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Over the past few decades, multilevel research has elucidated the basic neuroanatomy, neurochemistry, and molecular neurobiology of the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). The circadian timing system is composed of a large number of cellular oscillators located in the SCN, in non-SCN brain structures, and throughout the body. Cellular-level oscillations are generated by a molecular feedback loop in which circadian clock genes rhythmically regulate their own transcription, as well as that of hundreds of clock-controlled genes. The maintenance of proper coordination within this network of cellular- and tissue-level clocks is essential for health and well-being.

Chronic disruption of rhythms (CDR) impacts sleep and can result in circadian misalignment of physiological systems which, in turn, is associated with increased disease risk. Exposure to repeated or severe stressors also disturbs sleep and diurnal rhythms. Prebiotic nutrients produce favorable changes in gut microbial ecology, the gut metabolome, and reduce several negative impacts of acute severe stressor exposure, including disturbed sleep, core body temperature rhythmicity, and gut microbial dysbiosis. In light of previous compelling evidence that prebiotic diet broadly reduces negative impacts of acute, severe stressors, we hypothesize that prebiotic diet will also effectively mitigate the negative impacts of chronic disruption of circadian rhythms on physiology and sleep/wake behavior. Male, Sprague Dawley rats were fed diets enriched in prebiotic substrates or calorically matched control chow. After 5 weeks on diet, rats were exposed to CDR (12 h light/dark reversal, weekly for 8 weeks) or remained on undisturbed normal light/dark cycles (NLD). Sleep EEG, core body temperature, and locomotor activity were recorded via biotelemetry in freely moving rats. Fecal samples were collected on experimental days –33, 0 (day of onset of CDR), and 42. Taxonomic identification and relative abundances of gut microbes were measured in fecal samples using 16S rRNA gene sequencing and shotgun metagenomics. Fecal primary, bacterially modified secondary, and conjugated bile acids were measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Prebiotic diet produced rapid and stable increases in the relative abundances of Parabacteroides distasonis and Ruminiclostridium 5. Shotgun metagenomics analyses confirmed reliable increases in relative abundances of Parabacteroides distasonis and Clostridium leptum, a member of the Ruminiclostridium genus. Prebiotic diet also modified fecal bile acid profiles; and based on correlational and step-wise regression analyses, Parabacteroides distasