep often becomes a major source of stress in its own right. studies that have systematically examined incidence and severity of insomnia symptoms during a global pandemic are unavailable despite ubiquitous anecdotal reports and cautions from health professional regarding the immunosuppressive effects of poor sleep. however, in a recentlypublished study conducted between january 29 and february 3, 2020, c. zhang et al. (2020) surveyed medical staff responding to the covid-19 pandemic in china using the insomnia severity index (isi; morin, belleville, bélanger, & ivers, 2011) . more than a third of workers (36.1%) endorsed symptoms indicative of clinical insomnia and those with insomnia reported elevated levels of depression. insomnia is well-known to herald the onset of depression both acutely and years later even among those who have never been depressed (baglioni et al., 2011) . studies directed at uncovering precise mechanisms of affective risk during the covid-19 pandemic must therefore consider the presence and severity of insomnia symptoms. the covid-19 pandemic also has upended daily routines and associated 'cues' that serve to maintain regular sleep schedules. working from home, altered mealtimes, increased sedentary behavior, social distancing, and increased "screen time" are only some of the changes that hold potential to disrupt circadian rhythms that govern sleep-wake patterns. other factors such as social activities also can affect sleep-wake patterns. the human internal circadian clock j o u r n a l p r e -p r o o f runs slightly longer than 24 hours and therefore needs to be 'entrained' to the 24-hour day via internal and external cues (czeisler et al., 1999) . sunlight is the most potent exogenous cue that aligns our internal rhythm to the external environment, but quarantine measures and greater time spent indoors means that many individuals are receiving inadequate dosages of light exposure. although public health guidelines center on sufficient sleep duration (watson et al., 2015) , sleep timing is equally critical for overall health and well-being. misalignment of the sleep period with the body's 'biological night' is routinely linked with a host of serious risks, including anxiety, depression, suicide, cardiac events, and several forms of cancer (baron & reid, 2014) . healthcare workers who are working long hours and night shifts during the covid-19 pandemic are therefore a particularly high-risk group for circadian shifts and associated comorbidities. considering sleep's role in immunological function, this represents an area of priority for future research. the intersection of covid-19 with pre-existing chronic medical illness (e.g., cardiovascular disease, diabetes, hiv) raises additional challenges to the patient for managing multiple treatment cascades. these challenges are exacerbated by the poorer survival and disease course for patients with underlying medical conditions (emami et al., 2020) which in turn seems to be driving, in part, the alarming covid-19 racial disparity (laurencin & mcclinton, 2020) . the overlapping epidemic of covid-19 with hiv, for example, presents unique challenges for hiv access to care, hiv treatment engagement, and prevention. infection or if it exacerbates the likelihood of poor covid-19 outcomes. however, people living with hiv may have other comorbidities, such as cardiovascular disease and chronic lung disease, j o u r n a l p r e -p r o o f that increase the risk for a more severe course of covid-19 illness (guaraldi et al., 2011; guo et al., 2020) . there is also a concern that individuals who are immunocompromised, such as those with hiv, may be at greater risk for severe covid-19 symptoms (cdc, 2020a; duffau et al., 2018) . in the u.s., most people living with hiv (plwh) are tested, linked to hiv care, well engaged in antiretroviral treatment, and achieve hiv viral suppression thus ensuring their optimal health and protecting the public health by containing onward transmission (cdc, 2020b). however, structural and individual barriers to treatment and prevention create enduring inequalities and significantly increase the risk of infection, reduce access to, and engagement in, hiv care, and compromise participation in hiv biobehavioral prevention among particular risk groups. gay and bisexual men (particularly hispanic and african american men) are most impacted by hiv and account for nearly 70% of new hiv cases. hiv incidence rates in the u.s. are also significantly higher for those who are homeless or living in poverty (denning & dinenno, 2020) . with respect to individual barriers to care, plwh are disproportionally affected by traumatic life experiences, anxiety, depression, and substance use (brandt et al., 2017; nanni, caruso, mitchell, meggiolaro, & grassi, 2015; c. o'cleirigh, magidson, skeer, mayer, & safren, 2015) . each of these also have been associated with poorer engagement in hiv care, worse antiretroviral medication adherence, and poorer hiv disease course. their co-occurrence and interaction significantly increases both the risk for hiv infection (mimiaga et al., 2015) and poorer hiv disease management among those already infected (harkness et al., 2018; pantalone, valentine, woodward, & o'cleirigh, 2018) . these mental health barriers to full engagement in hiv care may well be exacerbated by increased levels of covid-19 specific anxieties and j o u r n a l p r e -p r o o f increases in general health-related anxieties. the requirements of social distancing also may contribute to feelings of isolation and loneliness which may in turn contribute to increased depression or depression-related withdrawal. both anxiety-related avoidance and depressive related withdrawal will likely have negative consequences for self-care generally and for hiv care specifically. these increases in distress will occur at a time when access to behavioral health services is already severely restricted. some plwh who become co-infected with covid-19 will already be struggling with hiv disease management (e.g., missed medical appointments, sub-optimal medication adherence) and may require additional supports to manage care and treatment at a time when many routine supports may not be available due social distancing and lack of routine medical services. protecting access to care and treatment among those already struggling with the complexities of the hiv care cascade who must now manage the additional burdens of the covid-19 illness is a robust clinical concern. here, we underline the importance of community (carrico et al., 2020) and health worker based approaches (operario, king, & gamarel, 2020) to hiv treatment and protecting access to care through innovative and virtual care models. many of those at risk for being lost to care during this covid-19 pandemic also may be vulnerable to perceived stigma (krier, bozich, pompa, & friedman, 2020; logie, 2020 ). many will have multiple stigmatized identities with respect to hiv status, covid-19 status, substance use, sexual or gender minority status, and others. keeping our community members and peers involved in our service delivery will help ensure our treatments are delivered in stigma-free contexts. empirical support for integrated treatment platforms that address mental health (ironson et al., 2013; safren, o'cleirigh, skeer, elsesser, & mayer, 2013) and substance use issues (mimiaga et al., 2019; safren et al., 2012) to protect engagement in hiv treatment and j o u r n a l p r e -p r o o f prevention (mayer et al., 2017; conall o'cleirigh et al., 2019) are available to guide these initiatives. in addition, protecting access and supporting engagement (virtual or otherwise), to mental health and substance use treatment will be critically important. these approaches may be particularly key for protecting access to hiv prevention services (i.e., hiv testing, access to preexposure prophylaxis [prep]) for those at risk for hiv. access to these services may be particularly important for those whose behavioral risk profiles and risk appraisals may be disturbed because of the impact of social distancing on usual patterns of substance use or sexual behavior. although much remains unknown about covid-19 and the mental health consequences of the pandemic, it is likely that regular physical activity offers protective effects. regular physical activity reduces risk of and helps manage conditions that appear to increase risk of adverse outcomes of covid-19 (e.g., obesity, cardiovascular disease, diabetes; lee et al., 2012) , and improves immune function (nieman & wentz, 2019) which likely positively affects the progression of covid-19. it also buffers the effect of stressors and (in part thereby) can prevent the onset of mental health conditions (harvey et al., 2018; jacquart et al., 2019) . further, diminished physical activity can disrupt sleep quality (buman & king, 2010; youngstedt & kline, 2006) , which increases susceptibility to infection and mental and physical illness (see sleep section). hence, establishing or maintaining a regular physical activity habit has the potential to mitigate the impact of the pandemic both at a personal and societal level. establishing and maintaining a regular physical activity habit has proven to be challenging. indeed, only 24% of adults meet the guidelines set forth by the department of health and human services (whitfield et al., 2019) . the covid-19 pandemic has impacted j o u r n a l p r e -p r o o f several factors, including a change in the daily routine and increased stress and anxiety, that can affect the intent of or ability to engage in behavior change. it is important to acknowledge the relationship between factors such as stress or changes in routine and physical activity participation can vary in strength or direction (i.e., negative or positive) depending on the individual and their context. for example, for some routine changes have created barriers for exercise participation, while for others changes to the daily structure have opened opportunities to engage in regular exercise. similarly, stress and anxiety at the "right" level can be motivating for some make exercise part of their daily routine, but when stress and anxiety become overwhelming, automated emotion action tendencies often cause people to move away from healthy (coping) behaviors such as exercise (otto et al., 2016) . importantly, such relationships may further vary within and across individuals depending on other individual difference variables (e.g., risk factors, protective factors, [mental] health diagnosis) and contextual factors (e.g., job loss, financial stress, isolation). research aimed at understanding the relationship between covid-19 and physical activity mostly likely will benefit from considering the importance of individual differences and the influence of contextual factors. comprehensive assessment batteries and statistical models that include the testing of these complex moderation effects are key. this perspective that acknowledges nuance in the relationship between covid-19 (pandemic) and physical activity also will aid efforts to develop or fine-tune intervention programs for physical activity uptake. the covid-19 pandemic, although still ongoing and presently under investigated from a behavioral health perspective, is apt to impart acute and potentially chronic exacerbations in psychological symptoms and disorders, addictive behavior, and health behavior and chronic j o u r n a l p r e -p r o o f illness. across various phenotypes overviewed in the current essay, previous scientific work and theoretical models predict covid-19, regardless of acquisition of the virus, has and will continue to have a strong negative psychological impact on negative mood states, various forms of substance use, and sleep, chronic illness, and physical activity. although many of these relations would be expected, theoretically, to be negative, select subgroups will certainly adaptively respond to covid-19 related stress (e.g., improve their physical fitness, improve self-care routines, quit/reduce maladaptive behaviors that place them at risk). in this final section of the paper, we discuss sociocultural considerations, developmental issues, and the role of individual difference factors for covid-19-related psychological, addictive, health behavior and chronic illness. we conclude by offering an integrative covid-19 model that could be used to guide research focused on the stress-related burden of the pandemic. certain subpopulations and contextual factors (e.g., loss of work) are likely to signify a vulnerability gradient for covid-19 in terms of mental health, addictive behavior, and health behavior. although there are numerous possible sociocultural factors that could be relevant, we highlight first responders and medical professionals, economic adversity, and racial/ethnic factors as three prototypical factors of public health importance. of all the sectors of the population, first responders and front-line healthcare professionals are arguably at the greatest risk for at least acute disruptions in anxiety, stress, and negative mood. first responders and healthcare professionals at the front line of the covid-19 pandemic have at their core mission to protect and preserve life (prati & pietrantoni, 2010) . these groups, although engaging in a diverse range of specific occupational activities (e.g., direct medical care, transport, public safety j o u r n a l p r e -p r o o f enforcement), share in common that they are among the first to respond to the covid-19 crisis and take primary responsibility for attending to covid-19 related health issues. first responders and healthcare professionals are undoubtedly experiencing emotionally challenging and unpredictable situations that can place their lives in danger. the acute emotional effects of managing covid-19 cases is likely to be amplified by heavy work schedules and reduced access to and isolation from social support systems (e.g., self-isolation after finishing a shift). it is likely that first responders and healthcare professionals working with covid-19 cases in hospitals will be exposed to potentially traumatic events, the greater-than-usual experience of life-threatening situations, working with emotional strain related to isolation of patients from their families (e.g., compassion stress in the form of offering emotional support to patients in a manner that family or caregiver of patients would typically offer), and exposure to the struggle to life and death. these experiences are apt to challenge the coping resources of even the most seasoned professionals, which can result in higher degrees of anxiety, stress, and depressed mood (lafauci schutt & marotta, 2011) . such elevated stress levels are likely to be related to changes in cognition and physical health, including emotional exhaustion, fatigue, sleep dysfunction, and problems with interpersonal relationships (kronenberg et al., 2008; lane, lating, lowry, & martino, 2010) . cognitive-based beliefs about personal safety and health can be altered and memories of potentially traumatic events engrained (setti & argentero, 2014) . collectively, the covid-19 related stress burden, as discussed in several sections of the current essay, will have a high likelihood of being related to increased risk of anxiety and depression for first responders and medical professionals working at the front line. moreover, consistent with past literature of these populations, the regulation of affect will be associated with addictive and health behavior to modulate such affect (e.g., physical activity, substance j o u r n a l p r e -p r o o f use). although some regulatory behavior will be adaptive (e.g., increasing sleep where possible to aid in recovery, engaging in regular physical exercise), others may be less adaptive (e.g., smoking to reduce stress) and promote the risk for other health problems (e.g., physical illness). economic adversity. economic hardship related to covid-19 is already evident at numerous levels of analysis, including job loss, reduced earnings, higher debt relative to assets ratio, inability to pay mortgage and bills, meeting governmental guidelines for poverty status, and worry about financials resources going forward due to the turbulent nature of the economy. past work has shown that economic hardship is related to behavioral health problems, including psychological disorders, addictive behavior, physical health problems, and interpersonal dysfunction in adults and children (k. j. conger et al., 2012; sareen, afifi, mcmillan, & asmundson, 2011) . for instance, economic adversity has been linked to reduced social competence and elevated physiological markers of stress (k. e. bolger, patterson, thompson, & kupersmidt, 1995; evans & english, 2002) . further, economic hardship is related to selfregulation capacity and the corresponding difficulty in dealing with additional responsibilities. for example, past work has found limited socioeconomic resources are related to harsher parenting behavior and greater substance use (r. d. conger & donnellan, 2007) . the negative effects may be particularly profound when economic hardship is severe or chronic (dearing, mccartney, & taylor, 2001; magnuson & duncan, 2002) . the totality of worsening economic conditions for individuals and families in the larger context of an uncertain economic future are apt to be related to elevations in anxiety, stress, and depression as well as other negative emotional states (e.g., anger, frustration, fatigue; newland, crnic, cox, & mills-koonce, 2013) . such emotional symptoms and problems are likely to be related to elevations in substance use and other maladaptive behavior (e.g., less supportive interpersonal behavior, less affection) and j o u r n a l p r e -p r o o f may exacerbate chronic health conditions. other work has found that these processes also disrupt social interconnections (scaramella, sohr-preston, callahan, & mirabile, 2008) . primary care givers who have children home from school, are unlikely to be able to work at their full capacity even with added flexibility in schedules. although certain occupations have decreased activity, many have not. therefore, it could be expected that for individuals with added responsibilities of educating their children at home occupational stress may be greater compared to those without such responsibilities. further, it is possible that the accumulation of occupational responsibilities that are not addressed for persons with additional educational responsibilities will accumulate and make it more challenging to recover when going back to 'normal,' resulting in a greater degree of occupational stress. grappling with lower socioeconomic states related to covid-19 will, for certain segments of the population, offer an additional psychological challenge. indeed, past work has repeatedly documented that lower socioeconomic status is related to adverse health outcomes for chronic illness and mortality rates (adler et al., 1994; adler, boyce, chesney, folkman, & syme, 1993) . moreover, harms faced by people who cannot afford not to work in dangerous settings can exacerbate the psychological and health risk associated with coid-19. further essential workers are more apt to be persons of color (handerson, mccullough, & treuhaft, 2020) . certain groups will be more likely to recover than others, which past work indicates is related to poorer health outcomes even at higher socioeconomic levels (kraus, borhani, & franti, 1980) . moreover, research has found that lower socioeconomic persons experience more chronic stress and negative life events (stansfeld, north, white, & marmot, 1995) . additionally, lower socioeconomic status is related to cognitive biases for threat (chen & matthews, 2001) , which engender greater degrees of interpersonal conflict and heightened negative emotional states j o u r n a l p r e -p r o o f (matthews et al., 2000; stansfeld, head, & marmot, 1997) . it would be expected that such negative emotional experiences will be related to maintained direct relations with poorer health behavior and health outcomes (mcewen & stellar, 1993) . in fact, research has consistently found that lower socioeconomic status is related to greater degrees of anxiety, stress, and depression when compared to those higher in socioeconomic status (mcleod & kessler, 1990) . this heightened stress reactivity may be at least in part attributable to having fewer resources. consequently, those struggling with a lower socioeconomic status due to covd-19 may be more contexts in which they must utilize their emotional resources and be less likely to be in a sociocultural context wherein such resources can be replenished (holahan, moos, holahan, & cronkite, 1999) . this perspective is in line with past work that has found that when persons are exposed to chronic stress, emotional resources are challenged, and there is a greater risk for future emotional distress (n. bolger & zuckerman, 1995; ensel & lin, 1991) . there is broad band evidence that significant health disparities exist for persons of racial/ethnic minority in the u.s. and beyond prior to covid-19 for psychological, addictive behavior, and health behavior as well as chronic illness. for example, african american/black individuals experience a disproportionate burden in disease morbidity, mortality, disability, and injury (mechanic, 2005; mensah, mokdad, ford, greenlund, & croft, 2005) . indeed, african american/black individuals remain significantly and consistently more at risk for early death than do similar non-latinx white individuals (williams, neighbors, & jackson, 2003; williams, yu, jackson, & anderson, 1997) ; overall early death rates of african american/black individuals are comparable to those observed among non-latinx whites in the u.s. decades ago (levine et al., 2016; williams & jackson, 2005) . differences in prevalence and rate of growth of chronic illness are not accounted for solely by j o u r n a l p r e -p r o o f exposure to lower income environments (franks, muennig, lubetkin, & jia, 2006) . indeed, social determinants of health (e.g., racism; krieger & sidney, 1996) , addictive behavior (e.g., tobacco use; sakuma et al., 2015) , and stress represent robust and consistent factors related to health inequalities among african american/black individuals and those from other underrepresented racial/ethnic groups. the covid-19 pandemic has appeared to strike racial and ethnic minority populations (e.g., african american/black) hard and with possible longerterm consequences. for example, less access to health care services for chronic illness, addictive behavior, and mental illness could exacerbate covid-19 related symptoms or promote a greater degree of stress-related burden associated with the pandemic (e.g., worry that loved ones, if infected, cannot access care). consequently, addictive behaviors (e.g., smoking, alcohol misuse) and health behaviors (e.g., disrupted sleep, emotional eating) may be used in the short-term to cope with such covid-19 related stress, increasing the longer-term risk for more severe negative emotional symptoms and health complaints (e.g., pain) and chronic health problems (e.g., obesity). additionally, situations characterized by mass fear and confusion, such as the current pandemic, also can elicit a human instinct to resolve the confusion and mitigate the fear by identifying a culprit for the introduction or spread of the disease (bard, verger, & hubert, 1997; bromet, 2011) . asian american persons are one group that has been singled out as responsible for the covid-19. the misdirection of fear and/or anger related to covid-19 toward a racial or ethnic group instead of the disease, however, can perpetuate fear and contribute to racism and stigma. several reports have already documented the rise in violent crimes and discrimination experienced by asian american persons related to covid-19 beliefs (e. liu, 2020) . covid-19 specific language, such as referring to covid-19 as 'the chinese virus,' has created a platform j o u r n a l p r e -p r o o f to propagate stigma and discrimination towards asian americans. it is likely that stigma and discrimination experienced by asian americans in response to covid-19 will increase emotional distress, coping-oriented addictive behavior, and may alter health behavior or exacerbate chronic illness. it would also be remiss to not call explicit attention to the fact that societies marked by greater economic and social inequality experience far more medical, psychological, and social pathology than do societies where such wealth inequalities are less pronounced (wilkinson & pickett, 2006 . further, such adverse effects occur across social classes, not merely among the most disadvantaged. yet, the adverse effects of economic (and thus social) inequality hurt everyone, although the poorest or most marginalized are affected the most (pickett, kelly, brunner, lobstein, & wilkinson, 2005; wilkinson & pickett, 2008) . there are far-reaching implications for psychological health, addictive behavior, and health behavior from a developmental perspective. for children, despite covid-19 appearing to have less severe symptoms and lower mortality rates than other age groups, are among the highest risk groups (sinha et al., 2020; zimmermann & curtis, 2020) . estimates suggests that there are over 1 billion children not in school (cluver et al., 2020) . the economic impact of covid-19 will likely be related to greater risk for children to be utilized to offset such financial hardship (e.g., selling merchandise on the street, forced begging for food and goods) and be a more likely to be abused (campbell, 2020) . for example, it is possible that children will be more likely to be used for child labor and be exploited for sexual behavioral and experience corresponding risk for sexual disease and pregnancy as well as serious psychological distress. interpersonal violence and child abuse will affect children at a significant rate, especially under j o u r n a l p r e -p r o o f conditions wherein there is no oversight from educational systems due to quarantine. world health organizations are already predicting an increase in children who will be orphaned and exposed to abuse and neglect (cluver et al., 2020) . child abuse is less likely to be detected during the covid-19 pandemic because the reduction or lack of child protection agencies monitoring cases, and teachers less able to detect signs of abuse. further, children who received meals at school through government programs such as the national school lunch program may now no longer have access to nutritious food, which can negatively impact their development. the lack of structure from schooling and missed education will have a lasting impact on well-being and apt to be related to increased anxiety, depression, and stress about educational attainment and progress going forward (van lancker & parolin, 2020) . although on-line school may help offset some of these challenges, disparities will exist for those who are most vulnerable, including those who lack internet access or cannot afford technology. older children and young adults may be more likely to drop out of school to help offset family needs. children and youth also may be engaging in more on-line behavior in general or due to emotional distress (e.g., loneliness due to social isolation) and be increasing the chance for solicitation from others who prey on their emotional vulnerabilities (peterman et al., 2020) . lacking access to physical activity due to quarantine protocols may reduce fitness levels and immunological response as well as decrease psychological wellbeing (rundle, park, herbstman, kinsey, & wang, 2020) . children and youth in juvenile systems, such as orphanages, already were exposed to high density living conditions and often lack access to proper medical or psychological care. the covid-19 pandemic is likely to place pressure on such systems (e.g., more children) and the physical environments of these settings may be amenable to the spread of infection. likewise, refugee or otherwise displaced children and youth often live-in high-density environments j o u r n a l p r e -p r o o f wherein social distancing is challenging if not impossible. further, lack of access in these settings to cleaning supplies and water can catalyze the spread of covid-19 or even the basic fear of acquiring the virus. to the extent the covid-19 challenges the medical system, it is possible other forms of medical care necessary for child welfare (e.g., routine exams, immunizations) will be reduced, as was the case during other pandemics such as ebola (mupere, kaducu, & yoti, 2001) . collectively, covid-19 places an enormous stress on children and youth, placing them at an increased risk for psychological disturbances and physical health vulnerability (j. j. liu, bao, huang, shi, & lu, 2020) . covid-19 also will affect ranges of the lifespan, including adults and older adults. the well-publicized health risks for older adults place an obvious psychological and health pressure on this group. older adults are among the most likely to have a chronic illness (e.g., diabetes, cancer, cardiovascular disease) and consequently they maintain an increased vulnerability to deteriorating health and death from covid-19. however, even in the absence of exposure to the virus, the fear and worry about contracting the disease is apt to be significant for this group, especially when in homecare facilities such as nursing homes or hospitals (armitage & nellums, 2020) . this group also is at significant risk for lacking transportation for food, which could challenge the quality of nutrition and have a negative effect in immunological function. similarly, older adults are among the least physical active groups, which again, will have the potential for decreasing psychological wellbeing and immunity. although not specific to older adults, the potential for disruption in grief and loss of others also is a significant psychological stressor. during the pandemic, regular methods of grieving such as funerals have been limited if not all together impossible. the inability to grieve with others or as traditionally done may spur escalation in psychological distress (e.g., sadness, j o u r n a l p r e -p r o o f depression) and complicate the grief process (wallace, wladkowski, gibson, & white, 2020) . to the extent that grief is impaired, individuals may engage in maladaptive addictive behaviors (e.g., alcohol misuse) to cope with the aversive experiences. similar types of emotional reactions may occur when parents are separated from their children due to quarantine protocols and disruptions in travel (e.g., cannot travel to see children located in another region). there are several individual difference factors at a psychological level of analysis that will place people at an increased or decreased risk for psychological problems, addiction, and poor health behavior, and chronic illness during and after the pandemic. research over the past few decades has theorized and found consistent empirical support for emotional symptoms and disorders as well as addictive behavior being explained by individual differences in transdiagnostic processes (sauer-zavala et al., 2012) . transdiagnostic factors may contribute to onset, maintenance, and exacerbation of emotional symptomatology and addictive and health behavior. a core aspect of transdiagnostic models is that they seek to identify basic processes underlying multiple, usually comorbid, psychopathologies or addictive behavior. one set of transdiagnostic factors relevant to covid-19 may be those that are "reactive" vulnerabilities; that is, individual differences that reflect a heightened emotional response to stressful stimuli. such vulnerabilities influence emotion experience by enhancing or diminishing the normative response to emotion stimuli and states, resulting in an excess or deficit, respectively, beyond typical emotional functioning; or altering the type of response to emotion stimuli and states (gratz & roemer, 2004; reiss, 1991; zvolensky, bernstein, & vujanovic, 2011) . in both instances, such reactive processes may be maladaptive because they serve to j o u r n a l p r e -p r o o f reinforce the intensity and frequency of future emotional symptoms. for example, when faced with negative emotion states, individuals with an emotional vulnerability factor that limits their capacity to handle distress may be more apt to execute behaviors that preclude habituation to negative emotion states, which could ultimately increase the intensity of future negative affect and solidify beliefs and learned responses that interfere capacity to adaptively respond to distress. to illustrate, a transdiagnostic factor that may be especially relevant to covid-19 related stress responsivity, substance use, and physical health is anxiety sensitivity (taylor, 2014) . anxiety sensitivity is a malleable, cognitive-affective factor reflecting the tendency to respond to interoceptive distress with anxiety (mcnally, 1989) . anxiety sensitivity is related to, yet distinct from, negative affectivity and trait anxiety (keough, riccardi, timpano, mitchell, & schmidt, 2010) . anxiety sensitivity has demonstrated racial/ethnic, gender, age, and time invariance (ebesutani, mcleish, luberto, young, & maack, 2014; farris et al., 2015; jardin et al., 2018) . given covid-19 can produce physical sensations and even when not infected, covid-related stress can elicit a range of interoceptive sensations, persons higher in anxiety sensitivity may be more be emotional reactive to such stimuli and engage in behavior to dampen stress symptoms (e.g., using tobacco, alcohol). for example, persons may interpret the onset of aversive bodily sensations (e.g., runny nose, cough, fever) as intolerable or catastrophic, exacerbating the experience of such interoceptive symptoms. further, interoceptive symptoms might be particularly salient to persons with higher anxiety sensitivity who are prone to health inequalities (e.g., racial/ethnic minorities, persons in financial stress), as they may be more apt to perceive these internal sensations as uncontrollable because resources to regulate symptoms (i.e., adaptive cognitive and behavioral skills) are likely diminished due to chronic stress exposure j o u r n a l p r e -p r o o f (e.g., low socioeconomic status, discrimination). in turn, persons higher in anxiety sensitivity may be motivated to use substances to reduce emotional and interoceptive distress, elevating their chance for physical illness and compromised immune system function. although this illustrative example represents only one of many possible transdiagnostic amplifying factors, it draws attention to the fact that individual differences in psychological processes are apt to play a central role in the relation between covid-19 related stress and mental health, addictive behavior, health behavior, and chronic illness. individual difference factors also may play roles in offering resilience to covid-19 related stress. that is, individual differences may contribute to the likelihood of a resilient response to covid-19 in the short and long term. thus, in addition to the many situational and contextual factors, individual difference factors will likely shape the level of resiliency to covid-19 pandemic. here, it is likely individual difference factors that de-amplify stress responses will play a central role in offsetting relative risk for psychological, addictive, and health behaviors problems and exacerbation of chronic illness (pidgeon & keye, 2014) . as with affect amplifying factors, such as anxiety sensitivity, there most certainly is a range of factors of potential importance, including flexible coping repertoires, mindfulness, self-efficacy, selfcompassion, and proneness to experience positive affect. to illustrate, individual difference in the capacity to accept difficult covid-19 related stress may offset the potential escalation of anxiety, stress, and depression and mitigate the need for addictive or unhealthy behaviors (e.g., emotional eating) to delimit aversive internal experiences (ranzijn & luszcz, 1999) . consequently, the corresponding risk for health complaints or worsening of chronic health conditions can be offset. indeed, there is a large theoretical and empirical literature that suggests the capacity to accept difficult emotions experiences is related to psychological well-being and j o u r n a l p r e -p r o o f adaptation. for example, one of the reasons meditative practice is related to decreased stress is via change emotional acceptance (teasdale et al., 2002) . this type of work has robust implications in efforts to intervene on covid-19 related stress in the immediate context and for those that struggle to regain stability and growth in the future in terms of mental health, addictive behavior, and health behavior. despite the present lack of systematic empirical work on covid-19 in terms of behavioral health problems, there is good theoretical basis from past scientific work to hypothesize that covid-19 related stress burden, due to a myriad of sources, may play a major vulnerability role in terms of mental health, addictive disorders, and health behaviors as well as chronic illness. for some, the stress-related burden of covid-19 may elicit fundamental changes in risk potential and serve as a fertile basis for future behavioral health problems. for others, the ability to adapt to covd-19 will offer a different course; one that is characterized by greater stability, speed of recovery, and growth. further, it is important to recognize that the adaptation process to covid-19 related stress is apt to be non-linear in many instances. that is, contextual factors (e.g., future life stressors, access to social support) can influence the degree of risk for future problems. research described in this essay provides a basis to develop a theoretical model that could be used to evaluate covid-19 related stress burden on psychological, addictive, and health behaviour problems. we therefore begin this section by briefly outlining a general model that can be used as a heuristic for understanding the complex issues at hand. see figure 1 for a graphical depiction of the model. in general, we predict individual differences in affect amplifying and de-amplifying factors will predict the course of psychological, addictive behavior j o u r n a l p r e -p r o o f and health behavior and chronic illness even when considering differences in exposure to covid-19 experiences (e.g., time of quarantine, acquisition of virus). we would predict, based on past work that transdiagnostic affect amplifying factors will influence addictive and health behaviour, which in turn, will increase (or decrease if de-amplifying) the risk of chronic illness and psychological problems and their comorbidity. further, we can expect that this type of perspective will be moderated by daily stress in the future and access to stress-dampening resources (e.g., social support). accordingly, certain subgroups more prone to greater and more chronic stress, such as first responders and racial/ethnic minorities and orphaned children, may be particularly vulnerable. this conceptual model predicts that the associations which exist between are reciprocal and dynamic. although the model offered here is purposively general and is offered only as a heuristic, it is presumed that there is, in fact, specificity between specific affect amplifying and deamplifying factors, moderators, mediators, and various forms of psychological and chronic illness. that is, a specific type of individual difference factor like anxiety sensitivity is linked to a particular type of problem (e.g., anxiety disorder, worsening of a chronic respiratory illness, severity of hazardous drinking) via a specified mediating process (e.g., smoking, sleep disruption) in the context of certain moderating variables (e.g., higher levels of covid-19 stress burden). the core idea being that the underlying mechanism in this hypothetical example may be quite different from that explaining other problems. the above theoretical model requires empirical testing, and if it is confirmed, one next logical step would be to intervene in it to reduce the burden of mental health, addictive disorders, poor health behaviours, and chronic health conditions related to covid-19 stress burden. ideally, this type of intervention approach would target the root of the pathway, including affect j o u r n a l p r e -p r o o f amplifying (i.e., decreasing levels) and de-amplifying (i.e., promoting growth). however, intervention efforts sit in the fact that the healthcare system will continue to shift and adapt to treatment delivery, including the uptake of digital health technologies. digital health, including mobile health (mhealth), telemedicine/telehealth, and health information technology (e.g., mobile phones, wearable sensors), can be used to develop scalable interventions to promote adherence public health guidelines for mitigating the spread of covid-19. they also can be combined with greater attention to affect amplifying (i.e., decreasing levels) and de-amplifying (i.e., promoting growth) factors that govern many psychological, addictive, and health behaviour processes. here, there is great opportunity for growth of digital health interventions to offer standalone clinical grade therapeutic tactics and as an adjunct to face-to-face interventions. this type of work can close the gap in access to care and offer evidence-based interventions to large segment of society. for example, digital interventions can be used to combat resistance to public health measures at the level of individuals and institutions with a consideration of individual difference factors that affect emotional and behavioral self-regulation. indeed, the public's response to public health measures is itself a potential risk and protective factor for many of the psychological, addictive, and health behavior problems reviewed in this essay. the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing. there is a need for financial and social investment in research to better understand how covid-19 affects the onset, maintenance, and relapse potential for some of the most common, costly, and chronic behavioral health conditions in the general population. further, there is a need for the study of the role of psychological processes, addictive behavior, and health behavior in terms of the onset and maintenance of j o u r n a l p r e -p r o o f covid-19 infection and stress burden. there most certainly will be a demand for preventative and intervention efforts for managing the impact of covid-19 among individuals with elevated negative mood symptoms and disorders, addictive behavior, and certain health behaviors (e.g., sleep disorders) and chronic illness. this work is important to offset the current and projected burden to personal, system, and societal entities, and for providing a theoretical and empirical knowledge base for future pandemics. we presented a heuristic model, which posits that covid-19 related stress and mood, addictive, and health behavior may, in fact, exacerbate each other via several distinct mechanisms. future research in this emerging area has the potential to refine both theory and application with respect to covid-19 and its relation to affect, addiction, and health behavior as well as chronic disease. j o u r n a l p r e -p r o o f • the public health impact of covid-19 on psychological symptoms and disorders, addiction, and health behavior is substantial and ongoing • an integrative covid-19 stress-based model could be used to guide research focused on the stress-related burden of the pandemic • this work could provide a theoretical and empirical knowledge base for future pandemics j o u r n a l p r e -p r o o f how covid-19 may increase domestic 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potential conflicts of interest and to significant financial contributions to this work zvolensky receives personal fees from elsevier, guilford press, and is supported by grants from nih he receives research support from nih, texas higher education coordinating board, rebuild texas and greater houston community fund. he receives travel support and honorarium from iocdf for training in ocd treatment schmidt is supported by the military suicide research consortium (msrc), department of defense, and visn 19 mental illness research, education, and clinical center buckner receives funding from the u.s. department of health & human services' graduate psychology education (gpe) program (grant d40hp33350) smits reports grants from cancer prevention and research institute of texas; personal fees from big health, ltd., personal fees from aptinyx, inc., personal fees from elsevier vujanovic receives book royalties from routledge press and is supported, in part cleirigh is supported by grants from the nih and the centers for disease control and prevention we confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed he/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. we confirm that we have provided a current, correct email address which is accessible by the corresponding author and which has been configured to accept email from mjzvolen@central key: cord-017817-ztp7w9yh authors: land, walter gottlieb title: cell-autonomous (cell-intrinsic) stress responses date: 2018-03-28 journal: damage-associated molecular patterns in human diseases doi: 10.1007/978-3-319-78655-1_18 sha: doc_id: 17817 cord_uid: ztp7w9yh in this chapter, the role of cell-intrinsic stress responses is examined which include autophagic processes, the oxidative stress response, the heat shock response, the unfolded proteins response, and the dna damage response. autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy) is a self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components are delivered to the lysosome for recycling and degradation. the oxidative stress response is directed against any oxidative stress and is mediated by antioxidative defense systems including antioxidant enzymes such as superoxide dismutase, detoxifying enzymes such as glutathione peroxidase, and energy-dependent efflux pumps. the heat shock response is induced upon exposure of cells to any stress condition and characterized by emission of heat shock proteins which operate as damps to maintain and restore homeostasis. the unfolded protein response is induced by any stress of the endoplasmic reticulum that is perceived by three sensor molecules. under remediable endoplasmic reticulum stress conditions, the sensors trigger signalling pathways to resolve this stress. however, in severe irremediable endoplasmic reticulum stress, the unfolded protein response may lead to pro-inflammatory and pro-apoptotic responses resulting in regulated cell death. finally, the dna damage response is induced by any dna damage that occurs in a variety of exogenous and endogenous conditions. when successful, this stress response leads to dna repair and is associated with the emission of various damps which contribute to restoration of homeostasis. when unsuccessful, the dna damage response, like the unsuccessful unfolded protein response, can result in regulated cell death, either in form of apoptosis or necrosis. together, the ultimate goal of all the stress responses is to maintain cellular homeostasis and ensure cell integrity. when they fail, the incidence of regulated cell death is frequently observed. as comprehensively described in part ii, prms are specifically involved in the recognition of mamps and damps. as will be discussed in part vi, each of these recognition receptors can trigger distinct signalling cascades in innate immune cells that modify their gene expression to create and execute efferent innate immune responses that involve (1) production of inflammatory mediator substances such as cytokines and chemokines, (2) phagocytosis, and (3) cytotoxicity, as well as, as described in part viii, may elicit and shape antigen-specific adaptive immune responses. beyond this well-characterized mamp/damp engagement of prms leading to a variety of downstream efferent cellular and humoral responses, the innate immune defense program also depends on cell-autonomous, that is, cell-intrinsic, responses which counteract any stressful insult [1] . constitutive cell-autonomous immunity mobilizes pre-existing molecules and processes in order to primarily and quickly defend the cell and the host against infectious and sterile injury. hence it can be considered as the very first line of innate immune defense. here, the role of constitutive cell-autonomous responses will be examined, whose involvement in the innate immune defense to stress and injury has only been appreciated within the last few years. the focus of this brief overview will be mainly directed toward cellular stress responses. the term autophagy comes from the greek words "phagy" meaning eat and "auto" meaning self. autophagy is an evolutionarily highly conserved self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components such as defective/damaged or redundant organelles or protein aggregates are delivered to the lysosome for recycling and degradation. there is convincing evidence indicating that activation of the autophagic process is promoted by mamps and/or damps [2, 3] . in more simple words, autophagy is a classical cellprotective and cell-autonomous process of the innate immune system aimed at maintaining and restoring homeostasis at both the cellular (cell-intrinsic) and organismal (cell-extrinsic) level [4] . although autophagy was initially identified in mammals, a significant breakthrough in our understanding of how autophagy is controlled came from the analysis in the genetically tractable yeast system. pioneering work from ohsumi's group showed that the morphology of autophagy in yeast was similar to that documented in mammals [5] . (as known, ohsumi received the nobel prize in physiology or medicine 2016.) in fact, the discovery of the autophagyrelated genes in yeast has significantly advanced the understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. many yeast genes have mammalian homologues, confirming that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum [6] [7] [8] [9] . notably, a panel of leading experts in the field of autophagy has recently published a new definition of several autophagy-related terms based on specific biochemical features [10] . accordingly, in the following, three types of autophagy are briefly sketched including macroautophagy, microautophagy, and, in mammals, chaperone-mediated autophagy. each of them fulfils very specific tasks in intracellular degradation. there is general agreement on two main features that characterize bona fide, functional autophagic responses, irrespective of type: (1) they involve cytoplasmic material; and (2) they culminate with (and strictly depend on) lysosomal degradation [10] . thus, although autophagy substrates can be endogenous such as damaged cellular organelles or exogenous such as viruses or bacteria escaping phagosomes, autophagy acts on entities that are freely accessible to cytosolic proteins. this property is essential in order to distinguish between autophagic responses and branches of vesicular trafficking that originate at the plasma membrane, which also culminates in lysosomal degradation. such endocytic processes include phagocytosis, receptor-mediated endocytosis, and macropinocytosis, that is, processes which will be dealt with in part vi, sect. 22.6. of note, however, some forms of autophagy and the endocytic pathway interact at multiple levels, and the molecular machinery responsible for the fusion of late endosomes (also known as mvbs) or autophagosomes with lysosomes is essentially the same [11] . as stressed [10] , the strict dependency of autophagic responses on lysosomal activity is necessary to discriminate them from other catabolic pathways that also involve cytoplasmic material, such as proteasomal degradation [12] . thus, the 26s proteasome (box 18.1) degrades a large number of misfolded cytoplasmic proteins that have been ubiquitinated (for (poly)ubiquitination, see box 18.2) as well as properly folded proteins that expose specific degradation signals, such as the socalled n-degrons [13] . on the other hand, the proteasome system shares some substrates with different forms of autophagy whereby these two catabolic pathways differ drastically in their final products. thus, proteasomal degradation results in short peptides that are not necessarily degraded further but may flow into additional processes including but not limited to antigen presentation/cross-presentation at the plasma membrane, thereby generating mhc-ii and mhc-i epitopes (compare part viii, chap. 31). by contrast, lysosomal proteases fully catabolize polypeptides to their constituting amino acids which eventually become available for metabolic reactions or repair processes. together, as summarized [10] , bona fide functional autophagic responses navigate cytoplasmic material of endogenous or exogenous origin to degradation within lysosomes (or late endosomes, in specific cases). the binding of many ubiquitin molecules to the same target protein. in its simplest form, ubiquitin can be attached to the target protein as a single moiety resulting in monoubiquitination. ubiquitin itself can be ubiquitinated, resulting in the formation of ubiquitin chains attached to the target protein: polyubiquitination. polyubiquitination of proteins is the triggering signal that leads to subsequent degradation of the protein in the proteasome. ligases play a central role in polyubiquitination. ligases are enzymes that catalyze the synthesis of polyubiquitin chains. ubiquitin conjugation requires typically box the proteasome is a common complex for all living cells, needed to recycle and eliminate unwanted proteins. in analogy, it resembles a chaff-cutter. this molecular machine provides a pathway that is involved in many cellular levels such as protein degradation, antigen processing, cell cycle, apoptosis, and dna repair. the 26s proteasome that is present in the cytoplasm and nucleus is usually formed by one 20s proteasome complex and two 19s proteasome complexes, which are composed of proteases and structural units. the 26s proteasome is a giant protease responsible for the regulated degradation of polyubiquitylated proteins (see box 18.2) . it consists of at least 33 distinct subunits and is arranged into two modules: core particle containing catalytic sites and regulatory particles. the cylinder-shaped proteolytic core is the 20s core particle, which is capped at one or both ends by 19s regulatory particles. further reading: wehmer m, sakata e. recent advances in the structural biology of the 26s proteasome. int j biochem cell biol 2016;79:437-442. basically, the term macroautophagy is often used when describing autophagy in general. the phenomenon is characterized by its typical morphological features which involve dedicated vesicles that can occupy a considerable part of the cytoplasm. typically, macroautophagy is one type of autophagic processes in which the substrates are sequestered within cytosolic double-membrane vesicles termed autophagosomes. the substrates of macroautophagy include superfluous and damaged organelles, cytosolic proteins, and invasive microbes. mechanism of formation and regulation of macroautophagy are very complex and complicated processes that are outlined here in a considerably simplified way. macroautophagy involves the sequestration of cytoplasm via a double-membrane intermediate structure termed the phagophore which matures into an autophagosome; the latter compartment fuses with a lysosome allowing degradation and recycling of the cargo [14] . in more detail, the process begins with the formation of a membrane of unknown origin, the initial phagophore or isolation membrane. the phagophore then expands, surrounds proteins or organelles, sequesters cytoplasm, and, on completion, develops into a large double-membrane transport vesicle, the autophagosome. subsequently, the autophagosome fuses with a lysosome containing acid hydrolases and releases its contents into the lytic acid hydrolases-containing compartment as part of single-membrane vesicles, termed autophagic bodies. the fused compartment where the autophagic body and its contents are degraded is called an autophagolysosome or autolysosome ( fig. 18.1) . notably, the process of phagophore expansion three classes of enzymes. e1 (ubiquitin-activating enzyme) hydrolyzes atp and forms a thioester-linked complex between itself and ubiquitin. e2 (ubiquitin-conjugating enzyme) receives ubiquitin from e1 and forms a similar thioester-linked intermediate with ubiquitin. e3 (ubiquitin ligase) finally binds both the e2 and a substrate and catalyzes the transfer of ubiquitin to the substrate. ubiquitin itself is often a substrate for further ubiquitylation, which results in the formation of so-called polyubiquitin chains. ubiquitin has seven lysine residues, and depending on the lysine residue used for ubiquitin-ubiquitin chain formation, the polyubiquitin chain can signal different functions. proteins modified by lysine-48 (k48)-or lysine-29 (k29)-linked chains are usually degraded by the proteasome. in contrast, modification by k63-linked chains or by a single ubiquitin moiety (monoubiquitylation) seems to trigger other functions, e.g., protein sorting, gene expression, and dna repair. further reading: callis j. the ubiquitination machinery of the ubiquitin system. arabidopsis book 2014;12:e0174. provides tremendous flexibility and capacity with regard to cargo, allowing entire organelles to be deleted via autophagy; however, this flexibility also means that autophagy must be tightly controlled in order to prevent inappropriate degradation, which could lead to cell death (for relevant papers, see [6] [7] [8] [9] [14] [15] [16] . intensive studies have been carried out in the past two decades to understand the mechanism and regulation of autophagy. the biogenesis of autophagosomes needs the ordered intervention of autophagy-regulated (atg) proteins that act on different modules. thus, more than 30 atg genes have been identified in human that orchestrate the complex membrane dynamics involved in autophagic sequestration. these atg proteins act sequentially in three macromolecular complexes involved in the three successive stages of autophagy. initiation of autophagy requires the unc-51like kinase 1 (ulk1)-atg13-fip200 (also known as rb1-inducible coiled-coil 1) complex, whereby the kinase activity of ulk1 is controlled by the kinase mammalian target of rapamycin (mtor) in mtor complex 1 (mtorc1), which is sensitive to rapamycin [9] . the next process, membrane nucleation, requires the beclin1/ class iii pi3k complex, which also plays a major role in membrane trafficking and restructuring involved in autophagy [15, 17] ; the final process refers to the elongation, expansion, and closure of the phagophore membrane/autophagosome which mainly relies on atg8/microtubule-associated protein 1 light chain 3 (lc3) lipidation. in fact, atg8/lc3 lipidation is regarded as a hallmark of autophagy and is established by a covalent linkage of cytosolic lc3 to the lipid phosphatidylethanolamine on the surface of the autophagosome [7, 9] . of note, in addition to the cytoplasmic ptm of various atg proteins, recent studies have explored the transcriptional and epigenetic control of autophagy [18] . notably, in human cells, tfeb (for transcription factor eb) and zkscan3 (for zinc finger with krab and scan domains 3) were shown to be implicated in playing a crucial role in autophagy regulation [19, 20] . also, there is growing evidence in support of the notion that histone modification/dna methylation acts as an alternative approach for long-term autophagy control [21] (for histone modification, see part vi, sect. 24.2.3) . also recently, a new ampk→skp2→carm1 (for: ampactivated protein kinase; s-phase kinase-associated protein 2 (p45); coactivatorassociated arginine methyltransferase 1) regulatory axis was reported that incorporated cellular nutrient sensing with transcriptional as well as epigenetic control of autophagy [22] . as concluded by xu and klionsky [14] , "…this ampk-skp2-carm1 signalling axis integrates the various levels of autophagy regulation including cell signalling, and transcriptional regulation as well as epigenetic modification. epigenetic and transcriptional regulation provides an energy-saving approach for control and also create an enduring memory in preparation for future adverse events. thus, this study has deepened our understanding of how autophagy can be controlled in a holistic manner by pathways linking a multitude of regulation mechanisms. given the extensive involvement of autophagy in human diseases, this work also presents potential directions for novel therapeutic intervention." indeed, besides its beneficial function in controlling cellular homeostasis, macroautophagic pathways when disrupted can have severe consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases [23] . of note, macroautophagy can be divided into two subtypes depending on the organelle that is targeted for autophagic degradation; thus, the process of mitophagy corresponds to autophagy of mitochondria, whereas the term er-phagy refers to autophagy of the endoplasmic reticulum (er). both processes deserve a few more words in the following subsection. the term mitophagy corresponds to cargo-specific autophagy of mitochondria, a process which mediates the selective removal of mitochondria [24, 25] . the aim of mitophagy is to eliminate mitochondria, either to regulate their number to adjust to metabolic demand or to explicitly remove those that are damaged in terms of a quality control. mechanistically, mitochondria are selectively recruited into isolation membranes, which seal and then fuse with lysosomes to eliminate the trapped mitochondria. as discussed [24] , mitophagy is preceded by so-called mitochondrial fission that divides elongated mitochondria into pieces of manageable size for encapsulation and also controls segregation of damaged mitochondrial material for selective removal by mitophagy. the term er-phagy (also called micro-er-phagy) refers to a process of distinct selective degradation of er membranes and proteins in the lysosome under stress, and this is independent of the core autophagy machinery [26] [27] [28] (for er stress, see sect. 18.5) . studies on yeast showed that er-phagy is characterized by the fact that stress-induced er whorls are selectively taken up into the vacuole, the yeast lysosome. import into the vacuole was found not to involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that er-phagy is topologically equivalent to microautophagy [27] . recent studies on yeast provide evidence suggesting that the atg proteins atg39 and atg40 are specific receptors for this pathway of er-phagy [28] . at this point, it also appears worthwhile to mention that a more recent study on yeast revealed a novel er quality-control pathway, namely, the so-called macro-er-phagy. first results from this study suggest that this pathway delivers an excess of integral-membrane proteins from the er to the lysosome for degradation and, typically, requires the core autophagy machinery [29] . the brief overview about macroautophagy provides another typical example of innate immune responses which, when controlled, operate in a beneficial homeostatic way but, when uncontrolled, may lead to severe pathologies. for other forms of phagocytic responses, this phenomenon has not been investigated sufficiently. clearly, mitophagy also plays a key homeostatic role in mitochondrial quality control. upregulation of mitophagy has been shown to mitigate excessive mitochondrial accumulation and toxicity to safeguard mitochondrial fitness. hence, mitophagy is a viable target to promote longevity and prevent age-related pathologies [25] . concerning the two types of er-phagy (micro-and macro-er-phagy), one has to state that research in this exciting field has just begun. several questions remain to be addressed, for example, what is the purpose of er-phagy and what are the underlying mechanisms. future studies will probably provide a clue to elucidating the molecular mechanisms and physiologic roles of er-phagy in other organisms. of note, besides mitophagy and er-phagy, other specific forms of phagocytic pathways have been described. they include -pexophagy as a macroautophagic response preferentially targeting peroxisomes -nucleophagy as an autophagic response selectively targeting portions of the nucleus -ribophagy as a specific autophagic response targeting ribosomes -aggrephagy as an autophagic response specific for protein aggregates -lipophagy in terms of selective autophagic degradation of neutral lipid droplets -bacterial xenophagy as a macroautophagic removal of cytoplasmic bacteria which have escaped the phagosomal compartment upon phagocytosis -viral xenophagy as a macroautophagic response targeting fully formed cytoplasmic virions or components thereof -proteaphagy in terms of macroautophagic responses specific for inactive proteasomes -lysophagy as a specific macroautophagic disposal of damaged lysosomes in mammalian cells for details of these specific forms of phagocytic responses, the reader is referred to the excellent comprehensive review article of galluzzi et al. [10] . in addition to macroautophagy, two other types of autophagy have been described called microautophagy and chaperone-mediated autophagy (cma). microautophagy together with macroautophagy plays, for example, a role in nutrient recycling under starvation. on the other hand, cma is known to contribute to the maintenance of cellular homeostasis by facilitating recycling of amino acids of the degraded proteins and by eliminating abnormal or damaged proteins, thereby exerting major regulatory functions in different pathophysiological scenarios such as metabolic regulation. here, a few aspects of these two types of autophagic responses are skimpily touched. by contrast to macroautophagy, the process of microautophagy is much less defined in mammals since most studies have been performed in yeast and plants. according to current models, the term refers to a collection of diverse processes. unlike autophagy, microautophagy does not involve the autophagosome-dependent degradation of cytoplasmic components but rather and characteristically relies on the direct engulfment of small portions of cytoplasm into lysosomes or late endosomes by invagination and inward budding of the lysosomal/endosomal membrane, a process that leads to their degradation [30] [31] [32] . though microautophagy is the least studied form of autophagy, a molecular signature of the process has begun to emerge and has led to the definition of microautophagy as a type of autophagy in which the cargo is directly internalized in small vesicles that form at the surface of the lysosome/vacuole or late endosomes (multivesicular bodies), respectively [10] . in addition to macroautophagy and microautophagy, there is another type of autophagy experiencing increased attention, the cma. characteristically, in cma, cargo delivery also occurs directly at lysosomes, but it does not require formation of vesicles nor membrane invagination. instead, the substrate proteins for this autophagic pathway cross the lysosomal membrane through a protein-translocation complex, that is, a process that requires protein interaction with the chaperone hspa8 (also known as hsc70) and association of hspa8 with a specific splicing isoform of lamp-2, that is, the lysosomal protein lamp-2a. thus, chaperone-bound autophagy substrates bind lamp-2a monomers on the cytosolic side of the lysosome, which stimulate the formation of an oligomeric lamp-2a translocation complex [10, [33] [34] [35] . essential functions that cma fulfils in cells include a contribution to amino acid recycling during prolonged starvation as well as quality control, directly linked to the ability of this pathway to selectively remove single proteins from the cytosol. for example, cma is up-regulated during oxidative stress where it contributes to the degradation of oxidized proteins (reviewed in [36] ) (see next sect. 18.3). of note, growing evidence demonstrates that malfunction of cma plays a vital role in the pathogenesis of severe human disorders. often, the mechanisms underlying the alterations of cma in these pathologies involve perturbations in the functioning of the cma translocation complex. both diminished and enhanced cma activities have been shown to associate with diseases, an observation that emphasizes the importance of a tight regulation of cma activity (highlighted in [36] ). as argued above, current knowledge about mechanism and physiological relevance of microautophagy in mammalian cells is hard to judge since most findings derive from studies in yeast. however, future studies aimed at identifying proteins controlling microautophagy-related vacuolar membrane changes in yeast will probably allow to search for homologues in mammals and then investigate their contribution to mammalian microautophagy. by contrast, research on cma has already made substantial progress. for example, the recent identification of a plethora of new cma substrates and deficiencies in cma associated with diverse human pathologies has expanded our understanding of the importance of cma in multiple cellular functions. in fact, the growing number of connections between cma and human diseases has already generated interest in modulating cma activity for therapeutic purposes. there is a close relationship between autophagy and mamps and/or damps in the cellular response to injury. in fact, autophagy cannot be restricted to an innate immune mechanism that controls intracellular homeostasis alone but has to be extended in terms of "immunological autophagy" to a process that is committed to control and regulate efferent innate and potential adaptive immune responses. the "medium" for achieving this goal is the mamps and/or damps which operate as a link between intracellular and extracellular events. this scenario is briefly touched in the following. growing evidence indicates that autophagy regulates release and degradation of damps-here in terms of inducible damps-including hmgb1, atp, and dna in several cell types [37] . for example, autophagic mechanisms reportedly promote and regulate the release and secretion of hmgb1 in a ros-dependent manner in fibroblasts, macrophages, and cancer as well as net-mediated release of hmgb1 in neutrophils [37, 38] . moreover, autophagy has been shown to be required for the liberation/active secretion of atp by dying cancer cells [39, 40] . in addition, autophagy was found to contribute to the regulation of the ddr at multiple levels, that is, a process associated with the emission of damps [37, 41] (for ddr, see sect. 18.6). via emission of damps, eventually, together with mamps, autophagy can amplify or even instigate mamp/damp-prm signalling leading to efferent innate immune responses. on the other hand, autophagy can inhibit pro-inflammatory signalling cascades. for example, the atg5-atg12 complex, a key regulator of the autophagic process, was shown to negatively regulate rlr signalling by direct binding to card domains of rig-i and interferon promoter-stimulating factor-1 (ips-1) [42] (compare part vi, sect. 22.3.6). moreover, as reviewed elsewhere [1] , autophagy has been found to inhibit both nlrp3 and aim2 inflammasome activation and subsequent production of pro-inflammatory cytokines il-1β and il-18 (for inflammasomes, see part vi, sects. 22.4.2 and 22.4.4). as a possible mechanism, the authors propose that inflammasome components and pro-il-1β are subjected to ubiquitination and subsequent degradation by autophagy, thereby leading to functional inactivation of inflammasomes. conversely, an increasing number of studies suggest that damps, including hmgb1, atp, and dna, are powerful stimuli and regulators to elicit autophagic responses [3, [43] [44] [45] [46] [47] [48] [49] [50] . for example, hmgb1 was demonstrated to be an important regulator of autophagy in various types of cancer cells and keratinocytes. mechanistically, the reduced form of the hmgb1 protein was proposed to be responsible for the promotion of autophagy in an ager/rage-dependent fashion [47, 48] . also, and of high interest, in a clinical study on patients with chronic hepatitis b, hmgb1-induced autophagy was found to maintain treg function during chronic viral infection [49] . moreover, in studies on a mini pig lung iri model, evidence was provided indicating that autophagy, when triggered by damps such as hmgb1 and hsp60 during iri, amplifies the inflammatory response through enhancing k63-linked ubiquitination of traf6 and activation of the downstream mapk and nf-κb signalling (for traf6, mapk, and nf-κb signalling, see part moreover, there is already first evidence suggesting a role of atp in the regulation of autophagy [50] . in addition, there are accumulating data indicating that cytosolic dna, dislocated as a result of dna damage, may contribute to the regulation of autophagy whereby the dna damage-regulated autophagy modulator 1 (dram1) appears to play a mechanistically crucial role [51] [52] [53] . the mechanisms involved in mamp/damp-activated autophagic responses have only partially been elucidated. in fact, there is convincing evidence suggesting that many mamp/damp-recognizing prms including tlrs (in particular endosomal tlrs), nlrs, and anti-dna receptors can activate autophagic responses by triggering specific signalling pathways (reviewed or discussed in [1, 2, 54, 55] ). the crosstalk between autophagic responses and damps represents a powerful instrument of the innate immune system to integrate and unify various tools for the promotion and regulation of injury-induced inflammation and, in the presence of nonself-or altered self-antigens, injury-induced adaptive immunity. thus, on the one hand, autophagy is known to promote and regulate the release of damps (though the exact mechanisms are still elusive); subsequently, damps via prmtriggered pathways participate in the regulation of inflammation. on the other hand, activation of prms by mamps and/or damps promotes autophagy activation through a mechanism that has been partially elucidated. in fact, an increasing number of findings suggest that this activation process is triggered by prms following recognition of mamps and/or damps. nevertheless, the precise molecular mechanisms by which prms modulate autophagy remain largely unknown. there is increasing evidence in support of the notion that mamp/damp-activated autophagic responses promote emission of damps which in turn support cellular homeostasis in the course of adaptive stress responses in healthy cells. notably, this cellular homeostatic effect may spread out and affect the whole organism via emission of autophagy-dependent damps. in other words, via damps, autophagy as a cell-intrinsic stress response can fortify its defending capability by providing a link to promotion and regulation of cell-extrinsic efferent innate immune and eventually subsequent adaptive immune responses. however, despite the fact that autophagy is one of the best-known cell-autonomous responses in innate immunity and has clearly been shown to counteract dangerous infectious and sterile cell stress, much is left unclear. one such issue concerns the definition of autophagy-dependent cell death. as discussed and summarized [10] , autophagy-dependent cell death can be defined as a form of rcd (see next chapter) that can be retarded by pharmacological or genetic inhibition of macroautophagy. in this context, as stressed by galluzzi et al. [10] , it is important to note that (1) specificity issues affect most, if not all, pharmacological agents employed so far for suppressing macroautophagic responses and (2) multiple components of the macroautophagy machinery have autophagy-independent functions. in view of these facts and findings, these authors recommend to favor genetic approaches and to test the involvement of at least two different proteins of the macroautophagy apparatus in a specific instance of rcd before etiologically attributing it to macroautophagy. other unclear issues refer to the specific modulation of autophagy by mamps and/or damps, the precise interaction of autophagy with innate immune signalling cascades, and the cooperation between autophagy and other physiologic cell-intrinsic and cell-extrinsic processes during scenarios of cell stress and tissue injury. efforts to solve these problems are of utmost importance in view of the fact that autophagy-when induced by excessive, chronic, or acute-repetitive emission of damps-can contribute to the pathogenesis of many human diseases, that is, acute and chronic, infectious, or sterile inflammatory disorders. at the respective places, they will often be mentioned in the following chapters as well as in volume 2. oxidative cell stress and tissue injury reflect most potent and omnipresent threats an organism is exposed to. though there is a robust defense response continuously operating, this kind of injury is known to contribute to the pathogenesis of many human diseases. how can this be? oxidative stress is caused by an imbalance between the production of oxidants such as ros on one side and the biological antioxidative defense system's ability on the other side to counter the oxidant levels with antioxidants, that is, to readily detoxify the toxic reactive species or easily repair the resulting damage. thus, it is the excessive production of ros-overriding the antioxidative capacities-that is pathophysiological and contributes to dysfunction, damage, and even death of cells. by contrast, generation of ros in physiological low/ moderate concentrations-operating as second messenger molecules and causing so-called oxidative "eustress" [56] -assists in intracellular signalling pathways and, thus, is essential for optimal cell functions and homeostasis of an organism. in other words, the biological effects of ros-beneficial or deleterious-considerably depend on the amounts of ros present and, in action, a phenomenon that is in agreement with the idea that cellular ros generation has characteristics of hormesis implying a dose-response phenomenon that is characterized by beneficial effects at low doses and deleterious effectivity at high toxic doses [57] . to guarantee this homeostatic function of ros, to keep these molecules within physiological limits, and to prevent their deleterious effects, that is, to maintain hormesis, a smooth running of the oxidative stress response is of utmost importance. accordingly, a few aspects of this critical stress response are addressed in the following. reactive oxygen species are produced from molecular oxygen as a result of normal cellular metabolism. to understand any discussion on a role of ros in host defense or human diseases, one should define free radicals. according to halliwell and gutteridge [58] , "a free radical is any species capable of independent existence that contains 1 or more unpaired electrons." an unpaired electron is one that occupies an atomic or molecular orbital by itself. radicals can be formed by the loss of a single electron from a non-radical, or by the gain of a single electron by a non-radical. in this sense, superoxide anions (o 2 ·− ), hydroxyl radicals ( · oh), peroxyl radicals (ro 2 · ), and alkoxyl radicals (ro˙) are oxygen radicals. of note, ros is a collective term often used by scientists to include not only the oxygen radicals but also some non-radical derivatives of oxygen such as h 2 o 2 , hypochlorous acid (hocl), ozone (o 3 ), and singlet oxygen ( 1 o 2 ). nitrogen-containing oxidants, such as no · are called rns. generation of ros is generally a cascade of reactions that starts with the production of superoxide anions. superoxide rapidly dismutases to h 2 o 2 either spontaneously (especially at the low ph) or catalyzed by sod. other elements in the cascade of ros generation include the reaction of superoxide with no to form the very toxic peroxynitrite, the peroxidase-catalyzed formation of hocl from h 2 o 2 , and the iron-catalyzed fenton reaction, leading to the generation of hydroxyl radical. the oxidants are produced endogenously as by-products or metabolites of various metabolic processes. multiple enzyme systems produce superoxide radicals and their derivatives including xanthine oxidoreductase (xor), the reduced form of nadph oxidases (noxes), and mitochondrial electron transport chain (etc)associated molecular complexes ( fig. 18 [61, 62, [64] [65] [66] [67] [68] [69] [70] mitochondrial etc is believed to be the main source of ros. in the following, some aspects of these three systems (other systems not mentioned here) are briefly touched, exemplified by and focused on their role as vascular sources of ros production as investigated on models of iri. xanthine oxidoreductase (xor), as a housekeeping enzyme, is probably expressed in all cells but primarily in surface epithelia such as capillary endothelial tissue of various organs. xanthine oxidoreductase, a complex molybdoflavoprotein, is the ratelimiting step in the catabolism of purines, where it catalyzes the last steps of purine metabolism: the conversion of hypoxanthine to xanthine and of xanthine to uric acid, with superoxide/h 2 o 2 generated as by-products. there is a definite role of xor in reperfusion of tissue and organs [59, 60] . for example, experiments performed in isolated rat hearts have demonstrated that radical generation and functional injury are decreased by inhibition of xor with oxypurinol. similarly, in human aortic or venous ecs, xor-mediated ros generation has been shown to be a central mechanism of oxygen radical generation upon postischemic reoxygenation [61, 62] . the nadph oxidases were initially considered as enzymes expressed only in phagocytic cells involved in host defense and innate immunity; however, recent evidence indicates that there is an entire family of noxes based on the discovery of gp91phox homologues. the family comprises seven members, including nox1, nox2 (formerly termed gp91phox), nox3, nox4, nox5, duox1, and duox2 [63] . three members out of the enzyme family are important sources of ros in the vasculature, namely, nox1, nox2, and nox4 [64] [65] [66] . today, noxes are perhaps the best-studied enzymes involved in ros production in the blood vessels. remarkably, different members of the nox/duox family engaged in iri are localized in various cells, that is, in vascular cells and phagocytes. this may lead to the notion that noxes in vascular cells are responsible for the first wave of ros production because vascular cells are first confronted with reintroduced molecular oxygen. as generally believed, there is, in fact, no vascular specific nox isoform but rather a complex expression of various nox isoforms in different cells and regions of the vascular system. nevertheless, in arteries from humans and animals, nox2, nox4, and a shallow level of nox1 have been consistently found to be present both as messenger rna and as protein [63] . altogether, the findings and data briefly described here make clear that vascular cells are equipped with efficient machinery able to efficiently produce ros. regarding the different enzymatic sources, superoxide radicals appear to be predominantly generated compared, for example, to hydroxyl radicals. mitochondria have been implicated as potential oxygen sensors by increasing the generation of ros which regulate a variety of hypoxic responses [67] [68] [69] [70] . in fact, mitochondria are increasingly recognized as lynchpins in the evolution of tissue injury during postischemic reperfusion. it is generally acknowledged that the majority of intracellular ros production is generated in the mitochondrial etc and its associated metabolic enzymes. however, very little is known about which mitochondrial sites are involved in physiological or pathological ros production under native conditions. of note, using inhibitors to manipulate the redox states of particular sites and prevent superoxide generation from others, at least ten different locations of superoxide/h 2 o 2 production in the etc and associated enzymes (krebs cycle, β-oxidation, etc.) have been identified in mammalian mitochondria. in fact, the relative and absolute contributions of specific sites to the production of ros in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely valid in cells and in vivo [71] . for example, superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (fig. 18.3) . complex i (nadh-ubiquinone oxidoreductase) accepts electrons from nadh; these electrons are carried to complex ii (the succinate dehydrogenase-coq oxidoreductase), where they are used to oxidize succinate to fumarate. afterward, electrons continue to travel down their electrochemical gradient to complex iii (the cytochrome bc1 complex (ubiquinol-cytochrome c oxidoreductase)), and subsequently to complex iv (cytochrome c oxidase); finally, the electrons are used to reduce molecular oxygen to water. thus, complex i and complex ii oxidize the energy-rich molecules nadh and flavin adenine dinucleotide h 2 , respectively, and then transfer the resulting electrons to ubiquinol that carries it up to complex iii (for competent articles, see [72, 73] notably, complexes i and ii generate superoxide within the mitochondrial matrix, whereas complex iii produces superoxide at the qo site, resulting in the release of superoxide into either the intermembrane space or the matrix. regarding complex i, it was recently demonstrated that inhibition of nd5, a subunit of complex i, suppresses the activity of this complex and thus ros production [74] . furthermore, data from another set of studies on complex i showed that stable down-modulation of its subunits grim-19 and ndufs3 decreased complex i activity that was associated with a significant reduction in the overall nadh oxidation rate but with an increased production of ros by the target cells [75] . similar results have been found in studies on complex ii: there is evidence suggesting that inhibition of complex ii on the level of subunits even leads to an increase in ros production. the phenomena can be explained by assuming that, if electrons provided in the course of the etc cannot efficiently be transferred to the next complex, they would leak out from the inhibited complex and generate ros [76] . the complex iii subunits rieske iron-sulfur protein (risp) encoded by ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide 1 (uqcrfs1), and ubiquinol-cytochrome c reductase binding (uqcrb) protein appear to play a crucial role in hypoxia-triggered mitochondrial ros generation (for rieske, see box 18.3). thus, it was shown that risp promotes the hypoxic stabilization of the transcription factor hif-1α protein [77] and uqcrb was found to mediate hypoxia-induced tumor angiogenesis via mitochondrial ros-mediated signalling [78, 79] . also, and of note, a mouse model to permit conditional deletion of the nuclear-encoded risp gene was recently developed to assess its role in hypoxiainduced ros signalling in the pulmonary circulation [80] . it was found that depletion of risp abolishes the ros response to hypoxia in isolated pulmonary the rieske protein is an iron-sulfur protein (isp) component of the cytochrome bc1 complex that was first discovered and isolated by john s. rieske and coworkers in 1964. the rieske iron-sulfur protein is an essential subunit of mitochondrial cytochrome bc1 complexes and, like the majority of mitochondrial proteins, is encoded by a nuclear gene and synthesized on cytoplasmic ribosomes as a precursor with a 32-residue amino-terminal extension. the iron-sulfur protein is then post-translationally imported into the mitochondria where it is inserted into the bc1 complex in the inner mitochondrial membrane. at first, the precursor is translocated via translocation contact sites into the matrix. there, cleavage to an intermediate containing an 8-residue extension occurs. the intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex iii. arterial smcs and isolated pulmonary artery segments. further, in this article, it was discussed that mitochondria are not the only source of ros during hypoxia. thus, studies using a genetic knockout of p47phox suggested that cytosolic nadph oxidase systems may also contribute to a hypoxic pulmonary vasoconstriction response during acute hypoxia [81, 82] . according to the authors' conclusion, the blockade of hypoxia-induced ros responses (in these studies observed with depletion of risp) suggests that the mitochondria may act as the initiators of ros production, which could be amplified by engagement of nadph oxidase systems elsewhere in the cell. such "ros-induced ros release" might permit small ros signals generated by mitochondria to activate ros signalling throughout the cell, thereby avoiding mitochondrial damage that might arise if the entire cellular oxidant signal originated from that organelle [83] (or even leading to excessive ros production?). indeed, the substantial advances in oxidative stress research of recent times, in particular, the specification of hypoxia-sensing ros-producing enzyme systems, will contribute to new therapeutic strategies to be applied in acute and chronic human diseases known to be influenced by oxidative stress. for example, discrimination of oxidative eustress, a fundamental process in maintaining health, from oxidative damage will improve clarity in developing "redox medicine" [56] . when the redox equilibrium of a cell is upset by pro-oxidant environmental stimuli, that is, when oxidative stress exists, an adaptive stress response takes place which can result in upregulation of antioxidant proteins and detoxification enzymes. these antioxidative defense molecules comprise the following [58] : (1) agents that catalytically remove free oxygen radicals and other reactive species, for example, sod, catalase, peroxidase, and thiol-specific antioxidants; (2) proteins that minimize the availability of pro-oxidants such as iron ions, copper ions, and heme, for example, transferrins, haptoglobins, hemopexin, and metallothionein; (3) proteins that protect biomolecules against damage (including oxidative damage) by other mechanisms, for example, hsps; and (4) low-molecular-mass agents that scavenge ros and rns, for example, glutathione, α-tocopherol, and (possibly) bilirubin and uric acid. in the past, it was fashionable to divide the oxidative stress response into three main tiers: (1) antioxidant enzymes including sod, catalase, glutathione peroxidase, and glutathione; (2) detoxifying enzymes such as glutathione peroxidase, glutathione s-transferase, aldo-keto reductase, and aldehyde dehydrogenase; and (3) energy-dependent efflux pumps. as a fourth defense system, the antioxidant nutrients such as vitamins e and c as well as carotenoids were appreciated [84] . it was generally accepted that the first line of enzymes is of enormous importance in limiting ros-mediated damage to intracellular macromolecules. for example, among the most important regulators of ros levels were the sod enzymes: cu/ znsod in the cytoplasm and outer mitochondrial space and mnsod exclusively in the inner mitochondrial space. mechanistically, superoxide is converted to h 2 o 2 and oxygen (o 2 ·− + o 2 ·− + 2h + → h 2 o 2 + o 2 ) by sod. peroxiredoxins and abundant catalase enzyme then scavenge h 2 o 2 , converting it to molecular oxygen and water. another example of a first-line defense molecule is trx. thioredoxin contains two adjacent -sh groups in its reduced form which are converted to a disulfide in oxidized trx. notably, it can undergo redox reactions with multiple proteins using the reaction trx however, it then turned out that these antioxidative principles were clearly not 100% effective at performing this task, as under normal physiological conditions, lipid and dna oxidation products can be detected in blood and urine. because certain compounds of the chemicals generated after an interaction of ros with macromolecules are highly reactive, there must be an equal necessity to detoxify these secondary oxidation products to prevent them from also damaging dna, proteins, and lipids. without the adequate detoxification of such products, an extended chain reaction will occur resulting in the degradation of cellular components and the ultimate death of the cell. this second line of defense against ros is provided by those detoxifying enzymes. finally, detoxified metabolites produced by these enzymes are eliminated from the cell by energy-dependent efflux pumps such as the glutathione s-conjugate transporter, also called the multidrug resistance-associated protein (mrp) [58] . today, one must state that these previous notions are incomplete. at first, it became apparent that members of the so-called cnc (for cap 'n' collar)-basic region-leucine zipper (bzip) family of transcription factors are principal mediators of defense responses to redox stress. in mammals, the cnc family members nrf1 and nrf2 were shown to be involved in the transcriptional upregulation of cytoprotective genes encoding a large number of diverse detoxification, antioxidant, and anti-inflammatory proteins (e.g., glutamate cysteine ligase, nadph-quinone oxidoreductase, glutathione s-transferases, and aldo-keto reductases) as well as enzymes with essential roles in cell metabolism [85] . more recent studies then revealed that these transcription factors, notably nrf2, are activated by keap1 as the primary negative regulator of nrf2, that is, a molecule that simultaneously operates as a sensor protein able to perceive dyshomeostatic subclass iic-4 damps, for example, in terms of redox changes reflecting electrophilic stress. it is worth to add here that six critical domains have been defined in nfr2 (neh1-neh6), and it is neh2, located at the n terminus of nrf2 that acts as the regulatory domain for the cellular stress response. actually, neh2 interacts with the cytoplasmic protein keap1 [86] . in the following, this very important damp-induced and gene-based antioxidative and cytoprotective system is addressed a bit more in detail. increasing evidence indicates that several redox-regulated gene products serve to protect cells from ros damage. the antioxidant response element (are), a cis-acting dna regulatory element or enhancer sequence is known to be activated by oxidative stress and to be responsible for the transcriptional regulation of several redox-regulated gene products. both nrf 1 and 2 bind to are and regulate are-mediated gene expression and induction. the molecule nrf2 is more potent than nrf1 in activation of are-regulated gene expression and is regarded as the principal transcription factor that binds to the are. this transcription factor is ubiquitously expressed and present in various organs and tissues including the kidney, muscle, lung, heart, liver, and brain (for reviews, see [87] [88] [89] [90] [91] ). as touched in part ii, sect. 5.3.2 and part iv, sect. 13.4.5, the nrf2-triggered antioxidant response is initiated by activation of keap1 that functions as a substrate adaptor protein for the degradation of nrf2 and serves as an intracellular sensor for redox changes reflecting the presence of subclass iic-4 damps (for reviews see [92] [93] [94] ). earlier studies had already shown that nrf2 is a bzip transcription factor that translocates to the nucleus after liberation under oxidative stress conditions from its cytosolic inhibitor keap1 [86] . in the nucleus, nrf2 was found to form dimers with the proteins maf, jun, fos, activating transcription factors 4 (atf4), and/or creb binding protein (cbp) and, in addition, regulates transcription by binding to the are upstream of a variety of cytoprotective and detoxification target genes to combat the oxidative stress [95] . thus, established nrf2-regulated genes reportedly included cu/zn sod, catalase, trx, trx reductase, glutathione reductase (gr), glutathione peroxidase (gpx), and ferritin l (ftl) [96] (ftl and ferritin h (fth) subunits are responsible for intracellular iron storage). all of these genes are involved in the response to oxidative stress. there are several other genes also known to be engaged in the response to oxidative stress that are not described here. recently, the molecular signalling mechanism involved in the keap1 ↔ nrf2 pathway has been further elucidated and specified. the core can be seen in an axis consisting of redox change (subclass iic-4 damps)-initiated → keap1-induced → nrf2-triggered → are-driven expression of antioxidant and detoxifying genes ( fig. 18.4 ) (discussed in [92-94, 97, 98] ). the complex and complicated sequelae of the pathway are simplified in the following text. under homeostatic and stress-free conditions, binding of keap1 to the nrf2 molecule leads to its polyubiquitination and subsequent degradation by the proteasomal pathway, thereby maintaining a consistent generation of nrf2 and retaining its very low levels in the cytoplasm. in this scenario, keap1 homodimer binds to a single nrf2 protein via a high-affinity so-called "etge" motif and low-affinity so-called "dlg" motif. the twosite recognition of nrf2 by the keap1 dimer is essential for polyubiquitination of nrf2 (also see part ii, sect. 5.3.2) (for polyubiquitination, see box 18.2) . in this sense, the keap1 ↔ nrf2 system can be regarded as a vital part of regulating cells under a homeostatic environment. however, in case of dangerous and threatening oxidative (and xenobiotic) stress, the system instigates a stress response that is characterized by a rapid and dramatic cessation of the keap1-dependent polyubiquitination process resulting in a rapid increase of nrf2 abundance. in fact, exposure to ros-mediated stress (or electrophilic stress) is thought to modify the reactive "iic-4 damps-sensing" cysteine residues in keap1, which is associated with a conformational change of the protein resulting in a loss of keap1 ubiquitination activity (fig. 18.4) . notably, as a cysteine-rich protein, the human keap1 possesses 27 cysteine residues, and they are all reactive to stress to varying degrees. among these sensor cysteines of keap1, c151 is best characterized. evidence from a number of studies has suggested that c151 is the most reactive and critical to the keap1 ↔ nrf2 stress-sensing response. even, there is already evidence from a first atomic-level view suggesting that the unique environment of cys 151 (besides cys171, cys273, and cys288) appears to be the critical residue of keap1 responsible for detecting increased levels of oxidative stress [91, 92, 97] . oxidative modification of cysteine sensors of keap1 leads to a loss of its polyubiquitination and degradation activity thereby stabilizing nrf2. consequently, fig. 18.4 the oxidative stress-induced keap1 ↔ nrf2 pathway. under non-oxidative homeostatic conditions, the sensor keap1is bound to the nrf2 molecule resulting in its polyubiquitination and subsequent degradation via the proteasomal pathway. oxidative stress modifies the ros-sensing cysteine residues of keap1 leading to loss of its polyubiquitination and degradation activity, dissociation of nrf2 that becomes stabilized and accumulates. then, nrf2 translocates to the nucleus and forms a heterodimer with the smaf transcription factor. the nrf2/smaf heterodimer binds to are and induces transcription of numerous cytoprotective antioxidant and detoxification genes. are antioxidant response element, keap1 kelch-like erythroid cell-derived protein with cnc homology (ech)-associated protein 1, nrf2 nuclear factor-erythroid 2 p45-related factors 2, smaf small musculoaponeurotic fibrosarcoma, ub-ub-ub poly-ubiquitin chain. sources: refs. [92-94, 97, 98] stabilized nrf2 accumulates in the cytoplasm, translocates into the nucleus, and forms a heterodimer with a smaf transcription factor (smaf, small musculoaponeurotic fibrosarcoma). thus, this accumulation of nrf2 in response to ros (and electrophiles) cannot be regarded as an induction in a strict sense but instead is a mechanism referred to as derepression, that is, from the rapid degradation-based repression. as highlighted and discussed [93] , there are two models of how to explain the cytoplasmic accumulation process of nrf2. the "hinge-and-latch" model holds that the modification of the sensing cysteine residues of keap1 reduces its affinity for nrf2 but does not result in release. instead, newly synthesized nrf2 is translocated to the nucleus to trigger the transcription of nrf2-dependent genes. the other model denoted as the "conformation cycling" model claims that keap1 uses a cyclic mechanism to target nrf2 for polyubiquitination and proteasomal degradation. an important feature of this cyclic mechanism is that it ensures regeneration of keap1 which allows the cycle to proceed. modification of specific reactive cysteine residues of keap1 may block the cycle of keap1-dependent nrf2 degradation allowing de novo synthesized nrf2 to accumulate. the subsequent transcriptional process in the nucleus has been specified as well. as partially mentioned above, the nrf2-smaf heterodimer binds to are or electrophile-responsive element (epre) and induces transcription of numerous cytoprotective genes. of note, recently, an extensive genome-wide analysis of the nrf2-smaf-binding sequence, that is, the are/epre, and the maf homodimerbinding sequence (the so-called maf responsive element or "mare") was conducted, and the differences between these elements were clarified. as a result, it was proposed that are, epre, and the nf-e2 binding sequence be collectively named cnc-smaf-binding elements (csmbe) [99] . interestingly, prms appear to be co-players in this scenario. thus, tlrs have been observed to induce nrf2 activation. remarkably, in a recent study, tlr agonists were shown to activate nrf2 signalling via reduction of keap1 [100] . the authors could demonstrate that tlr signalling-induced keap1 reduction promotes nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. further, tlr agonists were found to modulate keap1 at the protein post-translation level through autophagy. in fact, tlr signalling increased the expression of autophagy protein p62 and lc3-ii and induced their association with keap1 in the autophagosome-like structures. the keap1 ↔ nrf2 system as briefly described here is a robust oxidative stress response. its regulatory mechanisms, for example, stress-sensing mechanism, proteasome-based regulation of nrf2 activity, and selection of target genes have been elucidated mainly in mammals (for proteasome, see box 18.1). nevertheless, the pathway is now regarded as an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress across the tree of life. thus, the keap1 ↔ nrf2 system has been found to be also present in zebrafish, fruit fly, and caenorhabditis elegans indicating that its roles in cellular defense are conserved throughout evolution among vertebrates and suggesting that analogous defense systems are widely conserved throughout the animal kingdom [101, 102] . as briefly demonstrated in this subchapter, aerobic organisms have integrated antioxidant systems, which include damp-promoted generation of enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ros. however, when ros is produced in excess causing pathological conditions, the stress response against oxidative damage can be overridden. thus, oxidative stress is known to contribute to many pathological conditions, including cancer, neurological disorders, atherosclerosis, hypertension, ards, and chronic obstructive pulmonary disease, just to mention a few of them. plausibly, these disorders are motivation enough to search for new effective therapeutic options by harnessing the new insights into mechanisms of the keap1 ↔ nrf2 system. certainly, intense research is essential for a detailed understanding of the precise consequences of targeting keap1 for disease prevention and treatment. all the more so as the oxidative stress response is integrated into other forms of innate stress responses that will be further outlined in the following subchapters. the heat shock response-one of the most ancient and evolutionarily conserved cytoprotective mechanisms found in nature-is induced upon exposure of living cells to acute, subacute, or chronic stress conditions. this defense response is characterized by the expression of a group of phylogenetically conserved intracellular hsps, which possess the capacity to recognize structures commonly found in the interior of proteins and to bind such structures. thanks to this property, they form a chaperone network involved in correct protein folding, trafficking, and complex assembly (for reviews, see [103] [104] [105] [106] [107] [108] [109] ). in order to be released from engagement with proteins after folding and take part in further rounds of activity, hsp70 and other chaperones utilize an intrinsic atpase domain to hydrolyze atp and assume a free conformation [110] . once released, hsps mediate protective cellular defense mechanisms including regulation of apoptosis, the aim being to maintain and restore cellular protein homeostasis (proteostasis). exposure of almost any cell to heat shock most often leads to the rapid transcription, translation, and accumulation of a variety of hsps that increase to quite considerable levels when the stress is pronounced. in fact, these molecules are induced by various environmental insults that can cause protein denaturation and unfolding within the cells, leading to the formation of nonnative proteins and protein aggregates, thereby emitting dyshomeostatic damps. of note, besides their protective role in different intracellular compartments, hsps in terms of inducible damps can translocate to the cell surface to get exposed or are actively secreted via non-canonical pathways. in case of necrotic cell death, they are passively released in large amounts into the extracellular space to act as constitutively expressed damps (compare part iv, sect. 12.2.3 and sects 12.3.2.3 and 14.2.2.2). once emitted, hsps are sensed by classical recognition receptors and/or non-classical receptors (e.g., cd 91) [111] [112] [113] [114] [115] [116] [117] . interestingly, recent knowledge about similarities between allograft and tumor rejection has visualized that the processes of both iri to allografts [108, 118, 119] and therapy-mediated injury to tumors [120] [121] [122] [123] are characteristically associated with emission of hsps. in all eukaryotes, the hsr is primarily regulated and controlled by the hsfs, in particular, hsf1, a sequence-specific factor that binds upstream to heat shock elements in the promoters of target genes [124] (fig.18.5 ). for example, hsf1 is activated by environmental stress including oxidative and tumor-associated stress [125, 126] . notably, in all eukaryotes, hsf1 responds to such stress conditions by undergoing a monomer to trimer transition and becomes massively phosphorylated, leading to its acquiring ability to bind to dna rapidly and activate transcription [127] . there is at least preliminary evidence suggesting that intracellular perturbations reflecting dyshomeostatic damps may activate hsfs [128] [129] [130] [131] . such molecular alterations reportedly include changes in cytosolic ca 2+ concentration, for example, caused by increase of fluidity in specific membrane domains [128, 129] . interestingly, a recent study in support of these earlier findings provided evidence of the existence of a plasma membrane-dependent mechanism of hsf1 activation in animal cells, which is initiated by specific membrane-dependent trpv calcium channel-like receptors [130] . these findings lend support to the notion that heat sensing and signalling in mammalian cells are dependent on trpv channels, suggesting that these receptors may act as a major hsr sensor in different epithelial non-cancerous and cancerous cells, capable of triggering the cellular hsr [130] . in another line of experiments, trpv2 was demonstrated to mediate the effects of transient heat shock on endocytosis of human monocyte-derived dcs, suggesting a central role of trpv2 in mediating the cellular action of heat shock on these important cells of the innate immune system [131] (for trpv channel receptors, also compare part ii, sect. 5.3.6). induction of an hsr is not only mediated by sterile stress conditions but is also believed to be promoted by cell-invading viruses or bacteria. consequent emission of hsps in their role as damps may reflect a mechanism by which pathogens may contribute to sterile inflammation. for example, exacerbation of hepatitis b virus (hbv)-associated liver injury is reportedly characterized by an abnormal immune response that not only mobilizes specific antiviral effects but also poses a potentially lethal non-specific sterile inflammation to the host [132] . heat shock proteins may be the most extensively studied damps in the context of hbv infection. a number of hsps such as hsp70 and hsp90 have been reported to be supportive factors in the process of hbv replication, and selective inhibition of these hsps was proposed to be host-based anti-hbv strategies [133] [134] [135] . thus, as argued [132] , both infective and sterile inflammation may synergistically contribute to the exaggeration of chronic hepatitis, if the hbv cannot be cleared entirely. likewise, bacterial infections were also reported to promote induction of the hsr [136, 137] . for example, in studies on h. pylori and e. coli infection models, the initiation of an hsp70 stress response could be demonstrated [138, 139] . the hsr is recognized and accepted as a classical stress response in nearly all species across the tree of life. it reflects the desperate efforts of a cell to restore homeostasis and survive upon both infectious and sterile insults. its products, the hsps, operate as damps in commission of the innate immune system to reach this goal. notably, via this mechanism, an hsr, induced by infectious damage to a cell, can promote sterile inflammation. whereas a successful hsr upon stress leads to restoration of cellular homeostasis and cell survival, an unsuccessful hsr may result in rcd such as apoptosis [109] . this phenomenon will be resumed in the following subchapters. the er is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells. as a subcellular organelle in the control of proteostasis, it is responsible for calcium storage and lipid biosynthesis as well as the synthesis, correct folding, processing, and maturation of proteins as well as for the orchestration of their transport along the classical/conventional secretory pathway. the er delivers these components to their destination compartments which include the er itself, the golgi apparatus, the plasma membrane, and the extracellular milieu or the endocytic and autophagic pathways. plausibly, the multifunctional nature of this organelle requires a myriad of proteins, unique physical structures, and coordination with and response to perturbations in the intracellular environment. a series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support and execute these processes. perturbation of er-associated functions such as accumulation of unfolded/misfolded proteins, excessive ros production, hypoxia, calcium and glucose depletion, or viral and bacterial infections reflect stress of the organelle and result in activation of an er stress-coping response, the evolutionary conserved upr [140] [141] [142] [143] . for example, the processes of both iri-mediated cell damage/cell death [144] [145] [146] and induction of the icd of cancer cells [147] [148] [149] are characterized by demonstration of er stress that is almost always associated with oxidative stress and vice versa [150] . to cope with er stress, cells have the unique possibility to activate the upr, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers rcd modalities, depending on the level of damage. perception of any perturbation of the er is provided by three sensor molecules of the upr embedded in the er membrane: the perk, ire-1, and atf6. the upr-mediated recovery of er homeostasis mainly occurs through the perk-eif2α-mediated temporary shutdown of protein translation and the activation of a complex genetic program that aims to improve er quality control and adaptive responses [140] [141] [142] [143] (fig. 18.6) . accordingly, perk, devoted to perceiving dyshomeostatic damp-emitting er perturbations (so far, no clear evidence for ire-1 and atf6 in this respect), may be regarded as a new family of non-classical prms, at least in a broader sense. in nonstressed conditions, the three sensors of the er homeostasis are kept in an inactive state by the er-luminal binding immunoglobulin protein (bip). the protein bip, also known as glucose regulated protein 78 (grp78), is a member of the hsp70 family of proteins (specifically hspa5). it functions as a chaperone to selectively bind unfolded proteins in the er lumen by interacting with exposed hydrophobic 18 .6 simplified scenario model illustrating the er stress-induced three arms of the unfolded protein response. perception of any perturbation of the er is provided by three sensor molecules of the unfolded protein response embedded in the er membrane: the perk, ire-1, and atf6. perk perceives dyshomeostatic damp (subclass iic-4 damp)-emitting er perturbations (ire1 and atf6 still questionable). perk phosphorylates eif2a to up-regulate transcription factor atf4 that induces the expression of transcription factor chop. ire1a signals through its rnase via the splicing of xbp1 mrna. the active transcription factor xbp1s translocates to the nucleus. atf6 is exported from the er to the golgi complex to enter the nucleus as a potent transcription factor. together, these transcription factors of the unfolded protein response determine the cell fate by the regulation of distinct subsets of target genes toward recovery of er homeostasis and cell survival or the induction of regulated cell death in form of apoptosis and ferroptosis. in addition, the transcription factor atf4 is differentially translated, up-regulating genes participating in autophagy and other homeostatic pathways. atf activating transcription factor, chop cytidine-cytidine-adenosine-adenosine thymidine-enhancer-binding homologous protein, eif2α eukaryotic translational initiation factor 2α, er endoplasmic reticulum, ire1α inositol-requiring transmembrane kinase/endoribonuclease 1α, perk protein kinase-like eukaryotic initiation factor 2α kinase, upr unfolded protein response, xbp1 x-box binding protein 1. xbp1s, x-box binding protein 1 whereby the "s" stands for the spliced form of xbp1. sources: refs. [142, [153] [154] [155] [156] [157] [158] [159] [160] [161] residues on nascent peptides [151] . however, in conditions of er stress, bip is detached from these sensors allowing their activation to trigger pathways, collectively included in the term of upr. this stress response acts as a corrective path, capable of both increasing the er folding capacity and decreasing the incoming polypeptide load. of note, this downstream pathway of each of the three upr sensors appears to have an innate preference for a particular type of er stress. moreover, as reviewed [152] , upon dissociating from bip, each of the three sensors modifies the er to mitigate stress in its own unique way. for example, atf6 is often the first sensor to respond to er stress. once atf6 dissociates from bip, it is translocated to the golgi apparatus for cleavage. the cytosolic domain of atf6 is then free to move to the nucleus, where it moderates increased expression of several proteins involved in lipid biosynthesis and chaperones. this allows an increase in the volume of the er and provides more chaperone proteins to aid in folding, thus relieving some of the er stress. the other two sensors, ire-1 and perk, remain as integral er proteins but oligomerize and autophosphorylate following bip disassociation (autophosphorylation, a type of post-translational modification of proteins (see part vi, sect. 24.3); typical for this biochemical process, a phosphate is added to a protein kinase by itself). under remediable er stress conditions, the three sensors trigger signalling pathways to resolve er stress aiming at maintaining cellular integrity (fig. 18.6) . they are briefly touched in the following (for reviews and original articles, see refs [142, [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] .). for example, misfolded proteins are dislocated in the cytosol where degradation processes such as the er-associated degradation (erad) and autophagy will clear them, thereby reducing their potential toxicity. characteristically, erad is a protein quality control mechanism conserved in all eukaryotic cells and represents a critical arm of the upr, necessary to alleviate er stress. the erad mechanism results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. the erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups). the whole process includes transcriptional activation of a variety of er-associated chaperones and folding enzymes which include but are not limited to bip and the lectins calr, calmodulin (cam), and calnexin (cnx). a pathway that represents the most conserved branch of the upr is mediated by ire-1, a multifunctional protein that possesses kinase and endonuclease activities. upon activation, ire-1 aggregates and autophosphorylates, thereby activating its endonuclease activity to catalyze the unconventional splicing of x-box binding protein 1 (xbp1) via removal of a 26-nucleotide intron. this processing event changes the open reading frame of the mrna, resulting in the translation of the transcription factor now termed xbp1s ("s" stands for the spliced form of xbp1) (for splicing, see box 18.4) . production of xbp1s leads to upregulation of several genes involved in the upr's adaptive phase, for example, expression of er-resident molecular chaperones and protein folding enzymes. activation of perk leads to the phosphorylation of eif2α that is required for the initiation of translation. this factor inhibits global protein synthesis by inhibiting the assembly of the 80s ribosome, thereby reducing er load and promoting cellular survival. at the same time and under these conditions, the transcription factor atf4 is differentially translated, up-regulating genes participating in protein folding, amino acid metabolism and transport, autophagy, and oxidative stress resistance/redox homeostasis. under conditions of prolonged or severe er stress that the upr cannot resolve (see below), atf4 also contributes to apoptosis through the induction of the transcription factor chop (for cytidine-cytidine-adenosine-adenosine-thymidine-enhancer-binding homologous protein) and by enhancing oxidative stress and protein synthesis. finally, the third branch of the upr is initiated by atf6. the sensor atf6 is retained at the er under homeostatic conditions but translocates to the golgi apparatus under er stress where it is cleaved by the golgi-resident proteases site-1 protease (sp1) and sp2. this event leads to the release of atf6 n-terminal fragment, a potent transcription factor that moves to the nucleus, where it binds the er stress response element upstream of a subset of upr genes to activate their transcription. together with xbp1, this fragment regulates the expression of several genes with functions in protein folding, protein transport, and lipid biosynthesis, that is, genes involved in re-establishing er homeostasis. strikingly, there is a considerable interconnectedness of the er stress-promoted upr with other innate immune processes. for example, an increasing number of studies support the view that oxidative stress has a strong connection with er stress. during the protein folding process, ros are produced as by-products, leading to impaired redox balance conferring oxidative stress. as the protein in molecular biology, splicing is a modification of an rna after transcription in which introns are removed and exons are joined. thus, an intron is any noncoding nucleotide sequence within a gene that is removed by rna splicing during maturation of the final rna product; an exon is any part of a gene that encodes a part of the final mature rna produced by that gene after introns have been removed by rna splicing. this process is needed for the typical eukaryotic messenger rna before it can be used to produce a correct protein through translation. for many eukaryotic introns, splicing is done in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snrnps), but there are also self-splicing introns. folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and facilitate the production of misfolded proteins, causing further er stress [163] . moreover, this stress response functions as a productive source of damps. typically, hsps and calr, like other er chaperones, can translocate to the cytosol and eventually to the surface of cells. once exposed, these molecules can operate as subclass ib-1 damps to facilitate engulfment of antigens as mostly described in the context of induction of icd [148, [164] [165] [166] [167] [168] . via these mechanisms and by crosstalk with other molecular machines of the innate immune system including nlrp3 inflammasome activation via txnip (see part iv, sect. 13.4.6.3 and part vi, sect. 22.4.2.2) , the upr may contribute to sterile inflammation and immunity (for articles, see refs [153] [154] [155] [156] [157] [169] [170] [171] [172] ). also, several signalling cascades triggered by the three sensors are apparently potent inducers of autophagy at a cellwide level that-as mentioned above-normally has an adaptive/protective function and consists of the three major types: chaperone-mediated, macro-and microautophagy. interestingly, recent evidence has indicated that upr-induced autophagic processes are capable of alleviating the upr pointing to a crosstalk between these two innate immune defense mechanisms [173, 174] . strikingly, a complex relationship reportedly exists between autophagy and damps in cellular adaption to stress and injury and cell death characterized by a crosstalk between autophagy induction and secretion or release of damps. in fact, growing evidence indicates that autophagic mechanisms are involved in regulating release and degradation of damps including calr, hmgb1, atp, and dna in several cell types [37, 148, 175] . this scenario may contribute to the observation that autophagy is also able to shape a supportive cellular immune response [176, 177] . this kind of innate immune interrelationships is, for example, involved in mechanisms of both reperfusion-mediated cell injury and icd of cancer cells (see refs [40, [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] ). overall, depending on the duration and intensity of the stress, the upr engages different cellular pathways to restore and maintain cell survival, on the one hand, or trigger apoptosis, on the other hand. in cases of severe irremediable er stress, however, the balance is tipped in favor of pro-death signalling; that is, the upr, now mediated by different biochemical pathways, may lead to pro-inflammatory and pro-apoptotic responses resulting in catastrophic rcd (fig. 18.6) . while the precise pathways of apoptosis induced by er stress are not known, the up-regulated perk → eif2α → atf4 → chop pathway plays an essential role by reversing translational arrest, increasing generation of ros, and promoting calcium efflux from the er. together, these signals lead to cytochrome c release from mitochondria and loss of membrane potential, resulting in apoptosis [191] . in this context, it is interesting that one pathway of rn (ferroptosis; see sect. 19.3.3) apparently shares a partially overlapping machinery with er stress, suggesting a molecular interconnectivity between these two events [192] . the issue of er-stress-promoted upr is a parade example of an injury-induced innate immune response that can decide besides life and death of a cell. placed at the very beginning of defense processes of multicellular organisms upon injury, this stress response nicely reflects a hierarchy of damp emission (see part iv, chap. 16) . striking is also the existence of an inter-organelle communication, for example, between upr, inflammasome activation, and autophagic pathways which emerge as a homeostatic network determining the switch from adaptive life-saving programs to cell death under stress conditions, where specialized sentinels are localized at organelle membranes to induce the core apoptosis pathway. as briefly sketched in the next section, this innate immune defense response is not only directed against sterile stress but also against pathogen-mediated stress. in the previous section, induction of a upr was mainly exemplified by referring to reperfusion-mediated cell damage and icd of cancer cells, that is, instances of sterile stress. however, growing evidence is coming to light clearly indicating that viruses and bacteria also induce er stress, thereby activating a robust upr. in fact, the constitution of cellular stress responses is meanwhile regarded as the first line of defense against both viral and bacterial infection. however, the outcome is not always in favor of the host; the pathogen may also profit from this stress response, at least under certain circumstances. an increasing number of reports have recently been published on this emerging topic (such as refs [192] [193] [194] [195] [196] [197] ), the quintessence briefly being addressed here. there are several mechanisms described of how a virus can induce er stress. the central mechanisms of perturbation of the er during virus infection can be seen in the production of large amounts of viral proteins by the virus concerned. such accumulation of viral proteins in the er implies a challenge to the protein folding machinery which may cause er stress and, in turn, activate the upr resulting in restoration of the er homeostasis or apoptosis. so far, at least 36 viruses have been found to be able to induce er stress and activate the three upr stress signalling pathways [198] . moreover, er stress can be caused by viruses via other mechanisms, for example, as a result of er membrane exploitation, imbalance of calcium concentration, or sabotage/depletion of the er membrane during virion release. viral infections may activate these pathways resulting in the inhibition or promotion of viral replication. for example, the perk-mediated global translation shutdown is a very efficient antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [199] . also, the virus-related upr was found to trigger host inflammatory signalling cascade through innate immune signalling pathways that activate nf-κb and ap-1 transcription factors as a result of chronic er stress. in fact, overexpression of viral proteins in the er has long been known to activate these transcription factors which induce expression of pro-inflammatory cytokines such as il-6 [200, 201] . increasing evidence supports the notion that the upr signalling synergistically interacts with virus-induced signalling to produce inflammatory cytokines and type i ifns. in addition, other lines of studies have shed light on a role of nod1 and nod2 receptors in transducing virus-related er stress signals to elicit inflammation [197] . so far, however, a possible contribution of damps to the promotion of these signalling pathways has not been investigated. importantly, however, the effects of virus-induced upr have been observed not only to inhibit but also to potentiate viral infection. in fact, manipulation of the upr response has become an asset for many viruses to promote their translation, thereby leading to chronic er stress. in other words, during infection, viruses are capable of hijacking the host translational machinery and fill the er with viral proteins. for example, this is the case for many positive-strand rna viruses, which house the virus replication machinery in the protective er-membrane. in fact, viruses need host er to produce increased quantities of viral proteins to continue replication. intriguingly, as discussed [194] , many viruses have evolved strategies aimed at continuing the replication cycle. thus, viruses were shown to manipulate the host upr in various ways to stimulate protein synthesis capacity and to improve cell survival by inhibiting cellular apoptosis. in particular, the link between the upr and autophagy are intensely discussed to be involved in this scenario. these two systems may act dependently, or the induction of one system may interfere with the other [202] . thus, experimental studies could demonstrate that different viruses modulate these mechanisms to allow them to circumvent and bypass the host immune response or, worse, to exploit the host's defense to their advantage. according to current knowledge, rna viruses including influenza virus, poliovirus, coxsackievirus, enterovirus, japanese encephalitis virus, hcv, and dengue virus were shown to regulate these processes. for example, recent studies on hcv-infected hepatocytes confirmed the evidence that virus-associated er stress and upr are linked to cellular autophagy. thus, induction of the cellular autophagic response is reportedly required to improve survival of infected cells by inhibition of cellular apoptosis. moreover, the autophagic response was demonstrated to inhibit the cellular innate antiviral program that usually inhibits virus replication. nevertheless, as argued by the authors [194] , though hcv induces er stress and autophagy, their cause-effect relationship is not clear. notably, the upr is not only modulated by viruses! recent evidence indicates that this stress response plays multiple roles during bacterial infections as well. thus, as comprehensively reviewed by celli and tsolis [203] , the upr has been shown to be induced in murine lungs by m. tuberculosis (associated with apoptotic events) and also be correlated with helicobacter-induced gastric carcinogenesis. similarly, in vitro infectious models have revealed upr induction in macrophages and epithelial cells infected with either brucella melitensis and b. abortus or listeria monocytogenes. on the other hand, there is evidence suggesting that bacteria can subvert the upr for their own advantage. nevertheless, indications that bacteria can modulate this response are still somewhat sparse. one example refers to l. pneumophila that recruits components of the erad on its vacuole to mediate turnover of bacterial effectors on the vacuolar surface and uses the proteasome to generate amino acids necessary for its intracellular growth. interestingly, like with virus-induced upr, a bacteria-initiated upr may turn out to be of advantage for the host or in favor of the bacteria, but it is not clear in each case whether this response benefits the host or the pathogen. thus, as argued by celli and tsolis [203] , "modulation of er function during infection by intracellular bacteria can promote bacterial infection by providing a replicative niche, but at the same time the resulting disruption of the secretory pathway can provide a pattern of pathogenesis that aids the innate immune system in recognizing intracellular infection and in mounting an appropriate defence. however, considering the more rapid evolution of bacterial pathogens compared to their hosts, it is likely that bacteria have evolved to modulate the upr to their advantage during infection." the function of the er stress-induced upr appears to provide another impressive evidence in support of the notion that immunity is induced by both sterile and infectious stressful injury and not primarily by invading nonself. in fact, this stress response is at the forefront of any stressful injury and is dedicated to initiating involvement of further damp-promoted defense mechanisms. however, like all the other innate immune processes, the upr may become uncontrolled. together, this may be reason enough that this stress response is involved in the pathogenesis of many human systemic and organ-specific disorders. in fact, the list of human diseases that are pathogenetically associated with er stress and activation of the upr is steadily growing [204] [205] [206] . they include neurodegenerative and metabolic diseases, autoimmune disorders, and atherosclerosis. given such an ever-increasing list of diseases, it is not a surprise that chemical compounds and inhibitors targeting the upr signalling pathways with a high degree of specificity have been and will be further developed [207] [208] [209] . maintaining genome integrity and transmission of intact genomes is a condition sine qua non for cellular, organismal, and species survival [210] . this homeostatic integrity is threatened by dna damage that occurs in a variety of conditions including but not limited to ionizing radiation, chemical reactions, and viral infections, two of the most dominant conditions being oxidative stress and replication stress. these exogenous and endogenous factors induce diverse lesions in the dna such as nucleotide alterations (substitution, deletion, and insertion), bulky adducts, collapsed dna replication forks, ssbs, and dsbs (see refs [57, [211] [212] [213] [214] [215] [216] [217] [218] [219] ). in response to dna damage, cells initiate and activate a complex network of cellular signalling cascades that cooperate to sense and repair lesions in dna, denoted as the ddr. this stress response plays an important role in fighting against detrimental effects of cell stress and injury. it orchestrates many processes, including not only dna repair but also regulation of cell-cycle checkpoints, transcription of ddr genes, and autophagy ( fig. 18.7 ). the ddr is controlled by three pi3k-related kinases (pikks): the atm, the dna-pk, and the atr kinases (mostly nuclear proteins). all three pikks are enormous polypeptides with similar domain organizations and various common structural features. equipped with the capability to autophosphorylate (see above however, at the beginning of this dance, sensors have recently been identified which, as prms in a wider sense or even as a new group of recognition receptors, are capable of detecting dna damage-as manifested by the toxic dsbs or generation of ssdna to activate these three pikks. as already briefly touched in part ii, sect. 5.2.6.4, two highly conserved multiprotein complexes, mrn and ku, are considered the primary sensors of dsbs to subsequently activate atm and dna-pk [221, 227, 228] . in addition, ssdna is sensed by the recognition molecule rpa that then, analogous to ku, acts as a signalling and repair platform for downstream factors such as the prp19, an e3 ubiquitin ligase involved in pre-mrna splicing, and atr kinase [229] . other lines of studies have provided evidence suggesting that the protein prp19 itself may act as the primary sensor of rpa-ssdna to subsequently activate atr [224] (fig. 18.7) . in brief, upon dna damage, dsbs are recognized by the mrn complex. following recognition, mrn recruits atm to this dna lesion where it binds to the c-terminus of nbs1 as a component of mrn [230] . following binding, atm kinase is activated. however, the exact mechanism whereby mrn activates atm is still not fully understood (discussed in [226] ). recognition of dsbs is also realized by ku70/80 heterodimer that has been shown to bind broken dsdna ends preferentially [231] . it is the ku70/80 then that recruits the catalytic subunit of dna-pk (dna-pkcs) at the site of dsbs to form the dna-pk holoenzyme. the major role of activated dna-pk is to promote a peculiar dna repair mechanism called non-homologous end joining (nhej), a pathway that repairs double-strand breaks in dna [231] . in fact, nhej repairs most dsbs in mammalian cells. as its name implies, nhej involves ligation of two broken dna ends without needing a repair template [232] . in contrast to atm and dna-pkcs which respond primarily to dsbs, atr is activated by a much wider range of genotoxic stresses, for example, reflected by exposure of increasing amounts of ssdna as a consequence of compromised activity of replication proteins or nucleolytic processing of various forms of damaged dna. in fact, first evidence suggests that ssdna is initially sensed by rpa which then recruits and activates prp19 [229] . in turn, prp19 facilitates the accumulation of atr-interacting protein (atrip, the regulatory partner of the atr kinase) at dna damage sites, thereby activating atr [233, 234] . plausibly, the recent discovery of dna damage-sensing molecules such as mrn, ku70/80, and rpa calls for the definition of those molecules they recognize, that is, broken dna ends at the site of dsbs and ssdna. as outlined in part iv, sect. 13.4.2, they have been tentatively sorted into a subclass of cell-intrinsically emitted damps (subclass iic-1). future studies will have to assign their exact place in the world of damps. apart from those damps emitted in the nucleus, other lines of studies lend support to the suggestion that dna damage or dna replication stress, in case of unsuccessful dna repair, promotes release of aberrant dna structures into the cytosol in the form of ssdna and dsdna breaks/fragments which operate as cell-intrinsically emitted dislocated damps. they are sensed by the dna receptor cgas and probably other as-yet-not-identified dna receptors to activate sting-dependent pathways to promote defense pathways [235, 236] (compare part ii, sect. 5.2.6; part iv, sect. 13.4.3; as well as part vi, sect. 22.3.7). as discussed by the authors [236] , it is conceivable that cytosolic dna is released by dysfunctional mitochondria upon dna damage or generated during repair of damaged genomic dna. moreover, as shown in studies on tumor models, the ddr-through the activation of sting-mediated pathways-induces the expression of another class of constitutive damps which are exposed at the cell surface, namely, the mics and different ulbps [236] [237] [238] [239] [240] (compare part iv, sect. 12.3.3). of note, however, the ddr does not always result in a happy end. in fact, when unsuccessful in repairing dna damage, the ddr can lead to cellular senescence or-like a upr in case of irremediable er stress-ultimately induces an rcd, most often in the form of apoptosis, less frequently in the form of necrosis. such subroutines of rcd are presumably aimed at mitigating the propagation of potentially mutated cells leading to cancer or other age-related pathologies (fig. 18.7) [222, 223, 241]. the dna damage response must be regarded as an efficient cell-intrinsic defense process, which is sophistically connected with other stress responses such as autophagy that also plays a significant role in maintaining genome stability [242] and the er stress-induced upr [243] . also, of particular interest is the observation that three tiers of damps are involved in this pathway, starting with broken dna ends at the site of dsbs and ssdna in the nucleus immediately generated upon dna damage; followed by dna fragments dislocated in the cytosol and operating as class iic-2 damps to promote intracellular innate immune signalling; and finishing with the exposure of subclass ib-2 damps (e.g., mics) able to activate nk, nkt, and γδ t cells (compare part vii, sects. 27.2.2, 28.2.2 and 28.4.2). this phenomenon may again reflect a particular hierarchy in the work of damps to maintain homeostasis. in fact, genomic integrity is of utmost importance and key to human health, and this is retained by the ddr. this stress response, however, may fail in maintaining and restoring homeostasis. accordingly, dna damage has been observed to play a causal role in numerous human pathologies associated with genome instability or aberrant pikk function such as cancer, leukemia, premature ageing, and certain chronic inflammatory conditions. furthermore, there is growing evidence suggesting that uncontrolled ddr signalling is associated with various neurodegenerative diseases, as documented by recent work showing that atm inhibition alleviates pathologies in models of huntington's disease [244] . this is reason enough that ddr pathways have been and are being explored therapeutically to induce freedom from these diseases. in particular, small molecule inhibitors of atm, dna-pk, and atr are regarded as potential therapeutic agents, for example, as innovative drugs in cancer treatment [245, 246] . an increasing amount of publications in the international literature provides convincing evidence that cell-intrinsic, cell-autonomous stress responses are initiated by any damage to a cell, regardless of being of sterile or infectious in nature. the ultimate goal of those responses, which are all highly conserved among eukaryotic species, is to maintain cellular homeostasis and ensure cell integrity. again this knowledge is in support of the concept that any form of host defense is primarily directed against injury and not against microbes. autophagy in terms of "immunological autophagy" appears to act as the center of all stress responses that is committed to control and regulate efferent innate and potential adaptive immune responses. the 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the cytochrome bc1 complex key: cord-329206-xsxkn5my authors: vojtkovská, veronika; voslářová, eva; večerek, vladimír title: methods of assessment of the welfare of shelter cats: a review date: 2020-08-28 journal: animals (basel) doi: 10.3390/ani10091527 sha: doc_id: 329206 cord_uid: xsxkn5my simple summary: the welfare of animals in shelters draws the attention of both the scientific and general public. it is possible to assess the well-being of cats in shelters using tools that are based on indicators used to reveal problematic aspects of welfare. this review aims to provide an insight into available methods of assessment of the welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators. abstract: at any moment, there are millions of cats housed in foster care facilities for abandoned and stray animals for various reasons worldwide. care, management and regulation among these facilities differ. moreover, shelters can never substitute the full comfort of a good home for the animal, and the welfare of cats in shelters is a subject of discussion in many respects. cats are animals sensitive to changes; for most of them, placement in a shelter is a stressful experience because of changes in routine, environment and the presence of other animals. stress is reflected in changes in behaviour, causes fluctuations in physiological values and disrupts the immune system, which is a predisposition to the development or reactivation of disease. evaluation of the presence and intensity of negative impacts is possible through the use of evaluation tools based on indicators that help set the environment and management of keeping so as to disrupt the quality of life as little as possible. although a comprehensive and valid welfare tool that would evaluate animal-based and at the same time resource-based (or management-based) indicators of cats in shelters is not currently available, it is possible to use partial evaluation of individual welfare indicators to assess welfare. this review aims to provide the readers with an insight into current options of assessment of the welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators with an application in both practical and scientific contexts. in recent years, cats have become the most popular pet animals in western europe and the united states. their number in the european union reached over 103 million [1] and in the united states over 94 million [2] . the result is an increased focus on issues dealing with their well-being and behaviour [3, 4] . at any moment, there are millions of cats housed in shelters around the world [5] . for example, approximately 3.2 million cats enter shelters in the united states every year [6] . in canadian shelters, 600,000 cats were housed in 2011 [7] . in 2009, 131,070 cats entered shelters in the uk [8] , 33,719 cats were housed in shelters in spain in 2018 [9] and about 7400 cats are housed in shelters in sweden annually [10] . the traditional view that animals with unrestricted movement pose a risk to public health and safety, and that the solution is euthanizing them, has already been partially overcome. in most western countries, the approach to solving the problem has changed over the last 30 to 40 years; non-governmental organisations and in some countries, public authorities have invested in programs to increase the number of successful adoptions to minimise the need for animal euthanasia [11] . in order to eliminate euthanasia of animals in shelters, some countries (e.g., czech republic, italy, sao paulo, austria, india, taiwan, germany, costa rica) [12] have adopted the so-called no-kill policy. in these countries, shelter cats can only be euthanised for medical reasons. in other countries, on the other hand, the shelters themselves determine the functioning policy; for instance, some no-kill shelters refuse to accept animals that are sick, elderly or have unacceptable behaviour because they are bad candidates for adoption. however, this type of restrictive policy can also lead to worsening of welfare in an unwanted group of animals and overcrowding in shelters that have open admission policies [13] . all facilities providing temporary care should aim to reduce the animal's stay there to a minimum. a problem can arise if the no-kill policy orders facilities to accept all animals and keep them in a shelter until they are adopted by a new owner, returned to the original owner, die of natural causes or are put down for health-related or behavioural reasons. an animal that no one is interested in (often an older animal or an animal with a disability) can therefore legally stay in a shelter for a very long time. however, shelters are generally not designed as facilities that would be able to replace a new home in the long run; the animal generally has limited living space and access to resources, which it often shares with other animals with various medical histories. since a multitude of facilities does not have a sufficient amount of staff at their disposal, only a minimum of time is reserved for the care of each animal; ammons [14] reports an average of 15 min per day per animal. the common goal of most facilities is to temporarily provide the animals with a suitable space that takes into account nutritional, housing, health care and human contact requirements. the quality of care is a critical aspect of cat welfare in many facilities [15] . various factors of shelter environment can be stressful for cats (different care routine, lack of a familiar/bonded caretaker, veterinary treatments, increased infectious pressure, the presence of other animals, inadequacy in terms of space or poor environment and overall lack of control over the environment) [16] [17] [18] [19] [20] . the intensity and number of negative factors may reflect the condition of the shelter; in many countries, the system for dealing with unwanted animals is complex, lacks comprehensiveness and the cooperation between the state (or state facilities) and private facilities is not functional; likewise, the level of supervision in various types of facilities is different, sometimes even completely absent. the standard of maintenance and care of animals can vary significantly across facilities, as can their funding. insufficient funding may be a factor causing a number of other potential animal welfare problems [13] . animal welfare can generally be assessed from three different perspectives [21] . the first defines well-being through a biological point of view-the welfare of the animal is preserved if there is no deterioration in health and reproductive ability. the second view assumes that well-being depends on the ability to engage in natural behaviour. although this approach is traditional, today there is a tendency to attribute feelings and emotions to animals; trying to understand mental processes creates another perspective on welfare. the third view of the welfare concept, applied in the manuscript, involves the assumption that animals are sentient beings capable of experiencing positive and negative emotions [22] . because the subjective state of well-being is the result of the mental processes which are intrinsic to the animal in which they take place and invisible to the eye, its assessment is difficult. the fact that animal welfare is based on the subjective states perceived by the animal prevents us from its accurate assessment in spite of utilising any available methods. while current evaluation methods do not possess the capability (or this capability is limited) to provide direct insight into the mental state of the animal, at least the indirect methods are available [23] . the development of the first protocols for the practical assessment of welfare began in farm animals. efforts to evaluate welfare first led to the creation of some indicators of well-being; these focused on key aspects-health, behaviour, housing and management of keeping [22] . it is now customary to divide these indicators into two groups-a group of indicators, which directly relates to the evaluated animals (indicators of behaviours, health and physiological markers of stress [23] ) and a group concerning the resources of the environment in which the animal lives. it is appropriate to assess the well-being of the animal in terms of comparing the provided living conditions with the optimal environment, and in terms of the strategies that the animal must apply to cope with the difficulties and successfully adapt to the conditions [23] . the welfare quality project [24] , focusing on cattle, pigs and poultry, proposed a scientifically valid welfare assessment system and its four principles (good feeding, good housing, good health, appropriate behaviour) became a key basis in designing tools and evaluating the welfare of two other categories of animals-laboratory animals and companion animals. while in the past the general interest was concentrated on livestock (with the intensification of agriculture linked to the emergence of large-scale livestock farming, it was natural for questions pointing to the deteriorating living conditions of farmed animals to arise), attention has now also shifted to species that seemed previously least affected. unlike in cats, tools that assess the well-being of dogs have been the subject of several studies [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . in addition, evaluation tools have been developed to assess the behaviour of dogs in terms of its prediction in the home environment of the new owner. the tests are mainly aimed at detecting aggression [37] [38] [39] [40] [41] [42] [43] [44] [45] . the tools have been criticised because some authors [46, 47] consider them to be scientifically insufficiently validated to decide on the future of the animal (often, based on testing, the shelter decides to move the animal to another facility or euthanise it because of unacceptable behaviour or low adoption potential). shelters in none of the european union countries use an officially validated tool to assess the behaviour of dogs in shelters [48] . behavioural tests have also been developed in shelter cats [49] ; their main aim, once admitted to a shelter, is to discriminate socialised animals from unsocialised ones in order to preserve their welfare. temperament tests form another group of tools used in cat shelters; the goal is to assess the cat's temperament and find a compatible new owner [50] . however, the situation in evaluating the welfare of cats in shelters is different compared to dogs-at present, there is no comprehensive well-being evaluation tool usable in cat shelters. efforts to improve animal welfare are noticeable, however, e.g., within the european union, a number of member states have not laid down a legal obligation regarding any specific procedures to be performed after the animal is admitted to a shelter or during its stay in a shelter [48] . since there is no comprehensive tool for evaluating the welfare of cats in shelters, it is desirable to consider currently available tools that enable to assess individual aspects of welfare, i.e., to use the indicators that have been developed to evaluate the welfare of cats under specific conditions. the aim of the review was to provide the readers with an insight into welfare assessment tools, whose criteria may form the basis for the development of a complex, more comprehensive and valid tool evaluating the welfare of cats in shelters and summarise additional options of assessment of welfare of cats in shelters with an emphasis on behavioural, physiological and health indicators. welfare or quality of life (qol) are interrelated concepts and are often interchanged; according to some authors [51] [52] [53] [54] their definitions are identical. while the welfare concept has been presented only for the last few decades (one of the first definitions of welfare was introduced by broom in 1986 [55] ), the origin of the term 'quality of life' dates back to the days of plato and aristotle, who used the term to examine the conditions necessary to live a 'good' life [56] . the principle of the original concept, which became widely accepted, was to assess the quality of human life in terms of health aspects [57] . the term qol was later introduced into veterinary medicine; the concept began to apply when deciding between treatment and euthanasia of an animal in veterinary clinics [58, 59] . although there are several approaches to understanding the focus of the concept of qol, taylor and mills [57] have provided a general definition of qol applicable in various contexts. according to the authors, qol can be defined as an individual state that an animal perceives at a given moment in its existence; the condition should include a balance between the positive and negative attributes of life. cummins [60] adds that well-being is the result of many factors; it is a multidimensional construct. while tools for assessing farm and laboratory animal welfare are based largely on five freedoms [61] (freedom from hunger, thirst, pain, injury, illness, discomfort, fear and anxiety and freedom to express normal behaviour), the evaluation of companion animals is mostly focused on the use of health and behavioural parameters that can provide insight into the animal's internal state and its ability to cope with adverse conditions [62] . stress (especially distress) has an impact on the quality of life of animals [54] . in addition to the evaluation of behaviour and health, it is possible to evaluate the worsened welfare or the ongoing stress response by using physiological markers of stress [23] . it is challenging to select indicators that, in turn, represent positive emotions and good living conditions, i.e., indicators or conditions that reflect a good quality of life. the proposed indicators of positive emotions reflect the positive cognitive attitude of the individual; these are behaviours such as play, exploration, vocalisation, social behaviour and grooming [63, 64] . however, some of these indicators have not been fully validated in order to be usable in practice. in developing welfare assessment tools, reliability and validity are key to assessing whether the resulting concept actually measures what it was designed to [65] . reliability is expressed as a measure of repeatability and consistency [66] and can be measured within a single observer (intra-observer) and between multiple observers (inter-observer), within a test (test-retest) and between components which are the subjects of the test (internal-consistency) [67] . it does not mean that a tool that was previously validated in the original language is automatically valid when its language is changed or when it is used in other cultural contexts [68, 69] . before assessing reliability and validity, the purpose and standardisation of evaluation need to be addressed [67] , taking into account practicality and executability [65] . there is a risk that measurements that take a long time or are too complex will not be performed correctly [67] or will not be used at all. some of the practical problems in assessing welfare or qol are similar to those that arise in assessing people with communication and cognitive impairments. like them, animals cannot provide their own self-assessment [70] [71] [72] ; therefore, questionnaire methods such as those common in human medicine cannot be used to assess their quality of life [73] . questionnaires can be used as comprehensive tools containing objective records [74] on nutrition, level of activity, environment, social interactions and behaviour when filled out by a person closely related to the animal [75] , i.e., the caregiver of the sheltered cat. however, the disadvantage of most animal-based measurements is their subjectivity [76] . the caregiver is required to interpret the animal's expressions correctly, which is a problem if shelter staff are not sufficiently educated and trained-testing can yield anthropomorphised, biased findings. results can also be influenced by the principles and feelings of the individual who evaluates the animal [75] . traditionally, quality of life assessment can focus not only on the assessment of the animal itself but also on the assessment of resources [77] and management (thus a combination of several indicators is used for evaluation), as these remain constant and can be measured objectively (e.g., assessment of appropriate housing, nutrition and the human factor). an isolated assessment of resources is insufficient because it does not provide a real picture of the level of welfare, but only indicates the risk of its deterioration; although optimal management may be set up in the shelter facility and the animal may have all the necessary resources at its disposal, this does not automatically mean that it is healthy and provided with a high standard of welfare. a comprehensive welfare assessment tool should, therefore, be based primarily on the parameters of the animal, supplemented by other parameters. in some studies, the approach of assessing quality of life only from the point of view of health is maintained [25] . if welfare assessment is set up properly, it takes into account feedback on changes that affect the animal's welfare and allows for appropriate course of action to be taken thereon [78] . the majority of tools assessing the quality of life that are available today tend to focus on evaluating the lives of cats as patients with certain types of health problems, such as diabetes mellitus [71] , cancer (see [79] for the review of current methods of qol assessment in cats receiving chemotherapy), degenerative joint disease [80, 81] , heart disease [82, 83] , skin problems [84] or chronic disease in general [85] . these tools cannot be used for the evaluation of healthy cats, as they consist of disease-specific parameters. in addition to specifically targeted tools, efforts have been made to create more general tools that can be used in a variety of situations, facilities and contexts. their purpose is to define and differentiate normal behaviour and health and their deviations that an individual exhibits. some of these comprehensive tools (modified karnofsky score, chew [cat health and wellbeing] tool, owner completed measure of feline qol, catqol tool, the awag software, shelter quality and the shelter dog qol evaluation tool) could be considered for shelter cats' welfare assessment after some adjustments (these tools were developed for the use in different context, so they are not able to cover the full range of evaluation criteria requirements emerging from the shelter environment) or their principles could be used when creating a new assessment protocol. a qol tool that can be used to assess aspects of cat health and well-being without specific health problems is the so-called karnofsky rating scale, modified by hartmann and kuffer in 1998 [86] . it was originally designed to determine disease progression, treatment effects and the ability to perform normal daily activities in human cancer and hiv patients [87] . the karnofsky score (ks) modified for feline patients consists of two parts: the first part is the score given to the cat by the veterinarian (clinician's score) after assessing its state of health and body condition; the second part consists of a questionnaire containing questions about behaviours, which are answered by the owner [86] . a high degree of correlation was found between the results of individual observers when assessing behaviours [88] . lower inter-observer reliability for clinician's score was found, requiring additional modifications; a standardised, detailed approach with appropriate criteria combined with more specific training on how to use the scale may improve inter-observer consensus and consistency. the score obtained from questionnaire seems reliable, so it may serve as a reliable tool in the assessment of qol [88] . although the ks assessment method is not primarily designed for cats in shelters, it is one of the few tools that can be used in this regard due to its relative non-specificity. it should be mentioned that the protocol could only be used for animals that have been in a shelter for a long time; the caregiver completing the questionnaire must be able to correctly estimate the level of reported activities of the cat. the need to find a comprehensive but especially reliable tool for assessing mainly the clinical aspects of healthy cats also resulted in a study conducted by freeman et al. [89] , a group of american scientists. they first defined the attributes that cat owners consider relevant in health assessment; then they tried to create an evaluation tool (chew [cat health and wellbeing]) based on these attributes. parameters that are part of the assessment include easily determinable, animal-oriented items such as play, mood, energy, appetite, body condition and coat condition. the experiment resulted in the establishment of eight domains containing 33 assessment items. the authors mention that the final tool showed good validity, was able to discriminate overall health status, demonstrated good internal and test-retest reliability and is usable across various age categories of cats [89] , which is an important aspect for its use in shelters, as they usually house cats in various age categories. on the other hand, its use in shelter environment has not been tested. however, because the tool is based on two types of animal-based indicators (it involves behavioural and health indicators), its use as a comprehensive tool for assessing quality of life of shelter cats could be considered. a similar procedure (where its authors let the owners of cats determine, what behaviour they considered normal in the animals) was employed in tool creation by tatlock et al. [90] as well-23 evaluation items were developed in 15 areas-general health, interaction with humans, vocalisation, pain, sleeping behaviour, appearance, interaction with surroundings, toilet habits, gastrointestinal symptoms, hydration, appetite, weight loss, grooming, general happiness and physical activity level. authors reported strong internal consistency and test-retest reliability for all domains, indicating good reliability. in qol tools, it is customary for each rating item to have the same weight; however, it should be noted that the importance and impact of the items included on quality of life may vary, which should be taken into account when assigning weight and calculating the overall qol score. in contrast to the aforementioned studies, in addition to animal-based indicators (general state of health, food intake, behaviour), the qol tool (catqol) proposed by bijsmans et al. [91] takes into account management-based indicators and also the principle of evaluating each item included in the tool according to the frequency or severity with which it affected the cat's life. the catqol questionnaire was validated by taking all aspects of the psychometric analysis into account and assessing each question individually. the disadvantage of the questionnaire is that it contains questions on cat's experience during the previous week. thus, a cat could not be evaluated sooner than after 7 days spent in a shelter. in addition, the authors mention that the result obtained reflects the nature of the relationship between the owner and the cat to a certain extent. adamelli et al. [92] , who analysed the relationship between a person and a cat with an emphasis on quality of life in 62 animals, concluded that the qol of a cat may be more influenced by the characteristics of the owner than the characteristics of the cat. qol was rated low, moderate or high in this study using four questionnaires that analysed care, behaviour and characteristics of the cat and the owner, a simple physical examination and lexington's attachment to pets scale (laps) test. the laps test, developed by johnson, garrity and stalones in 1992 [93] , is probably the most widely used questionnaire for assessing the emotional bond between humans and animals [94] . although the described tools have been tested for future use by cat owners (in the home environment of cats), they represent at least a sketch or insight into the criteria that could be evaluated in the case of shelter cats. also, other tools that have been developed to be used in various animal species and contexts could be considered for cats in shelters. one such tool is the animal welfare grid (awag) software, which was originally designed to monitor animal welfare in research institutions (originally used in primates) [95] . the awag draws attention to the temporal component of any suffering, reflecting the cumulative lifetime experience of an individual. the system measures criteria covering four different parameters that impact animal welfare-health, psychological well-being, environmental quality and clinical and management procedural events. within each parameter, a number of indicators are evaluated on a scale of 1 to 10; a score of one indicates the lowest impact on welfare, whereas a score of 10 represents the highest impact on welfare. for each parameter, the software calculates the mean score, which is used to create polygon; the total area of the polygon presents the cumulative welfare score of an animal at a particular moment [95] . the awag tool can be used either predictively or retrospectively, including monitoring specific events or procedures affecting an animal or group of animals. the advantage is that software is freely available on the project website (https://github.com/publichealthengland/animal-welfare-assessment-grid/wiki) and that it is transferrable and can be used to manage the welfare of a wide range of species in a variety of settings [78] . some studies have been conducted using the awag, however no study has described its use in a shelter environment, therefore, no information on possible problems in this context is available. a potential problem with applicability in a shelter could be the evaluation of indicators itself. the scale contains 10 evaluation points, i.e., it requires ability of the evaluator to distinguish subtle changes and deviations between scores. this could be an issue in behavioural assessment of shelter cats; shelter staff usually do not have information on temperament of admitted cats, so distinguishing subtle changes in their behaviour could be challenging. the basic four principles (good feeding, good housing, good health and appropriate behaviour) used in the protocol evaluating the welfare of dogs in shelters [34] have general application in the assessment of welfare and are therefore applicable to almost any species of animal; they could therefore also be applied when assessing the welfare of cats in shelters. however, the individual evaluation criteria, of course, have to be adapted to the conditions in the cat shelters and to the cats themselves-the biological and ethological aspects of the cats should be taken into account in the evaluation criteria. the shelter quality protocol for evaluation of welfare of dogs that are older than 6 months and have been housed in a shelter longer than 2 months, developed in 2016 by the italian instituto zooprofilattico sperimentale dell'abruzzo e del molise, follows the criteria of reliability, validity and feasibility [57] . it is a tool that uses direct observation of animal responses while identifying important aspects of the environment and management to detect welfare risks [34] . observations are made at the level of the shelter, the cage and the animal. in developing the evaluation criteria, the authors were inspired by the approach of the welfare quality consortium [24] , which developed protocols for the evaluation of livestock welfare [96] . other principles have been incorporated by kiddie and collins [32] into the shelter dog qol evaluation tool-unlike the animal-based, resource-based and management-based approaches applied in shelter quality, the qol evaluation tool focuses only on animal assessment; the tool does not take into account environmental assessment and does not look at the management of the facility. the authors first compiled a list of behaviours that were associated with stress and behaviours that were associated with positive emotions according to literature and the experience of the shelter staff. the evaluation itself consists of 5 parts-3 parts require observation of the dog's behaviour from a distance, in which positive and negative behaviours are recorded, the 4th part evaluates the dog's reaction to a person during an interaction. in the last part, three parameters are evaluated by the observer-body condition-according to a 9-point scale, the presence of scurf and eye discharge. each evaluation item can receive a score of 1 or 0, depending on whether or not the occurrence of the observed trait or behaviour is recorded. kiddie and northrop [97] launched a project called 'assessing the quality of life of kennelled cats', which aims to create a similar rating system for cats in shelters. the final tool has not yet been published. in this section, we discuss the options of assessing three categories of animal-based indicators-behavioural (section 3.1), physiological (section 3.2) and health (section 3.3), which can be used to evaluate the welfare of shelter cats in a practical and scientific context. for welfare assessment, it is important to select behavioural variables relevant to the studied species as indicators and to take into account its evolutionary history. the domestic cat evolved into a carnivore with a solitary lifestyle, so the development of exaggerated behaviour was not necessary. assessing their well-being based on behaviour may seem all the more challenging [4, 98] . in cats, some degree of variability in behavioural responses to stressful stimuli was found, which is probably influenced by temperament and personality; even its inheritance is presumed [99] . in this section, we discuss the behavioural indicators that indicate impaired welfare of cats in shelters and methods that can be used in practical as well as scientific evaluation of behavioural responses of cats to stress (cat-stress-score assessment) and methods that are used to discriminate socialised and unsocialised cats (approach tests). in terms of the applicability of approach tests, we discuss tests usable in scientific (cat-approach-test, human-approach-test) and practical (feline spectrum assessment) contexts. in general, behavioural assessments can be perceived as subjective (namely considering the possibly subjective interpretation of observed behaviours by the rater) and are not always accepted as a valid indication of stress unless they are accompanied by the results of physiological measurements. the combination of physiological and behavioural data may provide credible evidence of the presence of stress [100] , but there are cases, when this statement is not valid (e.g., cat-stress-score does not correlate with the level of cortisol). however, shelter staff usually do not have sufficient resources to analyse physiological data, so it is useful to have an easy-to-use tool for assessing behaviour [101] . cats could find particular aspects of the shelter environment stressful, such as confinement, the proximity of other unfamiliar cats, handling by caregivers and changes in routine [15, 76, 98, 102] . on the other hand, a recent study by vitale and udell [103] found that shelter cats seek out human interaction (more than pet cats) so lack of human proximity may also be a stressor. cats respond to the presence of humans and prefer contacting with them with toys [104] . behavioural changes form the body's primary response to stress, serve as an adaptation mechanism and reflect the animal's internal state [105] . stress is a common aspect of life and is experienced by all living beings [106] . a problem arises if the amount of stress that an animal is experiencing crosses a certain line and becomes undesirable, and the coping strategies to handle stress have been exhausted [107] . although it has been found that the level of stress in sheltered cats decreases as they spend more time in the shelter [108] and that most cats adjust to the shelter environment within 2 to 5 weeks [109, 110] , some cats are unable to get used to it at all and thus remain in a state of chronic stress for a long time [105] . gouveia et al. [111] studied the differences in the behaviour of cats kept in shelters in groups with various densities and sex ratios for various lengths of time. cats that stayed in the shelter for a long time were less active and more involved in conflict situations. the presence of stress in cats is more often manifested by inactivity with inhibition of natural behaviour (reduced food intake, insufficient coat care, reduced frequency of playful behaviour [5, 112, 113] , persistent vigilance [15] , agonistic behaviour [17] and the need to hide [15] ) rather than any active manifestation of abnormal behaviour [4, 99] . the aforementioned indicators of good and impaired welfare, along with some others, are summarised in table 1 . general activity exploration of the surroundings normal occurrence reduced occurrence or absence of activity (rarely increased occurrence) [113, 114] behaviour associated with metabolic processes feeding normal occurrence reduced occurrence or absence of activity [5, [113] [114] [115] drinking normal occurrence reduced occurrence or absence of activity [113, 114] urination normal occurrence reduced occurrence or absence of activity; urination outside of the litter box and instead in other locations of the cage [114, 116] defecation normal occurrence reduced occurrence or absence of activity [114] comfort behaviour resting normal occurrence excessive vigilance [114, 117] sleeping normal occurrence reduced occurrence or absence of activity; feigned sleep; somnolence [110, 113, 114] grooming normal occurrence over-grooming, self-mutilation or reduced occurrence of grooming [113, 114] playing occurrence of playful behaviour (individual play, play with other cats, objects or people) reduced occurrence or absence of activity [114, [118] [119] [120] social interactions interactions with people positive interactions with people (seeking human presence, direct contact, staying in proximity); positive responses to human-initiated interactions absence of or negative response to a human-initiated interaction, particularly redirected aggression and some forms of affective aggression [17, 114, 121] interactions with conspecifics present; positive activities (rubbing, allogrooming, not avoiding contact) absent or negative activities: hostility, aggression, contact avoidance [109, 113, 114, 117, 121, 122] communication scratching normal occurrence reduced occurrence or absence of activity [114] facial marking normal occurrence reduced occurrence or absence of activity [17] urine spraying normal occurrence increased occurrence [17, 123, 124] other types of reported activities compulsive behaviour absence of compulsive behaviour presence of compulsive behaviour [125] [126] [127] hiding hiding as a normal reaction to fearful stimuli or as a part of playful behaviour effort to hide [15, 110, 114, 128] vocalisation normal occurrence excessive occurrence [114, 129] the level of stress is affected by the quality of housing; the inability to show the natural range of activities for a longer period of time can lead to stress, which applies especially to individually housed cats in cage housing [130] , often poor in enrichment, which is often used in shelters e.g., in the united states [131, 132] or in general within the quarantine. the level of stress can be partially regulated by providing an undisturbed, dark place where the cat can hide [133, 134] , which, of course, does not address the overall lack of stimuli and space to engage in natural active movement. on the other hand, no difference was found in the stress responses of cats when comparing the amount of space provided per cat (1 m 2 , 2 m 2 , 4 m 2 ) when the resources were the same [135] . this may indicate that it is not the size of the cage but its capacity to be utilised that has an impact. group housing, chosen by a number of shelters because of lack of space, on the other hand, is not entirely satisfactory from the point of view of infectivity (as there is a constant change in the composition of groups) and of social structure-in nature, cats form groups only under certain conditions; one of them is the sufficient availability of resources. in addition, cat colonies are usually comprised of females and their offspring [136] . grouping in shelters is therefore generally problematic when mutually unfamiliar and unrelated cats are housed together [137] . some individuals in a group may prevent others from accessing resources due to their territoriality. in addition, cats have been found to be able to form a linear rank order when living in group; higher-ranking cats tended to gain weight, whereas lower-ranking cats tended to lose weight [138] . escaping or avoiding another cat may be a problem, as it may not be possible in a confined environment. on the other hand, the advantage of group housing is an environment that usually provides cats with more enrichment. cats have more space available for movement and more control over the use of their environment [135] . uetake et al. [139] reported lower levels of activity in individual cage housing as opposed to group housing. this finding is in contrast to the findings of guveia et al. [111] -cats that were housed in groups with a high number of individuals were found to generally show lower levels of activity [111] . there may be several reasons why the findings are inconsistent. cats in any housing system are exposed to stress, but it comes from a variety of sources, and cats also have different options for coping with stress. as density is often related to stress, key information such as the total number of cats and the spatial characteristics of the room are often missing. another complication when comparing cats from group and individual housing is the fact that the groups are not equal to each other and the studies do not take into account the characteristics of individual cats and the treatment of each group at the shelter [140] . suchak and lamica [140] attempted to address this issue by creating a retrospective matched cohort of cats in two housing systems. cats housed in groups were matched with cats housed individually with the same characteristics (age, breed, sex, size and coat colour). authors found no difference in cats' experience while at the shelter. however, in this study authors sought to compare individually and group housed cats based on measures related to their management in the shelter and outcome; behavioural measures were not assessed. the discussion about which type of housing is less stressful is still ongoing and with ambiguous results [140, 141] . the first scale developed to assess behavioural responses to stress was the global assessment score, designed by sandra mccune in 1992 [98] . it contained a description of 10 levels of stress in cats [98] , the number of which was later reduced to 7 by kessler and turner in 1997 [76] ; they used it in their study to compare cats housed in a shelter individually, in pairs and in groups. the scale was named 'cat-stress-score' (css) and was well received by a wide range of professionals. it is currently a relatively simple and at the same time the most commonly used method for assessing behaviours of stress in cats [135, 142] . the scoring scale consists of 7 grades, from the state of 'total relaxation' (score 1) to the state of 'extreme stress' (score 7). the resulting grade is obtained from a brief observation of the cat's posture and behaviour [76] described in the ethogram developed by the british cat behaviour working group [143] . the authors suggest that a score below 3 is still acceptable because it represents only the basic level of stress present in any living animal. an increase above this level means a response to an acute stressor and does not present a problem as long as the score does not remain the same over time [109] . they also point out that the scale can be used in all housing systems except those in which the temperature does not reach at least 15 • c. at lower temperatures, animals naturally do not show a relaxed attitude, which could lead to skewed results [109] . the use appears to be reliable-mccune [98] mentions 100% repeatability among observers, kessler and turner [76] reported a 90% match among observers who received training in the use of the scale and a 75% match among untrained shelter staff. however, more research should be performed on how the css scale could be reconstructed for increasing its validity and reliability. the disadvantage of the method is that the evaluation is based on a very short assessment period, is subjective [144] and the results may be influenced to some extent by the presence of some factors (e.g., age, sex, neuter status). another significant disadvantage of the method is that the score does not correlate with the level of cortisol [101, 142] . no correlation was found in the combination of evaluating the behaviour using the css scale and determining the ratio of cortisol to creatinine in cat urine; nor was it found when comparing the css test result with cortisol metabolites in the faeces [101, 142] . the authors explained the absence of correlation by the fact that the ratio of cortisol to creatinine in urine was cumulative, while the behavioural score was dependent on environmental conditions at the time of assessment. it is not clear whether the intervals between scores are equivalent (i.e., whether, for example, a change from score 3 to score 2 presents the same level of stress reduction as, for example, a change from score 6 to score 5) and also whether the use of intermediate stages in assessment is possible (when the cat exhibits behavioural elements of two separate grades at the same time) [145, 146] . decreased cat activity and suppressed natural behaviours that may persist for long periods of time and are a sign of stress may be misinterpreted by an untrained evaluator as calm and contented [105] . in order to improve the sensitivity to detect subtle variations in stress levels, bradshaw et al. [102] added half steps between scores 2-4. on the contrary, cats that have impulsive personality achieve higher scores in certain aspects of the scale, which does not automatically mean that they are extremely stressed. if possible, because of the individuality of each cat, it is more appropriate to carry out the assessment at different times during the day or over the course of several days [50] . observers may also misjudge manifestations of oestrus resulting in increased scores; an example is an increased rate of vocalisation [147] . the css scale has been used to assess cat stress in shelters in a number of studies [5, 145, 146, 148] . it was found the score was highest in cats in the morning, and that the tool was not suitable for detecting pretended sleep and overly excessive stress levels [101] . in a study by broadley et al. [144] , the scale was used to compare the behaviours of cats (originating from multi-cat households and households with only one cat) after they were placed in separate cages in a shelter. cats that came from households without additional cats could experience higher levels of stress in the first days in the shelter than cats that had previously been accustomed to the presence of other individuals, in spite of limited direct visual contact with other cats. kry and casey [133] used a scale to examine the effect of enrichment of individual housing in shelter using a bc spca hide & perch tm box to achieve a lower score for cats for which this enrichment was available. other tests to assess behavioural responses are approach tests. these are used to evaluate responses of cats to unknown stimuli (unfamiliar humans or cats) approaching the cat to determine whether a cat is socialised or not. the individuality of a cat and socialisation are the most important factors influencing a cat's behaviour toward humans and new situations in general [99, 109, [149] [150] [151] . the socialisation period begins in cats at the age of 2 weeks and ends around 7 weeks of age. the experience of kittens in this period has a long-term impact on their development and behaviour. kittens handled during this period are more friendly to humans than kittens that did not come into contact with humans [152] . in general, many facilities have developed their own rating systems, but only a few use a specific procedure or guide [153] . these tests have certain limitations; any cat that enters an unfamiliar environment is prone to fear. even well-socialised cats can show fearful aggression or motivation to escape. as a result, it can be very difficult to initially determine which cats are unsocialised and which have the potential to be adopted or returned to the owner [49] . it was assumed that the stress manifestations of the new environment in socialised cats would present itself differently than the stress manifestations of the new environment as well as people in non-socialised cats. however, it is not possible to separate these two fears [153] . frightened cats may first begin showing their characteristic behaviour after a few days or weeks when stress levels begin to decline or when they are re-evaluated in a calmer, less stressful environment [76, 145] . slater et al. [49] found that a number of facilities euthanises a cat as soon as it is marked as unsocialised, often on the day of admission to the shelter, without providing time for acclimatisation. in creating an assessment tool for distinguishing between socialised and non-socialised cats, slater et al. [153] therefore selected a three-day observation period because previous research found that about half of the shelters were able to keep the cat in the facility for at least three days [49] and that some stress reduction can be expected during this period [109] . in shelters where cats are not provided with acclimatisation time, there is an increased probability that socialised animals will be accidentally euthanised and also the likelihood that the original owner will no longer be able to reunite with their animal. the opposite extreme is the situation where unsocialised cat is kept in a shelter for longer than is necessary for the assumption that it will 'settle' and prove to be socialised. keeping non-socialised cats in a shelter for longer than necessary has significant consequences for them in terms of deteriorating living conditions and is not recommended [153, 154] . in their approach study, kessler and turner [109] described the test they used to assess the degree of socialisation of cats towards other cats. in this test, authors assessed the cat's reactions to another cat (cat-approach-test). the test was based on a procedure in which a calm, neutered male that was socialised in relation to other cats was placed in a portable box at a distance of one meter from the test subject. both subjects were allowed visual contact with each other for 1 min. after this time has passed, the behaviour of the tested cat was rated on a scale of 1 (the cat reacts extremely friendly to another individual) to 6 (the cat reacts extremely hostile to other individuals). cats were considered socialised when they reached an average grade below 3.0 after eight consecutive trials. on the other hand, those individuals who achieved an average mark higher than 4.0 in a series of eight tests were considered unsocialised. in research studies, rather than exposing a cat to another cat, human approach tests are used. it is generally possible to assess the stress manifestations of cats caused by humans in two main ways; either the test subject is allowed to approach or not approach the experimenter [98] or the experimenter approaches the location of the cat [98, 109, 155] . a less commonly used method is to hold a cat in one's arms for a predetermined period of time [156] . the human-approach-test, which was modified by kessler and turner [109] on the basis of the stranger-approach test [99] , the experimenter approaches the cat's cage from the front, addresses the animal and remains in front of the cage for 1 min while touching the door with their hands. after the time has elapsed, the experimenter opens the door of the housing unit and closes it again. the cat's response during the test is rated on a 6-point scale (1 = the cat reacts extremely friendly to humans, 6 = the cat reacts extremely hostile to humans). as with the cat-approach-test, the authors suggested how to interpret the results; the cat that received an average grade greater than 4.0 in 8 consecutive trials was considered unsocialised to humans. the cat that received an average grade of 3.0 or less (friendly response to humans) in eight tests was considered socialised. when comparing the results obtained using the human-approach-test and the css rating scale, no correlation was found, which raises the question of reliability [121] and validity for both tools. a critical aspect of the tests is the fact that some cats can distinguish between 'a known and an unknown' experimenter and adjust their behaviour accordingly, although horsfield [157] suggests that the difference between cats' responses to known and unknown individuals is minimal in the tests. the individuality of a cat and socialisation are reported to be the most important factors influencing a cat's behaviour towards humans [99, [149] [150] [151] . the basic idea of the human-approach-test has been retained in many modifications used across various studies [12, 132, 133, 144, 158, 159] . one of them is, for example, the human-approach test proposed by arhant and troxler [12] , who applied the test to cats housed in groups (the original human-approach-test by kessler and turner [109] was used only for cats housed individually in cages). the experimenter approached the selected cat in the group in a slow, smooth motion and presented his hand; he stopped if the cat withdrew or when the experimenter's hand was already 20 cm far from the cat's head. direct eye contact with the cat was not maintained during the test. the cat was rated as an 'animal with which contact is possible' if the cat did not withdraw and observed the experimenter. commonly observed reactions included scenting in the direction of the experimenter, sniffing the hand and rubbing the hand. cats that withdrew during the test, exhibited any form of aggressive behaviour, or were hiding in hiding spots were identified as 'cats with which contact is not possible'. in groups of 10 cats, all cats in the group were assessed, in larger groups, a sample of 10 cats was randomly selected. if there were cats of similar appearance in the group, a smaller sample of clearly identifiable cats was selected because individuals were not marked. in contrast to the human-approach test by kessler and turner, which was used mainly in research studies, the feline spectrum assessment by american society for the prevention of cruelty to animals (aspca) was created for practical use in shelters. the tool consists of 4 evaluation items (greet, hand on cage and cracking the cage door, interactive toy, touch with wand). the animals are tested for three days; the test result is a numerical score that indicates whether the animal is likely to be socialised or not [160] . in addition, to maximise the chance for the cats to show their true natures, the assessment tool should be used four times per cat across three days. it is important that cats are not given a place to hide so they are visible during the assessment items. the disadvantage of the tool is that cats younger than 6 months old and group-housed cats cannot be evaluated. on the other hand, the advantage is that assessment generally takes no more than five minutes per cat to complete, requires a very basic understanding of feline behaviours and its guidelines are available for free on the internet [160] . for teaching purposes of the evaluators, instructional videos were also created. the principles of approach tests have been applied to other group of tests, which aim to reveal cats' personalities. studies show that many aspects of a cat's individuality are stable over time [152] ; this fact has led and continues to lead to new studies on the temperament of cats [116, [161] [162] [163] [164] [165] [166] [167] and on development of tools for assessing the temperament of cats in shelters (feline temperament profile (ftp) [50] , aspca ® 's meet your match ® feline-ality™ [168] and its modifications [169, 170] and other alternatives of testing [171] ). testing of cats' personalities contributes to the improvement of welfare as it increases the chance of compatibility between the lifestyle of the new family and temperament of cats, which leads to a reduction in the numbers of cats that are returned to the shelter [50] . another category of indicators that can provide insight into welfare of an animal includes physiological indicators, which are mostly used to detect a stress response. a stress response can be assessed either by determining laboratory parameters or by direct assessment of vital signs-most often by measuring blood pressure, heart rate, respiratory rate and rectal temperature. although these indicators can reliably determine the presence of stress, it seems their applicability in shelters is limited due to several reasons. the analysis of laboratory parameters is often too expensive for the shelter to afford and measurement of changes in vital functions requires specific equipment. in addition, at least minimal handling with an animal is necessary to collect data. this is a significant disadvantage; since measurements are performed to detect stress but handling itself can be stressful to an animal, the results may be skewed and interpreted incorrectly. in order to reduce the handling stress, the animals need to be trained to the measuring procedure; this, however, cannot be applied in shelters. methods discussed in the following subsections are typically used in research for validating tools, that are easier to use, cost less and are more applicable to shelter settings. activation of the sympathetic nervous system causes an increase in heart rate, respiratory rate, systolic blood pressure and a decrease in heart rate variability to facilitate the 'fight or flight' response [172] [173] [174] . as was already mentioned in previous sections, cats are extremely sensitive to handling, environmental changes and changes in routine; the result may be a change in physiological values [15] . changes in physiological values are an established indicator of distress in various welfare studies [172, [175] [176] [177] [178] including cats, [173, 174, [179] [180] [181] [182] ; for example, pratsch et al. [182] , used an ear thermometer to monitor body temperature. an increase in body temperature is an accepted indicator of stress [183] . the temperature was measured to detect stress during transport of the cat to the veterinarian; an increase in temperature of 0.5-1 • c was observed, as well as a difference in temperature between the right and left ear, with the temperature of the right ear being higher. the right ear was warmer also in cats with elevated cortisol levels after a visit to the veterinarian, indicating the presence of stress [180] . although changes in heart rate, respiratory rate and temperature may be sufficient stress indicators, their applicability in the shelters is low as the measures usually require special equipment and skills. measuring procedures (e.g., wearing a heart rate monitor or taking temperature measurements manually by inserting the thermometer into the rectum or the ear canal) can be a stressful experience for animals, so the handling procedure itself can cause a change in values. another disadvantage of direct measurements lies in the monitoring of elevations or decreases of values, which, however, is not specific only to stress (but also, for example, to illness or increased activity). therefore, the assessment of changes in vital signs is possible under conditions where the animal is healthy and does not have enough space to develop intensive locomotive activity, which could cause false elevations of values [98] . common methods of assessing the ongoing stress response include assessing hypothalamic-pituitaryadrenal (hpa) axis activity, most commonly by determining cortisol, adrenocorticotropic hormone (acth) and glucose [101, 180, 184, 185] . activation of the hpa axis by a stress stimulus induces the release of acth, which increases the synthesis and secretion of cortisol from the adrenal glands [186] . the assessment of physiological indices has an advantage over other evaluation methods in its quantification. the disadvantage is the existence of great individual variability in physiological responses. the levels can also vary over the course of the day-comparing and interpreting the results can, therefore, be difficult [187] . a stress response is complex; a single measurement of a parameter may not reflect subtle changes that occur in the body permanently, which may result in drawing incorrect conclusions about the extent of stress. factors such as age, animal history, physical individuality and the circumstances under which the samples were obtained must be taken into account when interpreting the levels of physiological indices [188] . another disadvantage is that the analyses are usually too expensive for shelters, so their use is almost exclusively limited to the scientific context. assessment of glucocorticoid levels is a common part of welfare assessment in many animal species especially in a scientific context [189] [190] [191] . different types of samples can be used to determine cortisol levels; their aspects in terms of practicality are summarised in a table 2 . for acute stress measurement, it is appropriate to examine blood plasma [192] , serum [193] and saliva [50] . chronic stress, on the other hand, can be detected using non-invasively obtained samples-urine [139] , faeces (determination of glucocorticoid metabolites) [164] and fur [194] . [190, 192, 195] serum acute; peak concentrations: 30-180 min after stressor exposure reflects the actual level of cortisol in the blood invasive (restraint or sedation of cat is necessary); sampling requires skills sampling procedure as well as diurnal patterns, temperature, activity levels may confound results [15, 193, 195] saliva acute; concentration: 2-15% of the total amount of cortisol in the blood less invasive prior training of cats on sampling procedure is necessary; a relatively big amount of sample is needed for analysis, which can be a problem to obtain (saliva production is reduced during the stress exposure) sample is easily contaminated by the intake of food and water or by blood from the oral cavity [50, 182, 191, [196] [197] [198] urine chronic; concentration: 15% to 18% of the total amount of cortisol in the blood, peak concentrations: 9 ± 3 h after stressor exposure non-invasive; detection of long-term stress is possible; easy sample collection in single housing; sample collection does not requires skills in group housing, the sample is difficult to associate with an individual as cats share toilets diurnal patterns, temperature, activity levels may confound results; sample is often contaminated by blood [101, 110, 115, 139, 199, 200] faeces chronic; concentration: 80% of the total amount of cortisol in the blood, peak concentrations: 24 ± 4 h after stressor exposure non-invasive; detection of long-term stress is possible; easy sample collection in single housing; sample collection does not requires skills in group housing, the sample is difficult to associate with an individual as cats share toilets diurnal patterns, temperature, activity levels may confound results [164, 195, [199] [200] [201] [202] fur chronic non-invasive; easy sample collection (does not requires skills); stability of the sample over time; does not require special transport and storage conditions; ability to determine the time period in which stress occurred in the animal inability of detecting changes in cortisol levels during short periods of time (hours/days) cortisol levels may vary depending on some factors-still in research [194, [203] [204] [205] in addition to stressors, changes in cortisol concentrations depend on other factors (e.g., sampling and animal handling procedures) [206] , which can be a problem in assessing acute stress. the animals should be accustomed to the collection of the necessary sample, otherwise, it is a stressful stimulus that causes a physiological change in the body [207] . the marginal methods used today are identifying the level of cortisol from plasma, serum and saliva. in saliva, cortisol is only found in its free form [198] . siegford et al. [50] described a collection procedure as a procedure in which the experimenter provided the cat with a cotton swab to chew until it was entirely soaked. for each 'soaking', the cat was rewarded with a treat. pratsch et al. [182] conducted a pilot study aimed at detecting cortisol in saliva-the goal was to investigate the method of saliva collection by cotton swabs and the effects of various food and feeding time on cortisol levels in saliva. all cats were familiarised with sample collection (although the authors do not mention how). only 23 of the 54 samples contained sufficient saliva (50 µl) for further analysis, so the authors concluded that the method could not be used in the planned study. not only saliva collection, but also collection of urine can be problematic as stress in cats can be manifested by urinary retention for the first 24-48 h after admission to the shelter [101, 115] . in addition, 25% of all cats admitted to the shelter have blood in their urine [101] , which can interfere with the samples meant to be used for analysis. the disadvantage of detecting glucocorticoid metabolites in faeces is the fact that they do not detect short-term stressful experiences. short-lasting or small fluctuations in the level of circulating glucocorticoids are mostly masked by metabolites accumulating in faeces and bile. faecal samples containing a detectable amount of metabolites reflecting one-time stress experiences may also be overlooked in the case of irregular sampling [195] . however, unlike some other samples the collection procedure of urine and faeces is relatively simple when using a two-layer cat litter box or a litter box with non-absorbent litter [199] (although with a large number of housed animals sharing toilets, collection from specific individuals may be a bit problematic without constant animal supervision). the analysis of urine and faeces to determine the level of stress has been conducted in various studies focused on cats in shelters. for example, the cortisol to creatinine ratio in urine was found to decrease during the cat's stay in the shelter [110] and to be significantly lower in cats in enriched shelters than in traditional shelters [101] . an increase in the ratio was found in individually housed cats compared to cats housed in groups [139] . identifying urinary cortisol levels was also used in a study by lichtsteiner and turner [208] , who found no difference in the amount of cortisol in cats housed individually and in groups. in similar studies, the same results were obtained by ramos et al. [165, 201] , who used faeces as a matrix to analyse the level of cortisol. the level of glucocorticoid metabolites in faeces in cats decreased with increasing time spent in the shelter [202] . determining the level of cortisol from fur is an innovative method of detection of long-term elevated cortisol in the body [209] , first used in 2002 in wild hyraxes (procavia capensis) [203] . although the process of cortisol incorporation into the hair is not entirely clear [210] , in general, it can be said that the free, unbound fraction of cortisol is incorporated into the hair pulp during its growth [211] from blood vessels through passive diffusion [210] . the cortisol-containing sebum secreted by the skin glands is thought to play a role [212] . to determine the presence of stress, the fur must be harvested from previously shaved areas [197] close to the skin and not by plucking to avoid blood contamination [213] . the area and size of the fur collection area vary across studies (in a study by finkler and terkel [194] , cat fur was collected from a 5 × 5 cm sacral area with small surgical scissors). the advantage of using fur as a matrix is especially the ability to determine the time period in which stress occurred in the animal [204] and the possibility of rapid sampling from a larger number of animals, which is an advantage for animals in group housing. another advantage is the fact that the stress caused by handling the animals during collection does not affect the concentration of cortisol in the collected fur [205] . although there was a doubt that the level of cortisol in fur does not represent its actual level in the body but only a kind of 'local level' present in hair follicles [214] , stalder and kirschbaum [215] found indications that hair cortisol reflects the systemic cortisol levels well and seems only slightly affected by the local follicle cortisol production. there was found a connection between the concentration of cortisol in faeces and fur [216] and between its concentration in saliva and fur [189] . however, there is evidence that some factors affect cortisol levels in fur -e.g., colour, which was confirmed in dogs [213] . debatable factors may be, for example, sex, age, state of health and various areas of the body [204] . accorsi et al. [216] found a significant correlation between agonistic behaviour and the level of cortisol in the fur, suggesting that a greater amount is found in the fur of aggressive cats. neutered cats also had lower levels of cortisol in their fur than intact individuals. interesting insights can be gained by combining the assessment of behavioural and physiological indicators of cat stress. for example, hiding was negatively correlated with cortisol levels, leading the authors to conclude that this may be important compensatory behaviour in an unpredictable environment [15] . behavioural assessment is probably a less sensitive indicator of stress than the cortisol-creatinine ratio. the behaviour must, therefore, be extreme for stress to be detectable. cats with higher cortisol levels have also been found to vocalise more and move less than cats with lower cortisol levels [50] . the spectrum of coping strategies is wide in cats [116] -while some show elements of stress behaviour but have physiologically adapted to new conditions, others have high levels of stress hormones, but altered behaviour is not noticeable. the welfare assessment protocol should also include parameters indicating deteriorating animal health [217] , including impairment in body condition score [218] . indicators should be selected to reflect the most frequently observable signs of health problems occurring in cats in shelters. the problems discussed in this section include signs of upper respiratory tract disease, sickness behaviours, gastrointestinal and skin problems, pain in general and changes in body condition. we discuss the methods of evaluating body condition in more details in section 3.3.2., because only for this indicator more than one validated tool that is well applicable in shelters was created. efforts to develop valid tools for assessing pain in cats are also intensive, however usability of most of them is low in shelters due to their specificity to the certain context of use or time-consuming procedure. in addition, many of these tools have not been validated yet. in the case of other indicators described in this section, we discuss the reasons why they need to be included in the health evaluation and what results have been obtained by studies that included or studied these indicators. admission of the cat to the shelter is a stressful experience that can suppress the activity of the immune system and reactivate the subclinical course of the disease (e.g., feline herpesvirus) [5, [219] [220] [221] . stress can inhibit the production of mucosal antibodies, particularly immunoglobulin a (s-iga) [222] , resulting in increased susceptibility to pathogens causing upper respiratory tract disease (urtd) [223] [224] [225] . after lack of space, urtds are the second most common reason for cat euthanasia in shelters in the united states [226, 227] . emotional state and health are interconnected; a decrease in immunoglobulin a (s-iga) secretion was found in stressed shelter cats [222] . it has, however, been proven that 'gentling' (gentle stroking of the head and neck area of the cat together with gentle vocalisation) at regular intervals can help improve the cat's mental health, increase s-iga immunoglobulin production and thus reduce susceptibility to upper respiratory tract diseases [16] . immunoglobin s-iga is essential for protection against pathogens that may be inhaled or ingested [228] and are highly concentrated in shelters [229] . higher concentrations of cats are associated with an increase in the prevalence of urtds [230, 231] . cats that have achieved a high degree of stress according to css are almost five times more likely to develop upper respiratory tract infections than cats with low levels of stress [5] . risk factors in the development of a urtd are the cat's age (the risk is greater in younger individuals [224, 229] , the number of days spent in the shelter and specific conditions in the shelter [224] . the development time of a urtd reflects the incubation time of urtd-causing viruses [220, 232, 233] . although shelters differ in the prevalence of pathogens, many cats show signs of disease after as little as 1 week spent in the shelter [5, 224] , according to dinnage et al. [227] up to a third of the shelter cats suffer from a urtd. therefore, it is appropriate to include an assessment of the absence or occurrence of eye and nose discharge among health indicators [5] . to evaluate upper respiratory tract infections, bannasch and foley [224] developed a three-point scale, the degrees of which express the severity of symptoms (1. absence of upper respiratory tract infection, 2. mild upper respiratory tract infection, 3. moderate to severe upper respiratory tract infection). in addition to urtds, as a result of events perceived as aversive by cats, the so-called 'sickness behaviours' (sb) occur [113, 115] . it is a term denoting a set of non-specific clinical but also behavioural symptoms, which include e.g., vomiting, diarrhoea, refusal of food, lethargy, fever, drowsiness and a general decrease in activity [234] . stella et al. [115] observed how unpredictable events (presence of dogs, change of caregiver, loud sounds, grouping of new individuals, irregularities in periods of light and dark, temperature fluctuations, transfer of cats) induced by experimenters influenced the occurrence of sb in cats. a negative impact of the events on the animals resulting in a demonstrably increased incidence of sb was confirmed by the authors [115] ; the inclusion of sb as an indicator of deteriorated welfare is therefore appropriate. similarly, for lower urinary tract diseases and idiopathic cystitis in cats, the relationship between the stressor (especially cohabitation with other cats, limited access to the external environment, sharing of feeding bowls and generally frequent changes) and the occurrence of the disease has been demonstrated [235] [236] [237] . gastrointestinal problems, vomiting and diarrhoea are often associated with stress [115, 238] . it seems that stress can affect the integrity of the intestinal barrier, causing an increase in its permeability and local inflammatory response [239] . a large number of shelter cats suffers from diarrhoea caused by dietary issues, stress or illness (gastrointestinal infection, inflammation or neoplasia [240, 241] . in a study by german et al. [242] 11.9% of all cats in the shelter had diarrhoea, in a study by andersen et al. [243] it was up to half of the cats. most cats had at least one feline or zoonotic pathogen in their faeces, with the most common pathogens in cats with diarrhoea being feline coronavirus and tritrichomonas foetus [243] . diarrhoea was most common in kittens and cats housed in a group [242] , and a higher prevalence of pathogens was also found in the faeces of group-housed cats [243] . in order to determine the severity of diarrhoea, faecal consistency can be assessed, which was the approach chosen in a study by german et al. [242] based on a 6-point scale (1 = severe diarrhoea, 6 = constipation). monitoring faecal consistency may be a useful health indicator of welfare, but in group housing, where multiple cats share several toilets, identification of an individual with diarrhoea may be challenging [243] . however, there are some procedures that could be used to mark faeces; in the study by griffin [244] , commercially available concentrated food colourings (known as bakers' pasters) were used as faecal markers in group-housed cats. cats consumed food colourings in canned cat food. colourings served as faecal markers by imparting a distinct colour to each cat's faeces, allowing identification in the litter box. in another study, to facilitate identification of faecal samples from females housed in pairs, each cat was fed a teaspoon of canned cat food daily with one animal receiving 4 ml green commercial food dye mixed into the food [245] . in addition to dyes, glitter and plastic beads were used to mark faeces in various felids in zoos [246] . the state and condition of the coat can serve as additional health indicators of welfare [247] , as they reflect changes in normal grooming behaviour, food quality, the presence of social conflicts and chronic diseases [248, 249] . there is a clear link between the skin and the nervous system [250] . dermatological diseases such as atopic dermatitis can be caused by stress, which is likely to cause itching [251] . disorders associated with excessive coat care (e.g., psychogenic alopecia) manifest in the form of alopecic areas on the caudal part of the body [126] . it was found that the worsened coat condition of shelter cats correlated with the average length of stay of a cat in a shelter (the longer the cat was in the shelter, the greater the risk of deterioration of coat quality), fewer toilets per cat and unpleasant odour [218] . similarly, the number of food bowls and their location were associated with a worsened condition of the coat-the monitored shelters, where the study was performed, an average of two bowls was assigned to three cats. the condition of the coat may indirectly indicate the occurrence of conflicts between cats, in which there are fights and, as a result, injuries. the presence of injuries, similar to the condition of the coat, can be included among the indicators of welfare [218] . in a study by arhant et al. [218] , a combination of assessment of health indicators (coat condition, body condition, eye and nose discharge) and behaviours was used to assess the welfare of cats in shelters housed in groups. the authors found that of all the monitored indicators, only two of them were stable over time (body condition and coat quality) as the composition of cats in the shelter was constantly changing. these two indicators correlated with repeated visits to the shelters. inter-rater reliability was found to be good for coat quality but only moderately good for body condition. to ensure better agreement between observers, the authors recommend improving the evaluation guidelines by providing pictures and more detailed instructions. authors suggested that body condition and coat quality are feasible and valid for assessing impaired welfare in cat shelters. to assess the welfare of stray cats and abandoned cats, a scale with five components related to cat health was used in a new zealand study [252] -body condition score (bcs), coat condition, presence of nasal discharge, presence of injuries and the ear crusting. the authors found no difference in the welfare of free roaming companion cats and managed stray cats. they also suggested further validation of the scale using a more detailed physical examination and a determination of biochemical parameters that would complement the overall picture of welfare. a problem in evaluating health indicators is that cats generally tend to hide the clinical signs of the disease [89] . some cats show no signs of pain at all, if they are supervised by humans, other animals, or in stressful situations [253] . the absence of indicators does not necessarily mean that the animal is healthy. with a large number of cats in a shelter, it can also be problematic to notice subtle deviations indicating deterioration in health, especially if the shelter caretaker has only a minimum amount of time to provide care to one animal. in shelters, there are animals with an unknown history; therefore, the caretakers have no way of determining 'normal behaviour', that is, the temperament of the given cat, and deviations in behaviour specific to each individual exhibited in case of deterioration. furthermore, in shelters where cats are housed individually in cage housing with minimal freedom of movement, the caretaker may not notice a reduction in activity or signs of pain, or he may not be able to distinguish an inhibition of activity due to stress from symptoms of an emerging disease [253] . group housing, on the other hand, is characterised by the placement of several cats in a single room containing equipment providing the cat with a place to hide [140] , which may prevent direct visual inspection of shy or frightened individuals. distinguishing between hiding as a manifestation of pain and hiding as a manifestation of stress can be problematic. tools based on animal observation are used in veterinary medicine to assess pain; observation is a non-invasive and effective method that can be used by different people and in different contexts [254] . several tools for detecting cat pain have been developed [255] , but only few are valid and reliable. an example of such a tool is the unesp-botucatu scale [256, 257] ; however, it is a tool applicable only if the cat has undergone an ovariohysterectomy. although cats are routinely neutered in most shelters, the unesp-botucatu scale, with its specificity, focuses only on the detection of pain after this procedure; the limited context of use thus excludes it from the detection of pain in general. however, the glasgow composite measure pain scale-feline (cmps-f) is available, which is a validated tool that can be used for assessment in a broader context, specifically in the presence of acute pain in cats [258] . in order to improve its discriminatory ability, it has been updated and its final version includes two additional pain-detecting markers [259] . in addition to assessing behavioural changes, detecting changes in facial expressions also has the potential to indicate the emotional experiences of animals and to provide valuable information about their internal condition. assessment of facial expressions using the so-called grimace scales can be a useful, valid and reliable tool for determining pain [260] . manifestations are objectively measurable using the 'facial action coding system' (facs) assessing the animal's facial movements (so-called action units-au). the system assigns codes to the activity of an individual muscle or group of muscles. in the case of cats, a specific coding scheme (catfacs) has been developed [261] . grimace scales are a simplified method of evaluating facial expressions that can be specifically applied to assess pain. in the case of cats, the feline grimace scale (fgs) was recently developed, which, according to the authors [262] , is a valid assessment tool for detecting the presence of pain in cats; to confirm the applicability of the tool in practice, evangelista et al. [263] applied the scale in assessing pain in cats after an ovariohysterectomy and found good applicability. the methodology was based on the evaluation of facial expressions using images and real-time scoring. the standard methodology for assessing facial expressions of pain using grimace scales is usually based on image scoring [264] ; screenshots are obtained from videos, but their analysis is time-consuming. in practice, pain should be assessed immediately and easily [263] . a change in weight can be another indicator of the welfare of cats in shelters. assessing body condition is a traditional and relatively easily applicable approach to welfare assessment. previous studies have shown a link between body condition, health, behaviour and welfare [5, 265] . there are several methods to describe the appearance of the body and the distribution of body fat, their practical aspects are summarised in a table 3 . [268, 269, 272] the most commonly used is the semi-quantitative body condition scoring (bcs), which uses visual and tactile assessment of the body. the body condition of the animal is sorted into three categories as underweight, optimal condition and overweight [273] . areas assessed in cats include the chest, hips, abdomen and waist [274] . a general problem in assessment can occur in long-haired cats when the silhouette of the body is not clearly visible. a 9-point scale has been well-received-individual levels correlate with the amount of body fat detected by dual-energy x-ray absorptiometry (dxa) [266] . to facilitate the assessment, the assessor may be provided with a series of illustrations of cat silhouettes-these reflect the characteristics of each point of the scale. the method has been validated and is considered reliable; scores determined by independent observers correlate [266] . the disadvantage of the scale is its inability to detect small deviations; therefore, it should not be the only method chosen to monitor changes in body weight [5] . at present, in addition to the 9-point scale, a simplified 5-point model of the bcs system is also used. its practicality seems to be better-the deviations between the stages are more noticeable, which in practice seems to be an advantage. each point on the scale represents a 7% increase in body fat. grade 3-3.5 represents optimal weight with 12 to 19% body fat [269] . 5-point scale has not been validated by dxa. however, good reproducibility was found when comparing results of experienced and inexperienced evaluators [269] . relatively new approaches to fitness assessment include the s.h.a.p.e. system (size, health and physical evaluation), which was developed to minimise variability in assessment across observers. it is an algorithm-based system, using the principles of existing scoring systems with a difference in grading-each category of body condition is assigned a letter from a (underweight) to g (obesity). the use of the rating scale does not require previous training of the observer [270] , so it could potentially be used by shelter caretakers. as with the 9-point scale, a strong correlation was found between densitometry and the s.h.a.p.e. system [270] . the disadvantage of s.h.a.p.e. and bcs is their relatively small informative value-they do not assess the state of muscle, only the storage of body fat [275] . musculature can be assessed using the muscle condition scoring (mcs) system; it contains four levels of evaluation-normal musculature, mild, moderate and severe muscle loss [276] . when verifying the applicability of mcs, good repeatability but only moderate reproducibility was found [271] . the animal's spine, scapulae, skull and wings of ilea are palpated during evaluation [276] . although the animal may appear to be overweight, it may also suffer from significant muscle loss; in contrast, animals with low a bcs score may show minimal loss of muscle [277] . overall, the system is mainly used in underweight animals that undergo muscle mass catabolism due to disease or injury [276] , and an example of the system's use is a study monitoring the changes of body condition in cats with hyperthyroidism [278] . the use of direct body weight measurement is an additional option that should be considered in body condition assessment. however, direct measurement, similarly to bcs, does not depict the ratio between individual body components (fat and muscle) [272] , so its use is not appropriate when monitoring these components is necessary. in addition, weighing procedure requires the use of equipment. on the other hand, the advantage is that the measurement is easy and quick. impaired body condition may predispose a cat to euthanasia [159] or to death in general [279, 280] . body condition assessment can provide useful information related to the health aspect of welfare, as stress is often associated with reduced food intake in cats [5, 113, 281] . some cats refuse to eat completely during the first days in the shelter [108] . weight loss due to anorexia has a serious effect on health because it increases the risk of hepatic steatosis [17] . in a study of 3219 cats in a shelter, 1.5% of cats were found to be underweight (which is equivalent to point 1 on the scale) [282] . in shelter cats, weight loss was observed during the first two weeks spent in the shelter [5] despite providing adequate food; authors of the study suggested the weight loss was probably caused by low food intake associated with stress (food intake and css were correlated). however, weight loss may also indicate a serious health problem [283] . in a study by tanaka et al. [5] , 59% of all admitted cats lost their original body weight in the first week in the shelter alone. an association between impaired condition and the development of upper respiratory tract diseases has also been found [5] , as well as an association between the level of body condition and the quality of housing [218] . the increased number of underweight cats correlated with housing with less than one resting place per cat, and housing with a lower number of hiding spots. cats that were given the opportunity to hide were able to adapt faster to the new conditions in the shelter, and their weight loss was not as pronounced as that of cats that did not have this option [108] . by being placed in a shelter, cats encounter a number of stressful stimuli, which can disrupt the state of their well-being. although the efforts of facilities to improve the quality of life of cats in shelters have increased significantly in recent years, as the overall interest in welfare issues has increased, it is necessary for experts to come up with new knowledge that would be applicable in practice. efforts to date have succeeded in enforcing certain measures based on a deeper understanding of the biology and behaviour of cats-for instance, practical improvements such as sound insulation of housing units from dog sounds and other stressful sounds, access to windows and natural light sources, sufficient number of hiding places and elevated locations or placement of housing units above floor level, giving cats a greater sense of security [105, 284, 285] . the current research also focuses on procedures and methods of management and enrichment of the environment (e.g., playing specific music to cats to relieve stress [286] , various forms of olfactory [287, 288] , cognitive [289, 290] , visual [291] , food [292] , pheromone enrichment [293] or social enrichment in the form of human interaction [103] ). another trend in the design of new facilities is group housing of cats, but opinions on it differ in the scientific community. in certain respects, this type of housing may be a form of social enrichment; it has been found that cats from multi-cat households had a higher level of stress after entering a shelter when placed alone than cats from single-cat households [101] . on the other hand, groups of cats may compete for the resources provided; in a study by dantas-divers et al. [294] , 143 conflicts over 4 days were recorded, of which 44 involved competition over resources. given the experiences that cats perceive as aversive in a shelter environment, the need to study appropriate welfare indicators and the development of new tools is immense. the aim of using welfare assessment tools is to reveal critical aspects of living conditions and at the same time establish care management so that disruption occurs as little as possible. as it is not entirely possible to satisfy each individual due to cats' unique temperament, personality, history and coping strategies and at the same time to combine the personnel, financial and value structure of the shelter, it is necessary to seek compromises. the disadvantage of evaluating individual indicators separately is the fact that the results provided by the indicators may not match [207] . while behavioural measurements are valuable and commonly used indicators because they can be used immediately, are non-invasive, and require a relatively short time to train the observer [295, 296] , physiological indicators are accurate, quantifying stress levels [187] and can demonstrate the presence of stress even if there is no external behavioural change. as deterioration of health is associated with welfare impairment, health indicators should also be included in the assessment [217] . although the assessment of environmental indicators has recently faded into the background and the focus has shifted to animals, they can also help to complete the overall picture of welfare. although shelters tend to develop their own welfare assessment procedures, there is no consensus among the indicators that should be part of the tools, let alone the validity and reliability of these procedures [297] . another fact is that shelters for cats are characterised by many significant differences across countries (e.g., in the number of animals-cats housed in large shelters versus small ones or home refuges; housing-individual 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context and culture-specific teachings and resources available to individuals, families, and communities. a social-ecological framework is used to differentiate the impact of exposure to traumatic stressors and the development of (or resistance to) ptsd, based on the individual’s or group’s (i) personal, unique physical characteristics, including skin color, racial background, gender, and sexual orientation; and (ii) family, ethnocultural, and community membership, including majority or minority group status, religious beliefs and practices, socioeconomic resources, and political and civic affiliations. while personal, familial, social, and cultural factors can be a positive resource contributing to safety and well-being, they also can be a basis for placing the person, group, or entire community or population in harm’s way or at heightened risk of developing ptsd. this is an adaptive response in one sense, providing an awareness and readiness to respond should the genocide or any associated forms of stigma, discrimination, or violence ever recur with impunity. however, it can also become a form of persistent hypervigilance similar to that seen in ptsd, placing a strain upon the individual's or group's daily life that may compromise their well-being. our discussion of the impact of exposure to traumatic stressors and ptsd on ethnoracial groups and individuals whose forebears have experienced historical trauma will bear this fact in mind. in addition, gender-based biases and beliefs, many of which are based on longstanding religious and cultural traditions, have caused women to be systematically discriminated against and subject to routine physical and sexual assault. genderbased discrimination and violence against females (whether intra-or extrafamilial) have been so widespread as to be implicated in what kristof and wudunn (2009) term "gendercide." in their recent book, they cite examples of selective abortions based on a fetus's gender and differential nutrition and care beginning in infancy also based on gender preference. they are then often followed by lifelong major disparities in education and restricted role and career opportunities for females as compared to males. unfortunately, even today, with all the advances that have occurred predominantly in western societies, these same issues remain in place around the world. the increased recognition of the underclass status of the majority of women and girls and the discrimination they face, along with the violence perpetrated against them (often seemingly with impunity), in countries around the world (whether industrialized and "advanced," or relatively primitive) has led to the recent development of major initiatives against global violence and discrimination against women. malala yousafzai, who was shot by the taliban for her espousal of universal education for girls, was awarded the nobel peace prize, the youngest recipient to date. the brutality of the attack against her was shocking, yet it served to highlight the traumatic threat to which many girls and women across the world are exposed when targeted for hateful acts by those who believe this is necessary to maintain the status quo and the subservience of females. discrimination and violence based on sexual orientation and transgender/intergender status are yet other sources of traumatic victimization that must be well recognized. sexual orientation is both a personal and social characteristic that is more complex than the gender that a person inherits based on inborn sexual characteristics. when socially ascribed gender and culturally promulgated expectations for gender-based activities, such as mating, are a mismatch to an individual's sense of his or her own true sexual preferences and identity, the conflict can be psychologically devastating. global initiatives therefore are underway to prevent or ameliorate the adverse impact of discrimination, stigma, and violence based on sexual orientation and identity providing an essential foundation for the basic liberties, freedom from assault, and the right to marry to gay, lesbian, bisexual, and transgendered (glbt) individuals. it should also be noted that boys and men are also subject to abuse and assault at rates that are not yet adequately researched. males may be more subject to violence when they are in a position of vulnerability of some sort due to being a member of a group that is targeted and/or of lesser status/lesser strength. depending on social, cultural, and other diversity issues in the traumatic stress field 505 their cultural background and its traditions and beliefs, individuals may also have "multiple vulnerability status"-that is, to be members of more than one group or to have characteristic that cause them to be even more susceptible to discrimination or victimization (i.e., adolescent black male in the united states; a baby born with physical or developmental disabilities in a culture that endorses selective resources to the ablebodied; a gay man or lesbian woman of color in a highly homophobic and racist society). age is yet another vulnerability factor dimension that has not received adequate recognition, with individuals at either end of the life span as most vulnerable. research has substantiated that children and adolescents are the most at-risk segment of the population globally (finkelhor, 2014) . victimization of the elderly and the lessabled/disabled members of the population is now documented as widespread in many societies and is increasingly under investigation. like other forms of abuse, victimization of the elderly and less-abled is often based on the victim's relative degree of dependence and his powerlessness to defend himself. the extent and impact of exposure to traumatic stressors experienced by each of these vulnerable populations is discussed in this chapter, as are the efforts of international non-governmental organizations (ngos) to provide them with resources to reduce their exposure to traumatic stressors or to mitigate the adverse effects of traumatic exposure and ptsd (box 11.1). box 11.1 key points 1. culture, ethnicity, gender, sexual orientation, and disability are potential sources of resilience, but they also may lead to chronic stressors such as social stigma, discrimination, and oppression, which can increase psychological trauma and ptsd. 2. cumulative adversities are faced by many persons, communities, ethnocultural minority groups, and societies that may lead to-as well as worsen the impact of-ptsd: • persons of ethnoracial minority backgrounds; • persons discriminated against due to gender or sexual orientation; • persons with developmental or physical disabilities; • economically impoverished persons and groups, including the homeless; • victims of political repression, genocide, "ethnic cleansing," or torture; • persons chronically or permanently displaced from their homes and communities due to catastrophic armed conflicts or disasters. 3. members of ethnoracial minority groups have been found to be more likely in some cases to develop ptsd than other persons, but in other cases they are less likely to develop ptsd (e.g., persons of asian or african descent). 4. members of ethnoracial minority groups often encounter disparities in access to social, educational, economic, and health care resources; it is these disparities that are the most likely source of the increased vulnerability of these persons to psychological trauma and ptsd. (continued ) to psychological trauma in the immediate or most distant past, or both. psychological trauma and ptsd occur across the full spectrum of gender, racial, ethnic, and cultural groups in the united states (pole, gone, & kulkarni, 2008) . psychological trauma and ptsd are epidemic internationally as well, particularly for ethnoracial minority groups (which include a much broader range of ethnicities and cultures and manifestations of ptsd than typically recognized in studies of ptsd in the united states; de jong, komproe, spinazzola, van der kolk, & van ommeren, 2005; de jong, komproe, & van ommeren, 2003) . the scientific and clinical study of ptsd and its treatment among gender and ethnoracial majority and minority groups is of great importance, especially given the disparities, adversities, and traumas to which they have been subjected historically (miranda, mcguire, williams, & wang, 2008 )-and to which they are still exposed in health and health care, education and income, and adult criminal and juvenile justice (ford, 2008) . although latinos (and possibly african americans) persons are at greater risk than european americans for ptsd based on available research findings (pole, gone, & kulkarni, 2008) , it is possible that the elevated prevalence may be due to differences in the extent or types of exposure to psychological trauma (including prior traumas that often are not assessed in ptsd clinical or epidemiological studies; eisenman, gelberg, liu, & shapiro, 2003) or to differences in exposure to other risk or protective factors such as poverty, education, or gender-based violence (turner & lloyd, 2003 , 2004 . in addition, there is sufficient diversity (in norms, beliefs, values, roles, practices, language, and history) within categorical ethnocultural groups such as african americans or latinos to call into question any sweeping generalizations about their exposure and vulnerability or resilience to psychological trauma (pole et al., 2008) . race, ethnicity, gender and sexual identity, and culture tend to be described with shorthand labels that appear to distinguish homogeneous subgroups but that actually obscure the true heterogeneity within as well as between different groups (marsella, friedman, gerrity, & scurfield, 1996) . one partial antidote for this problem is for clinicians and researchers to be curious about these issues and to ask study participants or clinical patients to self-identify their own racial, ethnic, and cultural background and to essentially educate them about their unique characteristics and associated belief systems and traditions (brown, 2008; brown, hitlin, & elder, 2006) . it also is important to carefully assess factors that are associated with differential exposure to adverse experiences (such as racial-ethnic discrimination) or differential access to protective resources (such as income, health care, education, police protection), rather than assuming that each member of an ethnocultural group is identical on these crucial dimensions. however, when systematic disparities in exposure to stressors or deprivation of resources are identified for specific groups, such as persons from indigenous culturesthe original inhabitants of a geographic area who have been displaced or marginalized by colonizing national/cultural groups-are found to have a generally increased risk of discrimination, poverty, addiction, family violence, and poor health (harris et al., 2006; liberato, pomeroy, & fennell, 2006) , it is crucial not to mistakenly conclude that those persons are less resilient than others when they are confronted with traumatic stressors. commonly, the very opposite is true: persons and groups who are subjected to chronic stressors or deprivations tend to be more resilient than others, but they also are more exposed to and less protected from traumatic stressors (pole et al., 2008) . racism and associated discrimination and mistreatment are particularly chronic stressors faced by many members of ethnoracial and other minority groups. racism may constitute a form of psychological trauma in and of itself, increasing the risk of exposure to psychological trauma, and exacerbating its impact by increasing the risk of ptsd (ford, 2008) . as of yet, few systematic studies have directly examined racism as a risk factor for exposure to psychological trauma, although the connection is increasingly recognized (carter & forsyth, 2009; hunter & schmidt, 2010; miller, 2009) . perhaps, the holocaust and other forms of genocide have been the most investigated to date. studies of survivors of the holocaust and other types of ethnic annihilation provide particularly graphic and tragic evidence of the infliction of psychological trauma en masse in the name of racism (staub, 2005; yule, 2000) . studies are needed that systematically compare persons and groups who are exposed to different types and degrees of racism in order to test whether (and under what conditions) racism is a form of, or leads to exposure to other types of, traumatic stressors (ford, 2008) . when racism leads to the profiling and targeting of ethnoracial minority groups for violence, dispossession, dislocation, or annihilation, the risk of ptsd increases in proportion to type and degree of the traumatization involved (pole et al., 2008) . for example, studies based in the united states (pole et al., 2008) and internationally (macdonald, chamberlain, & long, 1997) suggest that racial discrimination may have played a role in placing military personnel from ethnoracial minority groups at risk for more extensive and severe combat trauma exposure. one study found that self-reported experiences of racial discrimination increased the risk of ptsd among latino and african american police officers (pole, best, metzler, & marmar, 2005) . another study with asian american military veterans from the vietnam war era showed that exposure to multiple race-related stressors that met ptsd criteria for psychological trauma was associated with more severe ptsd than when only one or no such race-related traumas were reported (loo, fairbank, & chemtob, 2005) . this study more precisely operationalized racism than any prior study, utilizing two psychometrically validated measures of race-related stressors and ptsd. however, as in the pole et al. (2005) study, the stressors/traumas and ptsd symptoms were assessed by contemporaneous self-report, so the actual extent of racism experienced by the participants cannot be definitely determined. the loo et al. (2005) study also did not control for traumatic stressor exposure other than that which was related to racism. in order to extend the valuable work these studies have begun, it will be important to utilize measures based on operationally specific criteria for categorizing and quantifying exposure to discrimination (e.g., wiking, johansson, & sundquist, 2004 ) as a distinct class of stressors that can be assessed separately as well as concurrently with exposure to psychological trauma. research also is needed to determine to what extent the adverse outcomes of racial disparities are the direct result of racism as a stressor (e.g., racially motivated stigmatization, mistreatment, subjugation, and deprivation resulting in personal and community depression and destabilization), as opposed to the indirect effects of racism (such as microaggressions that accrue over time). racism can also indirectly reduce access to protective factors (adequate nutrition and other socioeconomic and community resources) that protect against the adverse effects of stressors (such as poverty, pollution, disaster) and traumatic stressors (such as accidents, crime, or violence). hurricane katrina and its aftermath provided just such an example. it is important to determine whether ptsd is the product of either the direct or indirect effects of racism, or both, particularly given its demonstrated association with other psychiatric conditions (such as depression, anxiety, and addiction) and with increased risk of physical illness (such as cancer and cardiovascular disease) in ethnoracial minorities (e.g., among american indians; sawchuk et al., 2005) . education is a particularly relevant example of a socioeconomic resource to which ethnoracial minorities often have restricted access as that as a protective factor mitigating against the risk of ptsd (dirkzwager, bramsen, & van der ploeg, 2005) and overall health status (wiking et al., 2004) . racial disparities in access to education are due both to direct influences (such as lower funding for inner-city schools that disproportionately serve minority students) and indirect associations with other racial disparities (such as disproportionate juvenile and criminal justice confinement of ethnoracial minority persons). racial disparities in education are both the product of and a contributor to reduced access by minorities to other socioeconomic and health resources (such as income, health insurance, adequate nutrition) (harris et al., 2006) . when investigating risk and protective factors for ptsd, it is essential therefore to consider race and ethnicity in the context not only of ethnocultural identity and group membership but also of racism and other sources of racial disparities in access to socioeconomic resources. although all ethnoracial minority groups tend to be disproportionately disadvantaged with regard to the more privileged majority population, particularly severe disparities in access to vital resources often are complicated by exposure to pervasive (both intrafamilial and community) violence and by the loss of ties to family, home, and community. when family and community relationships are severed-as occurs with massive political upheaval, war, genocide, slavery, colonization, or catastrophic disasters-racial and ethnocultural groups may find themselves scattered and subject to further victimization and exploitation. for example, there continue to be massively displaced populations in central and south america, the balkans, central asia, and africa. when primary social ties are cut or diminished as a result of disaster, violence, or political repression, the challenge expands beyond survival of traumatic life-threatening danger to preserving a viable life, community, and culture in the face of lifealtering losses and suppression of those very factors needed to maintain (garbarino & kostelny, 1996; rabalais, ruggiero, & scotti, 2002) . ethnoracial groups that have been able to preserve or regenerate core elements of their original cultural norms, practices, and relationships within intact or reconstituted families may actually be particularly resilient to traumatic stressors and protected against the development of ptsd. for example, persons of asian or african descent have been found to be less likely than those of other ethnocultural backgrounds to develop ptsd. whether this is due to factors other than ethnicity per se, such as having cultural practices and beliefs that sustain family integrity and social ties, is a question that has not been scientifically studied and should be a focus for research (pole et al., 2008) . a recurring theme is that the psychological trauma inflicted in service of racial discrimination may lead not only to ptsd but also to a range of insidious psychosocial problems that result from adverse effects upon the psychobiological development of the affected persons. when families and entire communities are destroyed or displaced, the impact on the psychobiological development of children and young adults may lead to complex forms of ptsd that involve not only persistent fear and anxiety but also core problems with relatedness and self-regulation of emotion, consciousness, and bodily health that are described as "complex ptsd" (herman, 1992) or "disorders of extreme stress" (de jong et al., 2005) . a critical question not yet answered by studies of ptsd and racial discrimination (pole et al., 2005) and race-related stress (loo et al., 2005) , as well as by the robust literature that shows evidence of intergenerational transmission of risk for ptsd (kellerman, 2001) , is whether racism constitutes a "hidden" (crenshaw & hardy, 2006) or "invisible" (franklin, boyd-franklin, & kelly, 2006) form of traumatization that may be transmitted across generations. recent research findings demonstrating highly adverse effects of emotional abuse in childhood (teicher, samson, polcari, & mcgreenery, 2006) are consistent with a view that chronic denigration, shaming, demoralization, and coercion may constitute a risk factor for severe ptsd and associated psychobiological problems. research is needed to better describe how emotional violence or abuse related to racism may (along with physical violence) constitute a form of traumatic stress and how this may adversely affect not only current but also future generations. a fully articulated conceptual model for the scientific study and social/clinical prevention and treatment of the adverse impact of psychological trauma and ptsd requires principles and practices informed by this diversity of factors, rather than a "black and white" view of race, ethnicity, or culture that misrepresents the individual's and group's heritage, nature, and needs. treatment preferences, in terms of characteristics of the therapist as well as the therapy model, differ substantially not only across but also within ethnoracial groups (pole et al., 2008) . as a result, it is not possible as yet-and may never be possible-to precisely prescribe how best to select or train therapists and design or adapt therapies to fit different ethnocultural groups and the individuals within them. a culturally competent (brown, 2008 (brown, , 2009a (brown, , 2009b approach to treating ptsd (ford, 2008) begins with a collaborative discussion in which the therapist adopts the stance of a respectful visitor to the client's outer and inner world-clarifying the client's expectations and preferences, and the meaning of sensitive interpersonal communication modalities (such as spatial proximity, gaze, choice of names, private versus public topics, synchronizing of talk and listening, use of colloquialisms, providing advice or education). ptsd therapists thus must avoid stereotypic assumptions and become both a host and guest in the client's psychic world in order to ensure that assessment and treatment are genuinely collaborative and sensitive to each client's ethnocultural traditions, expectations, goals, and preferences (parson, 1997; stuart, 2004) . at times, it is helpful to involve other members of the family or culture in assisting with the treatment. religious beliefs and spirituality are other dimensions of culture that have not yet been given sufficient focus in most psychotherapy but must also be assessed and understood by the therapist (walker, courtois, & aten, 2014) . cultural competence means many things to many people, and unfortunately, it is often mistakenly equated with being of the same racial, ethnic, cultural, religious, or national background as the persons involved in a study or receiving services, or knowing in advance exactly what each person believes and expects, how they communicate with and are most receptive to learning from others, and what their experience has been in relation to sensitive matters such as psychological trauma or ptsd. this is likely to be a serious mistake for several reasons. sharing some general racial, ethnic, cultural, or national features (or an apparently identical language or religion) is not synonymous with shared identity, knowledge, or history. even persons from as virtually identical backgrounds as monozygotic twins raised in the same family have substantial differences in physical and temperamental characteristics as well as often quite distinct social learning histories, and thus rarely if ever can reliably read one another's minds or exactly know one another's vulnerabilities and strengths. therefore, cultural competence should not be defined in terms of stereotypic assumptions about identity or prescience but instead based upon a respectful interest in learning from each person and community what they have experienced and how they understand and are affected by psychological trauma and ptsd. we should also note that the idioms of distress can differ by culture and tradition. professionals from industrialized nations and anglo cultures must be cautious and respectful in working with individuals and communities from other cultures that are challenged by ptsd in the aftermath of exposure to violence or disasters. before offering or providing education or therapeutic assistance, it is essential to become aware of how the potential recipients understand and prefer to communicate about traumatic stress and the process of healing from traumatization. the implication for psychometric assessment of psychological trauma and ptsd with clients of ethnocultural minority groups (hall, 2001; marsella et al., 1996) is that it is essential to carefully select protocols that do not confront individuals with questions that are inadvertently disrespectful of their values or practices (e.g., including peyote as an example of an illicit drugs in a native american tribe that uses it for religious rituals), irrelevant (e.g., distinguishing blood family from close friends in a group that considers all community members as family), or incomplete (e.g., limiting health care to western medical or therapeutic services, to the exclusion of traditional forms of healing). a systematic assessment of trauma history and ptsd thus should include not only a recitation of events in a person's life and symptomatic or resilient responses in the aftermath but how the person interpreted these events and reactions based on their cultural framework, beliefs, and values (manson, 1996) . interventions for prevention or treatment of ptsd typically have been developed within the context of the western medical model (parson, 1997 ; but see andres-hyman, ortiz, anez, paris, & davidson, 2006; hinton et al., 2005; hwang, 2006 , for examples of culturally sensitive adaptations). evidence-based ptsd treatment models are not necessarily incompatible with culturally specific healing practices and have in common the goal of fostering not just symptom reduction but a bolstering of resilience and mastery (see chapters 7 and 8). the integration of culturally specific methods and rituals in prevention or treatment interventions for ptsd, however, requires careful ethnographic study (i.e., observing and learning about the values, norms, beliefs, and practices endorsed and enforced by different cultural subgroups and their particular idioms (ways of describing and explaining) traumatic stress and ptsd) so the ptsd clinician and researcher can truly work collaboratively withrather than imposing external assumptions and standards upon-the members of the wide range of ethnic and cultural communities. in most cultures, girls and women are subject to discrimination in the form of limitations on their access to crucial socioeconomic resources. women earn 30-40% lower wages or salaries than men in most job classes in the united states (http://www.payequity.org/info.html) and europe (http://www.eurofound.europa.eu/ewco/2007/01/ es0701049i.htm). although girls and women are approaching parity with boys and men in access to education in most areas of the world (and exceed the enrollment of boys or men in secondary and college/university education), in sub-saharan africa and asia, women and girls are as much as 33% less likely to be able to enroll in education and to have achieved literacy as adults (http://www.uis.unesco.org/template/ pdf/educgeneral/uisfactsheet_2008_no%201_en.pdf). girls and women also may be systematically subjected to extreme forms of psychological and physical trauma as a result of their gender being equated with second-class citizenship and social norms that permit or even encourage exploitation. sexual exploitation of women and girls is an international epidemic, including abuse and molestation, harassment, rape and punishment of rape victims, forced marriage, genital mutilation, and sex trafficking or slavery (box 11.2). physical abuse or assault of women and girls is tolerated-and in some cases actually prescribed as a form of social control-in both mainstream cultures and subcultures that span the globe and include most religions and developed as well as developing or preindustrial societies. similar potentially traumatic forms of violence are directed at many glbt persons as a result of both formal and informal forms of social stigma and discrimination. epidemiological studies have been conducted with samples of glbt youth (d'augelli, grossman, & starks, 2006) and adults (herek, 2009) in the united states, suggesting that they are often subjected to potentially traumatic violence as a result of their nontraditional sexual orientation and behavior. instances of violence specifically related to sexual orientation include: • 5-10% of gay men and 10% of lesbians who were physically assaulted in the past year; • 20-32% of glb adults who were subjected to actual or threatened violence toward their person or a property crime at some point in their lives; • 9-11% of glb adolescents reported past incidents of physical or sexual violence. in contrast to the general pattern of stigma-related violence being directed toward girls and women, gay and bisexual boys (d'augelli et al., 2006) and men (herek, 2009) were more likely to report violent victimization or threats than lesbian or bisexual women or girls. the findings from the survey of glb adolescents suggest that stigma and victimization begin early in life, with physical and sexual attacks occurring as early as ages 8-9 years old. one in eleven glb adolescents met criteria for ptsd, box 11.2 "making the harm visible": sexual exploitation of women and girls women from every world region report that the sexual exploitation of women and girls is increasing. all over the world, brothels and prostitution rings exist underground on a small scale, and on an increasingly larger scale, entire sections of cities are informally zoned into brothels, bars, and clubs that house, and often enslave, women for the purposes of prostitution. the magnitude and violence of these practices of sexual exploitation constitute an international human rights crisis of contemporary slavery. in prostitution: a form of modern slavery, dorchen leidholdt, the coexecutive director of the coalition against trafficking in women, examines the definitions of slavery and shows how prostitution, and related forms of sexual exploitation, fit into defined forms of slavery. in some parts of the world, such as the philippines, prostitution is illegal but well entrenched from providing "recreational services" to military personnel. in "blazing trails, confronting challenges: the sexual exploitation of women and girls in the philippines," aida f. santos describes the harmful conditions for women and girls in prostitution in the philippines, with problems related to health, violence, the legal system, and services. in other regions, such as northern norway, organized prostitution is a more recent problem, stemming from the economic crisis in russia. in "russian women in norway," asta beate håland describes how an entire community is being transformed by the trafficking of women for prostitution from russia to campgrounds and villages across the border in norway. political changes combined with economic crises have devastated entire world regions, increasing the supply of vulnerable women willing to risk their lives to earn money for themselves and their families. aurora javate de dios, president of the coalition against trafficking in women, discusses the impact of the southeast asian economic crisis on women's lives in "confronting trafficking, prostitution and sexual exploitation: the struggle for survival and dignity." economic globalization controlled by a handful of multinational corporations located in a few industrialized countries continues to shift wealth from poorer to richer countries. in her paper "globalization, human rights and sexual exploitation," aida f. santos shows us the connection between global economics and the commodification and sexual exploitation of women and girls, especially in the philippines. structural adjustment programs implemented by international financial institutions impose loan repayment plans on poor countries, which sacrifice social and educational programs in order to service their debt to rich nations and banks. fatoumata sire diakite points to structural adjustment programs as one of the factors contributing to poverty and sexual exploitation in her paper "prostitution in mali." zoraida ramirez rodriguez writes in "report on latin america" that the foreign debt and policies of the international monetary fund are primary factors in creating poverty for women and children. these forces leave women with few options, increasing the supply of women vulnerable to recruitment into bride trafficking and the prostitution industry. (continued ) social problems such as sexual and physical abuse within families force girls and women to leave in search of safety and a better life, but often they find more exploitation and violence. physical and sexual abuse of girls and women in their families and by intimate partners destroys girls' and women's sense of self and resiliency, making them easy targets for pimps and traffickers who prey on those who have few options left to them. these factors are evident in many of the papers from all world regions in this volume, such as jill leighton and katherine depasquale's, "a commitment to living," and martha daguno's, "support groups for survivors of the prostitution industry in manila." government policies and practices also fuel the demand for prostitution, as they legalize prostitution or refuse to enforce laws against pimps, traffickers, and male buyers. in making the harm visible, we see how countries with governmental structures and ideological foundations as different as the netherlands and iran, both promote and legalize sexual violence and exploitation of girls and women. in "legalizing pimping, dutch style," marie-victoire louis exposes the liberal laws and policies that legalize prostitution and tolerate brothels in the netherlands. at the other extreme, religious fundamentalists in iran have legalized the sexual exploitation of girls and women in child and temporary marriages and the sexual torture of women in prison. sarvnaz chitsaz and soona samsami document this harm and violation of human rights in "iranian women and girls: victims of exploitation and violence." global media and communication tools, such as the internet, make access to pornography, catalogs of mail-order brides, advertisements for prostitution tours, and information on where and how to buy women and girls in prostitution widely available. this open advertisement normalizes and increases the demand by men for women and girls to use in these different forms of exploitation. donna m. hughes describes her findings on how the internet is being used to promote the sexual exploitation of women and children in "the internet and the global prostitution industry." in this milieu, women and girls become commoditiesbought and sold locally and trafficked from one part of the world to another. how do we make the harm of sexual exploitation visible? in a world where sexual exploitation is increasingly normalized and industrialized, what is needed to make people see the harm and act to stop it? the women in making the harm visible recommend four ways to make the harm of sexual exploitation visible: listen to the experiences of survivors, expose the ideological constructions that hide the harm, expose the agents that profit from the sexual exploitation of women and children, and document harm and conduct research that reveals the harm and offers findings that can be used for policy initiatives. reprinted with permission from the introduction to making the harm visible, edited by d. hughes 3-20 times the prevalence of children (copeland, keeler, angold, & costello, 2007) and adolescents (kilpatrick et al., 2000) in national samples in the united states. although gender and sexual orientation may seem intuitively to be simpler phenomena than race or ethnicity, in reality they are quite complex in terms of referring to not just biological characteristics but many aspects of psychological identity and social affiliations. being a female or a male, let alone gay, lesbian, bisexual, or transgendered/ intergendered, means many different things to different people. although more stable than changeable, sexual orientation and even gender may be changed for the same person over time. it is inaccurate to assume that all or even most people of a given gender or sexual orientation are identical or even similar without careful and objective assessment of how they view themselves and how they actually act, think, and feel. in relationship to psychological trauma and ptsd, therefore, the broad generalizations that have been suggested by research concerning gender and sexual orientation relate more to the way in which people of a gender or sexual orientation are generally viewed and treated (which varies, depending on the society and culture) than to inherent qualities of a given gender or sexual orientation (which is highly individual across all societies and cultures). the finding that girls and women are more often subjected to sexual and intrafamilial traumatic stressors, while boys and men more often experience physical, accidental, combat, and assaultive traumatic stressors is consistent with stereotypic sex roles that are found in many (but not all) cultures that assign females to the role of subservient helper and caregiver, while males are assigned to the role of leader and warrior. there are biological foundations for these differences-such as due to distinct levels of the sex-linked hormones estrogen and testosterone, and brain chemicals that differentially affect females and males (oxytocin and vasopressin; see chapter 5). however, biology need not dictate a person's or a group's destiny, so it is inaccurate to assume that males or females must always fill these sex role stereotypes, particularly when there are severe adverse consequences, such as the epidemics of abuse of girls and women and of boys and men killed as violent combatants or as the "spoils of war." stereotypes can be even more insidious and damaging in relation to sexual orientation. only in the past 3 decades has homosexuality been rescued from the status of a psychiatric disorder (as it was in the first three editions of the diagnostic and statistical manual). stigma and harassment evidently are still experienced, potentially with traumatic results when violent acts are tolerated or even encouraged, by glbt adults and youth. it is not surprising that the prevalence of ptsd is greater among persons with other than heterosexual sexual identities, and the extent to which this is the result of the pernicious stigma directed at such individuals in most cultures or of outright traumatic violence, or both, remains to be tested. persons with physical or developmental disabilities are another group of persons who unfortunately may be subjected to stigma and discrimination. physical disabilities are more common in developing countries than in more industrialized and affluent nations in which medical technology and accident and illness prevention have reduced the risk of severe injury or genetically based physical disabilities (mueser, hiday, goodman, & valentini-hein, 2003) . persons with physical disabilities may be at risk for exposure to traumatic accidents or maltreatment as children and as adults due to limitations in their abilities to care for themselves and live independently, particularly if they have cognitive impairment due to conditions such as mental retardation or serious mental illness. only one study that examined the prevalence of exposure to potentially traumatic events among physically disabled persons could be located. that was a national survey of women with physical disabilities by the center for research on women with disabilities (nosek, howland, & young, 1997) . on the one hand, the study found that disabled women were no more likely to report exposure to physical or sexual abuse than women without physical disabilities. however, in more detailed interviews with a subsample, more than 80% reported instances of abuse, on average two incidents per woman (each often lasting for a lengthy time period). for example, the report provides verbatim quotations: more than half of all respondents (52% with disabilities, 51% with no disability) reported a history of either physical or sexual abuse, or both, which is a substantially higher prevalence than that reported in epidemiological surveys of nationally representative samples of women. notably, women with disabilities were more likely than women without disability to report emotional abuse from a caregiver or family member and to have experienced all forms of abuse for a longer time period than women without disability. although ptsd was not assessed, women younger than 50 years old with spina bifida (39%), amputation, traumatic brain injury (tbi), or multiple sclerosis (>25%) were highly likely to be diagnosed with depression than women with no disability. in light of the extensive histories of potentially traumatic abuse and of depression, it appears that women with physical disabilities-particularly those in early to midlife adulthood with disabilities that involve progressive deterioration or mental or psychological disfiguration-may be at risk for having experienced traumatic interpersonal violence and other forms of abuse and suffering from undetected ptsd. tbi is a special case of physical disability because it involves physical injury that specifically compromises mental functioning. tbi ranges from mild (no more than 30 minutes of unconsciousness and 24 hours of amnesia) to severe (coma of at least 6 hours or amnesia for more than 24 hours). studies with adults and children of both genders who have sustained tbi demonstrate that they are as likely to develop ptsd as persons in equally severe accidents or assaults who have not (mcmillan, williams, & bryant, 2003) . fewer studies have been conducted with persons with severe than mild tbi, but they have not been found to be less likely to develop ptsd, as was originally hypothesized-due to not being able to experience or later recall the psychologically traumatic aspects of the injury as vividly as a person who does not lose consciousness or have amnesia. a subsequent study confirmed that adults with tbi were less likely to report acute traumatic stress symptoms immediately after the injury and to recall having felt helpless when interviewed several weeks later but that 3 months after the accident, they were equally likely to report ptsd symptoms as injury survivors with no tbi (jones, harvey, & brewin, 2005) . tbi definitely exacerbates, and indeed may cause, ptsd, as tragically is illustrated by the extremely high estimates of prevalence of ptsd among military veterans of the iraq and afghanistan wars with tbi. thus, ptsd warrants careful assessment when tbi has occurred. concerning developmental disabilities, similarly, only one published study of ptsd could be located (ryan, 1994) . in that study, 51 adults receiving services for learning disability were more likely than other adults (kessler, sonnega, bromet, & hughes, 1995) to report exposure to traumatic stressors (100% prevalence, on average two past traumatic events). however, they had no greater risk than adults in the general population when exposed to traumatic stressors. the most frequently reported types of traumatic exposures were multiple experiences of sexual abuse by multiple perpetrators (commonly starting in childhood), physical abuse, or life-threatening neglect. traumatic losses involving a caregiver or close relative or friend (including witnessing the death in several instances (such as witnessing a sibling dying in a fire, a close friend die during a seizure or an accident, or a parent commit suicide by shooting himself in the head with a gun)) were also reported by at least 10% of the participants. most of the learning-disabled adults who met criteria for ptsd had been referred for treatment for violent or disruptive behavior, typically with no psychiatric diagnosis or a diagnosis of schizophrenia, autism, or intermittent explosive disorder. when ptsd was diagnosed, major depression was a frequent comorbid disorder; yet, neither ptsd nor depression typically had been identified prior to the clinical assessment study. the findings of this study suggest that adults with developmental disorders often have been targets for abuse or neglect in childhood or have sustained severe traumatic losses and that their ptsd and depression tend to go undiagnosed as clinicians make their behavioral difficulties the focus of treatment services (box 11.3). poverty is an adverse result of having low "social status." this does not mean that a person or group is objectively deficient but rather that he or she is identified socially and politically as either not deserving or not possessing the social mandate to have access to resources such as money, safety, housing, transportation, health care and nutrition, education, and gainful employment. kubiak's (2005) social location theory states that each individual possesses identities within their society that are defined (2001) 1 show the adverse impact of undetected ptsd: "a 15-year-old girl with a learning disability has suffered early abuse of a physical and sexual nature, including neglect. she presented [for medical evaluation] in early childhood with behavioral problems of aggression. she settled in a residential school from age 12 to 13 before an act of arson. she later revealed that she had experienced inappropriate sexual behaviors with peers at school. she complained of intrusive thoughts and images, along with depressive symptoms. at times she shows sexually inappropriate behavior and self-harm." "a 40-year-old man with a moderate learning disability who had been sexually assaulted by a care[giver] presented [for medical evaluation] in [an] acute state with disturbance of appetite, sleep, loss of skills, and emotional numbness, but the abuse was revealed only months later. on being exposed to the perpetrator at a later date, he showed a deterioration in mental state with acute symptoms of anxiety, and later developed a depressive disorder requiring medication. his level of functioning never returned to that prior to the traumatic event." a third case illustrates the therapeutic gains that a ptsd perspective can provide: a 32-year-old woman with learning disabilities and pervasive developmental disorder had been diagnosed at age 11 with schizophrenia and subsequently had been diagnosed with schizoaffective disorder, bipolar disorder, and borderline personality disorder. for 20 years after the first psychiatric diagnosis and hospitalization, she had been psychiatrically hospitalized more than 50 times due to episodes of acute suicidality complicated by auditory command hallucinations (i.e., she believed she was hearing voices telling her to kill herself) and compulsive self-harm behavior (she used sharp objects to cut virtually every area on both arms and legs). treatment included high doses of antipsychotic, antiseizure, antidepressant, and antianxiety medications and two courses of electroconvulsive therapy, with periods of relative stabilization sufficient for her to live in an assisted living residential home and on two occasions to live in an independent apartment with in-home daily case management and nursing care. each period of improvement was relatively brief, lasting no longer than 3-6 months, at which time she experienced severe worsening in the apparently psychotic, depressive, and anxiety symptoms, requiring multiple rehospitalizations and progressive loss of social and cognitive abilities. for several years, family therapy was conducted, and the patient's history of traumatic stressors was assessed gradually in order not to lead to further destabilization. in addition to potential episodes of sexual assault as an adolescent and young adult, her mother disclosed that her biological father had been severely domestically violent during the patient's first 2 years of life, until the mother ended that relationship. when ptsd was confirmed and accepted by the treatment team and the patient and her family as the primary diagnosis, the patient felt that she finally understood why she was experiencing the cyclic surges in distress and was able to utilize affect by factors such as their race, socioeconomic class, gender, age, residential status, and legal status. the greater the number of oppressed identities that one possesses, the more likely one will be "poor," including not only low income but also living in neighborhoods plagued with high crime, gang violence, abandoned buildings, drugs, teen pregnancy, high unemployment rate, underfunded schools, housing shortage, food of limited nutritional value, and unresponsive police. thus, poverty fundamentally is a breakdown of the social order as well as a resultant deprivation of resources for some people. the relationship between low income and exposure to psychological trauma and ptsd has been studied primarily in relationship to women and families, including those who currently have stable housing and those who do not. morrell-bellai, goering, and boydell (2000) identify poverty as a core risk factor for homelessness, because the socioeconomic benefits provided by a diminishing societal safety net and the typically insufficient employment wages provided by marginal jobs force people to rely on an increasingly limited pool of subsidized housing or to become homeless. associated risk factors include a lack of education, lack of work skill, physical or mental disability, substance abuse problem, minority status, sole support parent status, or the absence of an economically viable support system (fischer & breakey, 1991; morrell-bellai et al., 2000) . snow and anderson (1993) found that the most common reasons for homelessness reported in a survey of men and women living "on the street" were family-related problems such as marital breakup; family caregivers becoming unwilling or unable to care for a mentally ill or substance-abusing family member; escape from a dysfunctional and/or abusive family; or not having a family to turn to for support. poverty and homelessness involve a vicious cycle in which socioeconomic adversities are compounded by the experience of homelessness, leading to psychological disaffiliation, hopelessness, and loss of self-efficacy, and often substance dependence (bentley, 1997; hopper & baumohl, 1994; morrell-bellai et al., 2000) -which thus tends to perpetuate poverty and homelessness. a recent study by frisman, ford, lin, mallon, and chang (2008) reported that 91% of a sample of very low-income homeless women caring for children had experienced at least one type (and on average, five different types) of psychologically traumatic events, usually repeatedly and over long periods of time, with one in three having experienced full or partial ptsd at regulation skills (taught using dialectic behavior therapy and trauma affect regulation: guide for education and therapy; see chapter 7). over the next year, her medications were carefully reduced to the lower therapeutic range for attentional problems and anxiety, with a sustained improvement in mood and social and cognitive functioning such that she was able to successfully work as a skilled volunteer in an assisted living center for older adults. some time in their lives. in addition, ford and frisman (2002) found that one in three of these homeless women with children had experienced a complex variant of ptsd involving problems with dysregulated affect or impulses, dissociation, somatization, and alterations in fundamental beliefs about self, relationships, and the future (i.e., "complex ptsd" or "disorders of extreme stress"; ford, 1999) . more than half of the sample had a history of either or both ptsd and its complex variant. exposure to violence and other forms of victimization begins in childhood for many homeless individuals, in part due to their exposure and the vulnerability of their living conditions (north, smith, & spitznagel, 1994) . rates of childhood physical abuse as high as 52% among homeless adolescents have been reported (maclean, paradise, & cauce, 1999) , and this figure may be on the low end. extremely poor women, whether homeless or not, have elevated rates of lifetime ptsd or other mental illness, and a history of such disorders is associated with having grown up in family and community environments with violence, threat, and anger (bassuk, dawson, perloff, & weinrub, 2001; davies-netzley, hurlburt, & hough, 1996) . however, homelessness per se may confer additional risk: homeless mothers and their children have higher lifetime rates of violent abuse and assault than equally impoverished housed mothers (bassuk et al., 1998) . thus, poverty puts people at risk for traumatic violence, but not having a stable residence compounds this risk and the likelihood of developing ptsd. victims of political repression, genocide ("ethnic cleansing"), and torture when political power is used to repress free speech and citizens' self-determination, there is an increase in the risk to members of that nation or community and its neighbors and associates of psychological trauma. domestic violence (see box 10.5) is a microcosm that shares much in common with large-scale political repression, because physical, psychological, and economic power is used to entrap, systematically break down, and coercively control the thoughts as well as the actions and relationships of the victim. on a larger scale, political repression involves similar psychological (and often physical as well) assaults by the people and institutions in power on the people, families, communities, and organizations that are deprived of access to political power and socioeconomic resources-and therefore also on their fundamental freedoms and values. without access to self-determination and the resources necessary to sustain independence, people are vulnerable to not just traumatic exploitation and violence but also to the traumatic loss of their intimate relationships, their families, their way of life, and their values (box 11.4). genocide (also described as "ethnic cleansing") involves the planned and systematic elimination of an entire collectivity of people, based on discrimination against them. historically, genocide has occurred often when conquering nations not only dominated and subjugated other nations but sought to eradicate their core culture and its leaders and teachers and to kill off or enslave the entire population. examples in the twentieth century include the armenian genocide in turkey, the holocaust box 11.4 the lost boys of sudan: complex ptsd in the wake of societal breakdown in the book what is the what?, by dave eggers (2005), valentino achak deng (a fictional character based upon a real person) provides an autobiography that includes his trials and tribulations in his current home in atlanta, georgia, after a traumatic journey of many years as a "lost boy" fleeing from his family's home in a rural village in southern sudan to refugee camps in ethiopia and kenya. valentino graphically describes a relentless series of traumatic experiences that include his village becoming a war zone, the deaths of family and friends, starvation and continual threats of being killed while traveling by foot with thousands of other "lost" children to escape sudan, witnessing brutal acts of violence by children as well as adults (e.g., a boy beating another boy to death in a fight over food rations), and being robbed and beaten unconscious in his own home in atlanta by a predatory african american couple. valentino is a good example of a person who suffers from chronic and complex ptsd, yet is extremely articulate, intelligent, and resourceful. valentino struggles with both unwanted memories and the need to keep his memories so that he ultimately can make sense of what has happened to him: what is the what? by writing his autobiography, he did what the therapy for children or adults with ptsd is intended to do: making sense of, rather than attempting to avoid, memories and reminders of traumatic experiences as a part-albeit horrible or tragic-of one's complete life story (see chapters 7 and 8). for example, in trauma-focused cognitive behavior therapy, the therapist helps the child to write (or in other creative ways to depict, such as by drawing pictures; using puppets, dolls, or action figures; or using collage or music) a "story" of what happened to them before, during, and after traumatic experience(s) and to share this "story" with a parent who can help the child with feelings of guilt and fear so that the traumatic memory can be "over" in the child's mind. because valentino was not able to get that kind of help, his autobiography as an adult (the book) is a kind of second attempt to achieve a sense of resolution by telling his story. but we see how this is very difficult to do when current life involves new problems and dangers that interfere with achieving a sense of safety. whether valentino succeeds in achieving some degree of emotional resolution about what he and his loved ones have suffered is an open question. what is clear is that he never stops trying to do so. it also is apparent that valentino's ethnic identification and heritage as an african man from the dinka tribe is very important as a protective factor enabling him to retain a small but significant fragment of his sense of personal identity and his intimate ties to his family and community. he experiences an odyssey as a victim fleeing the scene of horrific trauma, an initially reluctant but eventually drug-induced savage combatant, and a refugee "stranger in a strange land" when he is able to escape to what seems like an entirely different planet in the cosmopolitan urban setting of atlanta and the southern united states. it is the psychological trauma that he experiences on this odyssey, and the chronic stressors and societal breakdown and oppression that led him-and millions of others of all ages and a multiplicity of ethnocultural groups-on this journey of crisis and survival, and not his ethnicity or cultural background that is responsible for the profound symptoms of ptsd that he develops. inflicted on jewish people in europe by the nazis, the "ethnic cleansing" in bosnia and serbo-croatia in the 1980s, and the massacres and mass starvation and epidemics perpetrated in rwanda in 1994, in sudan beginning in 2003, and in somalia, kenya, and zimbabwe most recently. genocide was first used as a term in 1944 by raphael lemkin, combining the words genos, from the greek for "race" or "kind," and cidere, which is latin and can be translated as "kill" (brom & kleber, 2008) . in 1948, the term was adopted by the united nations general assembly and defined by the united nations convention on the prevention and punishment of the crime of genocide (cppcg) as follows: gregory stanton, the president of genocide watch, described "8 stages of genocide"; http://www.genocidewatch.org/aboutgenocide/8stagesofgenocide.htm. accessed 4/14/15: 1. classification-earliest stage, dividing people into "us" and "them" (the victim group). 2. symbolization-assigning particular symbols to designate the victim group members. 3. dehumanization-equating certain people with subhuman animals, vermin, or insects. 4. organization-militias or special units created for the purpose of genocide. 5. polarization-broadcasting of propaganda aimed at marginalizing the out-group. 6. preparation-out-group members are physically separated or confined in a "ghetto." 7. extermination-murder, starvation, infection, or other forms of inflicting pain and death. 8. denial-refusal to accept responsibility or admit wrongdoing, maintaining the self-righteous position that the victim group deserved annihilation and were subhuman. these stages are approximate and vary in each separate incident, but they demonstrate how genocide differs from other forms of even very horrific violence (such as war) because the aim is not simply to subdue, harm, or exploit but to dehumanize, exterminate, and annihilate. genocide thus involves several traumatic features, including loss of self-worth and allegiance to core values and institutions; prolonged pain and suffering; bereavement; terror and horror of annihilation; injury; helplessness while witnessing demeaning, cruel, and violent events; and confinement. survival responses to genocide are described by brom and kleber (2008) as: … a narrowing of functioning and awareness in order to maximize the chances of survival [often involving] psychic closing off (also called robotization-that is, acting and feeling emotionally and mentally empty or on "automatic pilot" like a "robot"), [and] regression-that is, feeling, thinking, and acting like a child (or in the case of children, like a much younger age than actual chronological age). often victims also experience a strong dependence on perpetrators who decide on life and death. the "muselman effect" … manifested by complete physical decrepitude, apathy, slowing of movement, and gradual disintegration of personality (including loss of the capacity for rational reasoning) may result when individuals have been exposed to long-term and extreme circumstances. an additional phenomenon that is well documented is the so-called "death imprint" resulting when substantial witnessing of death continues to haunt the survivor. these reactions closely parallel the symptoms of both asd (such as dissociation and regression) and ptsd (such as intrusive reexperiencing and emotional numbing). the adverse long-term effects of experiencing genocide are severe and pervasive. more than one in three survivors become clinically depressed and develop ptsd. the social support of caring family members (and for children, parents, or other caregivers) and relationships and activities that individuals to retain their spiritual or religious beliefs and their sense of self-respect are crucial protective factors against ptsd and depression. however, even the most resilient and socially supported person is likely to experience distressing memories and survivor guilt years or even decades later. studies with elderly holocaust survivors who are physically and emotionally very hardy (often well into their 80s and 90s) have documented significant persisting emotional distress and ptsd symptoms 60 or more years later (brom & kleber, 2008) . moreover, the offspring of holocaust survivors with ptsd are more likely than offspring whose parents do not have ptsd to themselves experience ptsd as adults (yehuda et al., 2000 (yehuda et al., , 2012 . genocide often involves physical hardships that compromise physical health and may lead to long-term illnesses and depletion of the body's immune system. for example, the physical exertion and pain involved in torture, untreated physical illnesses, insufficient sleep, starvation, exposure to extreme temperatures, and forced labor may accelerate the aging process (brom & kleber, 2008) . genocide also often includes separating individuals from their families and community groups. this not only deprives the survivor of crucial social support but engenders a sense of isolation, distrust, and shame and of being permanently psychologically damaged (herman, 1992) . survivors also are faced with a choice of holding to their allegiance to their family, nation, culture, and racial identity, despite the punishment inflicted by the perpetrators, or abandoning these basic commitments and rejecting themselves and people like them. faced with this impossible choice (as epitomized in william styron's classic novel, sophie's choice), survivors often believe that they failed utterly and let down not only themselves but their family and culture no matter how resiliently they coped and the integrity of their efforts. survivor guilt is an expression of a sense of grief, powerlessness, and failure, including questioning why they survived and others did not. torture. torture is a terrible special case of political repression that involves "malicious intent and a total disregard for the recipient's dignity and humanity. thus, torture is among the most egregious violations of a person's fundamental right to personal integrity and a pathological form of human interaction" (quiroga & jaranson, 2008) . the 1984 united nations (un) office of high commissioner for human rights established a "convention against torture and other cruel, inhuman or degrading treatment or punishment (cat)," which has been endorsed by 210 nations and defines torture as follows: for the purpose of this convention, the term "torture" means any act by which severe pain or suffering, whether physical or mental, is intentionally inflicted on a person for such purpose as obtaining from him or a third person information or a confession, punishing him for an act he or a third person has committed, or is suspected of having committed, or intimidating or coercing him or a third person, or for any reason based on discrimination of any kind, when such pain or suffering is inflicted by, or at the instigation of, or with the consent or acquiescence of, a public official or other person acting in an official capacity. it does not include pain or suffering arising only from, inherent in, or incidental to lawful sanctions. (http://www.unhchr.ch/html/menu3/b/h_cat39.htm accessed 01/19/09) an amnesty international 2000 worldwide survey found that 75% of countries practice torture systematically, despite the absolute prohibition of torture and cruel and inhuman treatment under international law. torture may be euphemistically referred to as "enhanced interrogation techniques" and condoned in order to obtain "intelligence" from designated enemies of the nation, although this is completely prohibited by the un resolution (quiroga & jaranson, 2008) . widespread controversy has attended the use of such techniques by the us central intelligence agency in response to the september 11, 2001, terrorism incidents, controversy that peaked in 2014 with the presidential decision to close the guantanamo bay military prison, and the release of the us senate report revealing and questioning the legality and morality of torture tactics used in interrogation and incarceration. basoglu, livanou, and crnobaric (2007) , in a sample from the balkan war (1991-2001) studied from 2000 to 2002, showed that the torture need not inflict physical pain in order to produce ptsd. psychological assessment of torture survivors was systematized by the istanbul protocol, a manual on the effective investigation and documentation of torture and other cruel, inhumane, or degrading treatment or punishment that includes modules for medical, psychological, and legal professionals united nations resolution 55/89 on december 4, 2000 (quiroga & jaranson, 2008) . the psychological problems most often reported by torture survivors are emotional symptoms (anxiety, depression, irritability/aggressiveness, emotional liability, self-isolation, alienation from others, withdrawal); cognitive symptoms (confusion/disorientation, memory and concentration impairments); and neuro-vegetative symptoms (lack of energy and stamina, insomnia, nightmares, sexual dysfunction) (quiroga & jaranson, 2008) . the most frequent psychiatric diagnoses are ptsd and major depression, other anxiety disorders such as panic disorder and generalized anxiety disorder, and substance use disorders. longer-term effects include changes in personality or worldview, consistent with complex ptsd (quiroga & jaranson, 2008) . the greater the degree of distress and loss of sense of control during torture, the greater the likelihood of ptsd and depression. resilience, through being able maintain a sense of personal control, efficacy, and hope while enduring torture, is associated with less distress during torture and lower risk of ptsd (quiroga & jaranson, 2008) . social, cultural, and other diversity issues in the traumatic stress field 525 however, quiroga and jaranson (2008) cited a study by olsen showing that 10 years after torture, physical pain was still prevalent even if torture was primarily psychological in nature. based on this finding and related studies, they conclude the following (p. 655): the most important physical consequence of torture is chronic, long-lasting pain experienced in multiple areas of the body. all [physical] torture victims show some acute injuries, sometimes temporary, such as bruises, hematomas, lacerations, cuts, burns, and fractures of teeth or bones, if examined soon after the torture episode. permanent lesions, such as skin scars on different parts of the body, have been found in 40% to 70% of torture victims. … falanga, beating the sole of the feet with a wooden or metallic baton, has been studied extensively. survivors complain of chronic pain, a burning sensation. … acute renal failure secondary to rhabdomyolysis, or destruction of skeletal muscle, is a possible consequence of severe beating involving damage to muscle tissue. this condition can be fatal without hemodialysis. … a severe traumatic brain injury that is caused by a blow or jolt to the head or a penetrating head injury may disrupt the function of the brain by causing a fracture of the skull, brain hemorrhage, brain edema, seizures, and dementia. the effects of less severe brain injury have not been well studied. treatment for torture survivors must be multidisciplinary and involves a long-term approach. several treatment modalities have been developed, but little consensus exists concerning the standard of practice, and treatment effectiveness has not been scientifically validated by treatment outcome studies (quiroga & jaranson, 2008) . a key element that is widely agreed upon is to pay careful attention to not inadvertently replicating in benign ways aspects of torture in the treatment (such as by pressing a survivor to recount traumatic memories without the survivor's informed and voluntary consent; by encouraging or discouraging political, family, and social activities except as initiated by the survivor; or by behaving in authoritarian ways rather than seeking to be collaborative with the survivor). it also is best to use medical, psychiatric medication, and psychotherapy modalities to address the ptsd symptoms of impaired sleep, nightmares, hyperarousal, startle reactions, and irritability. quiroga and jaranson (2008) also recommend using groups for socializing and supportive activities to reestablish a sense of family and cultural values, and supporting the traditional religious and cultural beliefs of the survivor. currently, nearly 250 torture survivor treatment centers exist worldwide, 134 of them accredited by the international rehabilitation council of torture victims (quiroga & jaranson, 2008) . most of these centers also involve the survivors' families and communities in developing shared approaches to recovery and reparation of the harm done to all. the controversy concerning the use of torture on detainees in the so-called "war on terror" has led to deep concern on the part of not only the public at large but specifically by mental health professionals. the issue is that psychiatry and psychology professionals who are in the military or consult to the military have been involved in the detention and interrogation of suspected terrorists at high-security facilities such as the military base at guantanamo bay and the military prison in iraq, abu ghraib. as a result, guidelines for mental health professionals working in these or similar facilities in which prolonged detention and interrogation may involve practices that constitute torture have been developed by a special committee of the american psychological association's division (56) on trauma psychology (box 11.5). the apa council of representatives … included in its "unequivocal condemnation" all techniques considered torture or cruel, inhuman or degrading treatment or punishment under the united nations convention against torture and other cruel, inhuman, or degrading treatment or punishment; the geneva conventions; the principles of medical ethics relevant to the role of health personnel, particularly physicians, in the protection of prisoners and detainees against torture and other cruel, inhuman, or degrading treatment or punishment; the basic principles for the treatment of prisoners, the mccain amendment, the united nations principles on the effective investigation and documentation of torture and other cruel, inhuman, or degrading treatment or punishment an "absolute prohibition against mock executions; waterboarding or any other form of simulated drowning or suffocation; sexual humiliation; rape; cultural or religious humiliation; exploitation of fears, phobias, or psychopathology; induced hypothermia; the use of psychotropic drugs or mind-altering substances; hooding; forced nakedness; stress positions; the use of dogs to threaten or intimidate; physical assault, including slapping or shaking; exposure to extreme heat or cold; threats of harm or death; isolation; sensory deprivation and overstimulation; sleep deprivation; or the threat [of these] to an individual or to members of an individual's family. psychologists are absolutely prohibited from knowingly planning, designing, participating in, or assisting in the use of all condemned techniques at any time and may not enlist others to employ these techniques. we have come to the conclusion that the united states' harsh interrogationdetention program is potentially trauma-inducing both in general (e.g., indefinite detention, little contact with lawyers, no contact with relatives or significant others, prolonged absence of due process, awareness that other prisoners have been tortured, lack of predictability or control regarding potential threats to survival or bodily integrity) and in terms of some of its specific components (e.g., prolonged isolation, waterboarding, humiliation, painful stress positions). in other words, these potentials for trauma extend beyond the narrow procedures that meet international definitions of torture. the evidence for risk of psychological trauma to detained enemy combatants is particularly compelling and well grounded in formal research, but there is also suggestive anecdotal and theoretical evidence of trauma induction in interrogators and the broader society. we were particularly struck by the fact that the potentially traumatic elements include not only activities designed to extract information from prisoners but also much of the detention process as it is currently conceived, beyond much oversight, or compliance with international law. given the pervasiveness of these traumatogenic elements, it is questionable whether psychologists can function in these settings without participating in, or being adversely affected by, heightened risk for trauma. nonetheless, as a group of psychologists with expertise in preventing traumatic stress and ameliorating debilitating posttraumatic sequelae, we believe that certain steps could … minimize the risk of psychological trauma. they are as follows: 1. we believe that the risk of traumatic stress and negative posttraumatic sequelae will be reduced if psychologists adhere to both the apa ethical standards and subsequent refinements of apa policies pertaining to interrogation, detention, and torture. such adherence would be more likely if the apa ethics code were revised to incorporate, as enforceable standards, the specific interrogation and torture-related policy refinements that have occurred since 2002. psychologists should promote situations that maintain the risk of traumatic stress at acceptably low levels and avoid situations that heighten the risk for traumatic stress occurring. among other things, this means that psychologists should not provide professional services in secret prisons that appear to be beyond the reach of normal standards of international law or in settings in which torture and other human rights abuses have been credibly documented to be permitted on the basis of local laws. it also suggests that psychologists should not support or participate in any detention or interrogation procedure that constitutes cruel or inhumane treatment or that otherwise has been shown to elevate risk of traumatic stress (e.g., prolonged isolation). 3. if psychologists work in settings in which detention and interrogations are conducted, then they should conduct or seek an assessment of the potential traumatic features of the treatment of detainees before, during, and after interrogation. this assessment can be informal or formal, depending on whether other systems of oversight are in place. this assessment should include an examination of the social psychological factors that could elevate risk of trauma. because not all psychologists have expertise in assessing traumatic stress risk and/or social psychological (continued ) factors, the assessment should be conducted by psychologists who have this specific expertise. such assessments could inform decisions not only by psychologists but also by others working in facilities in which detention and interrogation occur. it is recommended therefore that apa advocate for appropriate governmental authorities to appoint an independent oversight committee for each facility of this type and that the oversight committees include psychologists identified by apa as having relevant expertise. 4. if psychologists work in settings in which risk of traumatic stress is found to be elevated then they should (i) formally recommend alterations that could reduce the traumatogenic potential of the detention and interrogation process (n.b. some recommendations may be aimed at policy makers rather than local authorities); (ii) conduct or seek an assessment of posttraumatic stress symptoms and associated features (e.g., depression, dissociation) in detainees, interrogators, and other directly or indirectly involved staff; (iii) recommend appropriate psychological interventions for any detainees or personnel found to be suffering from clinically significant psychological difficulties; and (iv) refuse to participate in any activities that significantly increase risk of traumatic stress. if a psychologist working in such settings does not have specific expertise required to meet some of the above recommendations, then she or he should consult with psychologist(s) who have this expertise to make the appropriate determination. 5. because some detainee abuses have been credibly linked to an absence of appropriate training and/or expertise, psychologists should advocate for, participate in designing, and/or assist with providing appropriate and comprehensive training to all personnel involved in interacting with detainees. this training should include (i) clear ethical guidelines emphasizing the prohibition of causing harm and the importance of protecting detainee rights, (ii) a research-based overview of the nature and consequences of traumatic stress and posttraumatic impairment as they relate to the interrogation and confinement process and all parties involved in layperson terms with practical implications, and (iii) detailed review of research on false confessions, in layperson terms, with practical implications for enhancing the validity and utility of information gathered in the course of interrogation and detention. because not all psychologists have expertise in these specific matters, apa should develop standardized training materials that cover the current state of psychological knowledge and practices on these important topics, and ensure that these materials are regularly updated by qualified psychologists in consultation with experts from other fields such as law enforcement, the military, and human rights. 6. because protecting human rights reduces the risk for traumatic stress and posttraumatic impairment, psychologists should collaborate with legal, military, and other colleagues to advocate for due process for all detainees, including providing clear guidelines about finite lengths of detention prior to formal hearing or trial and enforcing the recent supreme court decision to reinstate habeas corpus and other international standards of human rights. psychologists' support for these actions should not come from a blanket support for adherence to law but rather from an informed judgment that these … laws reduce the risk for harm. psychologists should be prepared to disagree with any future international laws or us supreme court decisions that heighten risk for traumatic stress. box 11.5 continued 7. psychologists should support increased transparency during the detention and interrogation process. such increased transparency could reduce the likelihood of traumatizing practices, increase the likelihood that traumatizing practices will be identified and stopped as early as possible, and protect ethical psychologists and other workers within the system from being falsely accused of acting unethically. we recognize that this recommendation raises an apparent conflict with the goal of secrecy commonly endorsed by national security organizations. we concur that full transparency is unreasonable and counterproductive. yet, we do believe that increased transparency is a safeguard against traumatizing practices. though the details of resolving this conflict are beyond the scope of this task force's expertise, we believe that reasonable, knowledgeable intelligence experts, in consultation with psychologists, can construct a system of oversight that will both retain credible independence from the military chain of command and guard classified information. one suggestion may be to establish a greater presence of psychological expertise within a framework of oversight protection. 8. if psychologists are going to continue to be involved in interrogations, then it will be important to continue to segregate the function of interrogation consultant from that of mental health provider to reduce risk of perceived or actual betrayal by the detainee. it is unknown whether betrayal of trust due to dual roles can constitute a direct form of traumatization under these circumstances, but it is likely that betrayal in this context could exacerbate traumatic stress that occurs of other aspects of detention and interrogation (especially in light of the ways that such detention appears to disrupt attachment as outlined in the body of our report). maintaining separate roles also may enable the psychologist to more effectively assist detainees with traumatic stress reactions by fostering a trusting therapeutic relationship. 9. psychologists should advocate for extra protections for detainees who are from vulnerable populations such as minors, ethnic minorities, or other groups that have limited access to socioeconomic or political resources or are potentially subject to societal discrimination or prejudice because such groups may be more likely to receive coercive interrogations and/or excessive force and less likely to be sympathetically viewed by the general public. for this purpose, psychologists may work within sponsor/authorizing organizations to institute developmental, gender, and culture sensitivity trainings for interrogators and should review evidence concerning the impact of different forms of traumatic stressors and differential sensitivity to the interrogation/detention setting/process on different (and particularly vulnerable) ages, genders, and cultural backgrounds. such psychologists should, to whatever extent possible, guard against such information being used to exploit vulnerable populations and instead emphasize ways to enhance safety and psychological wellbeing in the interrogation process. if psychologists lack relevant expertise to meet the recommendations, … they should seek or advocate for outside expert consultation. 10. psychologists should collaborate with colleagues from a variety of professions and organizations, including the military and intelligence organizations, to conduct ethical research on several aspects of the detention and interrogation process, especially its potential for inducing trauma. recent reviews suggest that most of the interrogation procedures used today have not received recent rigorous study (intelligence science board, 2006) . furthermore, very little of the recent study has been directed toward understanding the psychological effects of interrogation on not only the detainees but also the people working within and outside the interrogation and detention system. political violence not only leads to traumatic harm to people while they are living in their communities but also often when victims are forced to flee their homes either to another country or while remaining within their country. refugees are defined by the united nations high commissioner for refugees (unhcr) as persons who have left their nation of origin to escape violence. article one of the 1951 united nations convention relating to the status of refugees defines a refugee as someone who "owing to well-founded fear of being persecuted for reasons of race, religion, nationality, membership of a particular social group or political opinion, is outside the country of his nationality and is unable or, owing to such fear, is unwilling to avail himself of the protection of that country; or who, not having a nationality and being outside the country of his former habitual residence as a result of such events, is unable or, owing to such fear, is unwilling to return to it" (unhcr, p. 16; http:// www.unhcr.org/home/publ/3b5e90ea0.pdf; accessed 19.01.09). therefore, refugees are distinct from both legal and illegal immigrants, economic migrants, environmental migrants, and labor migrants (weine, 2008) . refugees must involuntarily leave home, community, and family and friends, often with limited resources or preparation and usually without knowing whom they can trust and where they can find safe passage and a safe haven. thus, both prior to and during the displacement, refugees often suffer psychologically traumatic experiences, including having their community or homes attacked or destroyed due to war; racially, genderbased, or ethnically targeted genocide or terrorism; institutionally orchestrated deprivation and violence; along with torture, atrocities, rape, witnessing violence, fear for their lives, hunger, lack of adequate shelter, separation from loved ones, and destruction and loss of property (weine, 2008) . estimating the number of refugees is very difficult. a minimum estimate that is probably much lower than the actual number is calculated by the united nations based on the number of persons in resettlement camps or individually recognized by a host country. based on this definition, there were 16 million refugees worldwide in 2007 (www.unhcr.org) (figure 11 .1). in côte d'ivoire, and was quickly followed by others in libya, somalia, sudan, and elsewhere. in all, 4 .3 million people were newly displaced, with a full 800,000 of these fleeing their countries and becoming refugees. "2011 saw suffering on an epic scale. for so many lives to have been thrown into turmoil over so short a space of time means enormous personal cost for all who were affected," said the unhcr antónio guterres. "we can be grateful only that the international system for protecting such people held firm for the most part and that borders stayed open. these are testing times." worldwide, 42.5 million people ended 2011 either as refugees (15.2 million), internally displaced (26.4 million), or in the process of seeking asylum (895,000). despite the high number of new refugees, the overall figure was lower than the 2010 total of 43.7 million people, due mainly to the offsetting effect of large numbers of idps returning home: 3.2 million, the highest rate of returns of idps in more than a decade. among refugees, and notwithstanding an increase in voluntary repatriation over 2010 levels, 2011 was the third lowest year for returns (532,000) in a decade. viewed on a 10-year basis, the report shows several worrying trends. one is that forced displacement is affecting larger numbers of people globally, with the annual level exceeding 42 million people for each of the last 5 years. another is that a person who becomes a refugee is likely to remain one for many years-often stuck in a camp or living precariously in an urban location. of the 10.4 million refugees under unhcr's mandate, almost three-quarters (7.1 million) have been in exile for at least 5 years awaiting a solution. overall, afghanistan remains the biggest producer of refugees (2.7 million), followed by iraq (1.4 million), somalia (1.1 million), sudan (500,000), and the democratic republic of the congo (491, 000) . around four-fifths of the world's refugees flee to their neighboring countries, reflected in the large refugee populations seen, for example, in pakistan (1.7 million people), iran (886, 500) , kenya (566, 500), and chad (366, 500 many people displaced from their communities by violence remain in their home country. they are not considered refugees, but "idps": "people or groups … who have been forced to leave their homes" due to "armed conflict, situations of generalized violence, violations of human rights, or natural-or human-made disasters, and who have not crossed an international border" (www.unhcr.org). as of 2007, there were more than three times as many idps as refugees, an estimated 51 million worldwide, 26 million social, cultural, and other diversity issues in the traumatic stress field 533 due to armed conflict and 25 million due to mass natural disasters (www.unhcr.org). between one and more than 3 million idps were known to be in several countries in 2007, including colombia, congo, iraq, somalia, sudan, and uganda. azerbaijan, cote due n'orde, and sri lanka had more than 500,000 known idps each. at least another 3 million persons are considered "stateless"-that is, to not be citizens of any nation, in 2007. nepal and bangladesh have the majority of the stateless persons in the world, although nearly 3 million persons in those two countries were made citizens in 2007. palestine and iraq are the other countries with large numbers of stateless persons. there may be millions more stateless individuals, because only 54 countries assisted the united nations in its census of stateless persons in 2007 (www.unhcr.org). the impact of forced displacement often is not just extremely stressful but traumatic. refugees, idps, and stateless persons have few protections and often must live in confined camps or crowded public shelters, where they are vulnerable to assaults (including rape), robbery, and illness. many have witnessed horrific violence associated with wars, genocide, or other forms of mass armed conflict that caused them to flee. loss of family and friends due to violence or illness is common, as well as due to being separated with no way to communicate. studies have documented high prevalence levels of ptsd and depression among refugees or idps from armed conflicts in central america, southeast asia, the middle east, and the balkans (fazel, wheeler, & danesh, 2005; marshall, schell, elliott, berthold, & chun, 2005) at least 10 times higher than the 1-3% prevalence estimates from epidemiological surveys (see chapter 3). ptsd prevalence estimates that are more than three times higher than these very high levels, as high as 30-34%, have been reported among disabled central american refugees (rivera, mari, andreoli, quintana, & ferraz, 2008 ) and among afghan mothers (seino, takano, mashal, hemat, & nakamura, 2008) . other studies have more specifically investigated physical displacement in the traumatic stress experienced by refugees. displacement may involve many stressors, and a research review found that "living in institutional accommodation, experiencing restricted economic opportunity, [being] displaced internally within their own country [or] repatriated to a country they had previously fled or whose initiating conflict was unresolved" were particularly problematic. this review of 56 reports involving 67,294 participants (22,221 refugees and 45,073 persons who were not displaced) showed that displacement alone was associated with more severe mental health problems, including ptsd (porter & haslam, 2005) . in contrast to most research findings on the etiology (see chapter 3) and epidemiology (see chapter 4) of ptsd, "refugees who were older, more educated, and female and who had higher predisplacement socioeconomic status and rural residence also had worse outcomes" (porter & haslam, 2005, p. 602) . people become "internally displaced" as often due to mass natural disasters as to armed violence. in the united states, several hundred thousand people had to leave the new orleans area following hurricane katrina in august 2005. almost 400,000 received medical care at american red cross shelters within the next month (mills, edmondson, & park, 2007) . many displaced persons already were severely disadvantaged due to living in poverty (roughly 28% of the population of new orleans), having limited access to quality health care, and exposure to community violence (mills et al., 2007) . in a study of 132 adult evacuees from new orleans and surrounding parishes (56% men, average age 43 years old, 74% black, 82% low income (annual income less than $30,000), 67% reporting a prehurricane psychiatric disorder (33% depression, 20% anxiety disorder, 8% bipolar disorder)), most (95%) waited several days to be evacuated, and a majority reported sustaining minor to severe injuries (63%) and mild to severe illness (71%) in the hurricane or evacuation process. one in seven lost a loved one due to death in the hurricane or its aftermath, and most (85%) were separated from a family member for a day or more. many (70%) lost their home, two-thirds of whom were without property insurance. almost two in three (62%), particularly women, people with a prior psychiatric disorder, and those who recalled feeling their lives were in danger during the hurricane or its aftermath, were injured physically, or felt they had limited control over their current life circumstances, reported symptoms sufficient to qualify for a diagnosis of asd. natural disasters of several magnitudes greater have occurred in less developed and affluent areas of the world. for example, the tsunami that struck on december 26, 2004, in the wake of the sumatra-andaman earthquake killed an estimated 250,000 people along the coastlines of the indian ocean, including 130,000 indonesians. another half a million indonesians were displaced from their communities. studies of survivors of this tsunami from indonesian areas such as aceh and north sumatra (frankenberg et al., 2008) , as well as from thailand (van griensven et al., 2006) and sri lanka (hollifield et al., 2008) , have demonstrated that posttraumatic stress, anxiety, and depression are suffered by hundreds of thousands, and perhaps millions, of people who experienced psychological trauma due to the tsunami (box 11.6). box 11.6 refugee posttraumatic stress in the wake of mass natural disaster frankenberg et al. (2008) reported a unique study of the impact of a massive natural disaster: the indian ocean tsunami that struck the day after christmas 2004. unlike most research on mental health after disasters, this study began with a survey of a representative sample of persons in the host country (indonesia) almost 2 years before the disaster. this "national socioeconomic survey (susenas)" provided a registry of respondents and predisaster data on health and socioeconomic characteristics of people throughout indonesia. the "study of the tsunami aftermath and recovery (star)" attempted to recontact 39,500 persons interviewed in 585 communities by the susenas. the study also was able to determine the extent of damage caused to each community by the tsunami. the researchers got data from the national aeronautics and space administration's moderate resolution imaging spectroradiometer sensor collected 1 year prior to the tsunami, and again immediately after the tsunami, to assess the degree to which the pretsunami ground cover visible in the first image had been replaced by bare earth in the second image. communities with at least 20% loss of ground cover were classified as heavily damaged (15% of the 585 surveyed communities). those with some loss of ground cover were categorized as moderately damaged (50% of all locales), and 35% with no loss of ground cover were classified as undamaged by the tsunami. community leaders' and field observers' estimates of damage strongly correlated (r=0.84 and 0.79) with these satellite-based estimates. one in three of the survey respondents (average age 36 years old) heard the tsunami wave or people screaming. fewer sustained injuries (3%), lost a spouse (3%), lost a parent or child (5%), or witnessed family or friends "struggle or disappear" (6%), but 25% lost a family member or friend, 25% lost their home, and 15% lost their farming land, livestock, or equipment. posttraumatic stress was assessed by asking every respondent 15 years or older to answer seven of the items from the ptsd checklist (see chapter 6), as follows: since the tsunami, have you ever experienced (never, rarely, sometimes, or exposure to probable traumatic stress due to hearing the wave or screams, being injured, or seeing friends or family members "struggle or disappear," doubled the severity of pcl-c scores. consistent with this finding, compared to the sleep difficulties reported before the tsunami, after the tsunami, there was a large increase in the likelihood of sleep difficulties only in the most heavily damaged areas. pcl-c scores increased the most in the worst damaged locales, followed by the moderately damaged ones, with little change in the nondamaged communities. pcl-c scores averaged 5.77, 4.71, and 2.98 for the heavily, moderately, and undamaged areas, respectively, at the time of the interview and had been 33% higher at their peak after the tsunami (based on respondents' recollections). this is consistent with other studies that have reported persistent ptsd symptoms among the worst exposed persons but a substantial decline in ptsd symptom severity over time even among heavily exposed persons (see chapter 2). women reported higher pcl-c scores than men, but primarily only in the heavily damaged areas. age was a factor in all communities: persons younger than 30 years reporting an increase after the tsunami and persons 50 years and older reporting lower pcl-c scores after the tsunami. interestingly, respondents who had a parent alive before the tsunami had lower pcl-c scores after the tsunami, but marital status, education, and income were not related to posttsunami pcl-c scores. property damage also correlated with posttsunami pcl-c scores. (continued ) as frankenberg et al. (2008) noted, these findings probably understate the severity and widespread nature of the harm, including posttraumatic stress, caused by a massive disaster such as this tsunami. however, the study provides the strongest evidence to date that a disaster that is not only life threatening for many but that displaces tens or hundreds of thousands of persons from their homes, families, neighbors, and way of life has the strongest adverse impact on communities that are most directly affected. another study (van griensven et al., 2008) conducted 8 weeks after the tsunami in six southwestern provinces of thailand (where more than 8000 persons died or were unaccounted for and another 8500 were injured) included random samples of 371 displaced persons and 321 nondisplaced persons from the most heavily hit area and 368 persons from less damaged areas. even though the extent of death and destruction was less in the worst-hit areas of thailand than in indonesia, symptoms of ptsd were reported by 12% of displaced and 7% of nondisplaced persons in the most damaged area of thailand (and by 3% in the less damaged areas). anxiety or depression symptoms were reported by three times as many persons, with similar proportions depending on displacement and the severity of damage to the community. thus, this study adds to the findings of the study from indonesia by demonstrating that displacement from home and community was a factor in ptsd and related symptoms soon after a mass natural disaster. the thailand study also resurveyed participants from the worst-damaged area 7 months later (9 months after the tsunami) and confirmed that displaced persons continued to be more likely than nondisplaced persons to suffer from ptsd, anxiety, and depression symptoms. consistent with other studies (ford, adams, & dailey, 2007) of postdisaster recovery (including the indonesia study), as the first anniversary of the disaster approached, about 40% of each group had recovered sufficiently to no longer report severe symptoms. whereas the indonesia study examined the extent of damage to participants' homes and (for most) the source of their incomes (farmland, animals, and equipment), the thailand study inquired directly as to whether respondents had lost their source of income and found that loss of livelihood was the strongest correlate of ptsd, anxiety, and depression symptoms. thus, the thailand study showed that losing not only home or community but also one's ability to generate an independent income through gainful work may contribute to the development and persistence of ptsd and related anxiety and depression symptoms. the defining characteristics of becoming a refugee in the wake of disaster therefore include (i) exposure to life-threatening catastrophe; (ii) loss of or separation from family and friends, (iii) loss of home and community; and (iv) loss of one's personal or family livelihood. each of these factors may result in acute posttraumatic distress, and the combination of several places people at risk for persistent ptsd. as weine (2008) describes, resettlement of refugees is a substantial challenge not only for displaced persons themselves but also for the host country. relatively stable and affluent countries in asia (such as pakistan, due to afghan refugees), the middle east (such as lebanon and syria, due to palestinian refugees), and africa (such as kenya and south africa), as well as most european and british commonwealth nations and the united states, have had a large influx of refugees. the half of all refugees who are resettled in cities experience economic pressures due to poverty and low-wage work and must live in communities that are crowded, segregated economically and culturally, and often adversely affected by crime, gangs, drugs, aids, and troubled schools (weine, 2008) . another half of all refugees are resettled in suburban and rural areas, which are more isolated (www.unhcr.org). in either case, refugees often face prolonged separations from family, friends, and loved ones, as well as the burden of having to find a way to subsist while saving money to bring others to their new home and to provide support to those back home who have stayed behind. refugee children have additional needs and challenges, including having to survive life-threatening experiences without adult help or guidance and then, if they are fortunate enough to be permanently resettled, having to return to being a "child" with a new family, community, and culture (henderson, 2008) . refugee children often display not only the symptoms of ptsd but also behavior problems (such as control, aggression, or defiance of authority), profound bereavement, and developmental, learning, or educational delays or deficits that are understandable in light of their often chronic deprivations before and during displacement. however, children also can be particularly resilient in the face of the psychological losses and traumas of being a refugee, and often they are a key source of hope for their families in the resettlement process (weine, 2008) . many refugees have opportunities to receive mental health services, either in the context of a refugee camp or after resettlement, but many do not seek or utilize these services. survival; getting stable and predictable access to food, money, housing, transportation, and safety; renewing communication with friends and family; and sustaining or regaining connection to cultural and religious traditions, values, and practices may take precedence over mental health treatment (and may in fact be the best form of therapy for many, under the circumstances). in resettlement settings, clinical treatment for refugee trauma is typically organized through refugee mental health clinics or specialized torture victim treatment centers, with services including crisis intervention, psychopharmacology, individual psychotherapy, group psychotherapy, and self-help groups and activities (weine, 2008) . to deliver culturally appropriate services, many programs involve traditional healers, socialization or mutual support groups, multifamily groups, and culturally based activities (weine, 2008) . services also tend to be provided by staff who themselves are members of the refugees' ethnic community, in collaboration with traumatic stress specialists and mental health professionals. the traditional model of western professional "expert" doctor or consultant who unilaterally tells local staff or clients how best to do assessment, diagnosis, or treatment has been justly criticized as culturally insensitive and potentially harmful rather than helpful (weine, 2008) . instead, the joint experience and expertise of the refugee client, local professional and paraprofessional alike, traditional healers, and traumatic stress professionals are taken together in a team approach that validates the client's and local helpers' cultures and traditions. this approach enhances the providers' ability to make a true cross-cultural assessment of symptoms and diagnoses, to adapt interventions to reflect different cultural beliefs and practices, and to engage not just individual clients but families and communities in recovery from ptsd. such an approach is consistent with new theoretical views of refugee traumatic stress, which include "the concepts of cultural bereavement, cultural trauma, family consequences of refugee trauma, community trauma, and social suffering" (weine, 2008) . this more culturally grounded view of refugees' experiences of traumatic stress and recovery from ptsd has led to the development of innovative therapy approaches (such as incorporating personal testimony and reconciliation into treatment) that address refugees' psychological vulnerabilities but strongly acknowledge their (and their families' and communities') hopes and strengths (weine, 2008) . when mass catastrophes, whether human-made or "acts of god" in origin, including natural disasters such as tsunamis, tornadoes, hurricanes, floods, or earthquakes, or public health emergencies, such as aids, severe acute respiratory syndrome (sars), pandemic influenza, ebola, or human-made disasters such as terrorist attacks, airline crashes, ferry capsizes, and train derailments, cause tens or hundreds of thousands or even millions of people and families to experience psychological trauma, the resultant suffering and needs are generally beyond the capacities of traditional mental health services and other forms of government-sponsored services. ngos play a critical role supporting and assisting persons and communities affected by catastrophic disaster or violence, including providing psychological support through clinical and nonclinical behavioral health services (hamilton & dodgen, 2008) . ngo responses to the mental health needs of mass-disaster survivors are based on the core belief that "all disasters are local" (hamilton & dodgen, 2008) . this means that local responders such as law enforcement, police, emergency medical teams, and professionals from the health care facilities, schools, and government are invariably first on the scene and frequently remain involved for months or years afterward. when insufficient resources are available, a local community may request help from the country, state, or provincial governments, which in turn may request regional or national assistance from both government and private sectors. for that reason, ngos that provide assistance following disasters, such as the american red cross, the national voluntary organizations active in disaster (nvoad), the united way, and the salvation army do so through their local chapters, which organize the initial relief efforts to provide shelter, food, legal aid, health and mental health care, and humanitarian assistance. organized in 1970, nvoad is the umbrella organization coordinating all disaster relief services provided by volunteer organizations such as the american red cross throughout the united states. ngos also work closely with faith-based organizations (fbos) in the united states (such as catholic charities united states, church world service, lutheran disaster response, national association of jewish chaplains) within the national response framework of the federal emergency management agency (fema), which guides the nation's "all-hazards incident response" (hamilton & dodgen, 2008) . for example, american red cross disaster mental health (dmh) volunteers provide mental health services to people in shelters, while the church of the brethren provides crisis intervention to young children through their disaster child care program (hamilton & dodgen, 2008) . the american red cross is the most widely recognized ngo providing dmh services in the united states. in 1905, congress chartered the american red cross to "carry on a system of national and international relief in time of peace" to reduce and prevent the suffering caused by national calamities program (hamilton & dodgen, 2008) . in 1990, the american red cross established a formal dmh services program and began training licensed and certified mental health professionals to volunteer and assist other red cross workers to cope with and recover from the traumatic stress (or "vicarious trauma") of their disaster relief work. initially only licensed psychologists and social workers were permitted to become red cross dmh volunteers, but recently professionals from other disciplines, such as psychiatry and masters-level marriage and family therapy or counseling professions, also have become eligible. the american red cross has set up formal agreements with the american psychiatric association, the american psychological association, the national association of social workers, the american counseling association, and the american association of marriage and family therapy. the agreements provide that in the event of a mass disaster, the red cross will notify each professional associations to put out a call to their memberships for professionals who have completed red cross preparatory training and who can take time out from their ordinary work to serve as dmh volunteers for 2 weeks or more at red cross disaster services sites. the american red cross sets up and oversees family assistance centers for disaster-affected communities, provides crisis and grief counseling through its dmh volunteers, and coordinates with federal agencies such as fema and the national transportation safety board (for airline or mass transportation disasters) to provide child care services and interfaith memorial services. the red cross also works closely with disaster-focused ngos such as the national organization for victim assistance, disaster psychiatry outreach, and the international critical incident stress foundation, inc. (icisf). founded in 1989, the icisf trains mental health professionals, emergency responders, clergy, and chaplains to conduct critical incident stress management (see chapter 9) teams to support disaster services personnel. in 2005, the american red cross broadened the scope of dmh services to include assisting disaster-affected persons who are seeking red cross assistance, as well as red cross volunteers. all dmh volunteers now are trained in psychological first aid (see chapter 9) so that they will provide mental health services to disaster victims in an appropriately circumscribed manner that is therapeutic without attempting to conduct psychotherapy at a disaster relief site. two other freestanding programs participating in a dmh response are the green cross assistance program, which provides trained traumatology specialists and the association of traumatic stress specialists, an association of mental health professionals and paraprofessionals who assist survivors of psychological trauma (hamilton & dodgen, 2008) . a number of us ngos also work internationally to provide psychosocial support and traumatic stress counseling to survivors of disasters and mass conflicts. these include the international services of the american red cross, the united methodist committee on relief, church world services, green cross, action aid-the united states, the american refugee committee, the center for victims of torture, and doctors without borders (hamilton & dodgen, 2008) . the international federation of red cross and red crescent societies also assist many nations' red cross organizations in serving their own and neighboring countries. a 2006 analysis by the united states homeland security institute found that fbos and ngos had a significant beneficial impact during and after hurricanes katrina and rita, with mental health and spiritual support among 10 types of services (hamilton & dodgen, 2008) . the study reported that while fbos and ngos faced significant limitations and challenges in providing services, mental health and spiritual support was one of the three best-applied special practices, particularly services designed to preserve family unity within disaster relief shelters. hamilton and dodgen (2008, p. 451 ) describe how ngos can work together to meet critical needs in times of mass crisis, using the september 11, 2001, terrorist attacks in new york, washington, and pennsylvania, as a case in point: local mental health providers working in mental health settings mobilized quickly, but needs were expected to surpass local capability. the american red cross dispatched dmh providers from local and adjacent communities to provide mental health support and stress reduction assistance. national volunteers recruited from across the country arrived within a few days to augment that mission. concurrently, icisf-trained volunteers, some of whom were already part of military mental health systems, also arrived to provide assistance. other agencies, such as fbos, also organized support for victims. in washington, the military was the gatekeeper for volunteers and worked closely with the american red cross to coordinate mental health support. in new york, civilian authorities collaborated with the american red cross. as family assistance centers were set up to aid grieving families, national dmh volunteers continued providing mental health support. because the terrorist attacks created a crime scene, access was controlled and ngos needed official standing to provide assistance. incorporating lessons learned from 9/11, a similar event today would be different in several ways: all ngos and government agencies would organize their response under the national incident management system (nims) and the nrf, thus creating a more centralized, coordinated response and reducing overlapping or competing activities on the part of nvoads. because of ongoing coordination and outreach efforts since 9/11, a greater array of disciplines and specific types of expertise would be available through ngos. the benefits of these efforts were seen during the responses to hurricanes katrina and rita in 2005. personal and community characteristics that reflect ethnocultural, national, gender, age, and disability factors are crucial in defining the identity of every human being. when traumatic stress occurs in a person's or community's life, its impact is influenced by these identity factors. when identity is used as a basis for stigma, discrimination, or socioeconomic disadvantage, those stressors compound the effect of traumatic stressors and can be traumatic in and of themselves. by addressing the vulnerability that this combination in a scientifically and clinically responsible manner (alcantara, casement, & lewis-fernandez, 2013; c'de baca, castillo, & qualls, 2012; ghafoori, barragan, tohidian, & palinkas, 2012) to assist rather than stigmatize persons and communities (ruglass et al., 2014) , the traumatic stress professional can play 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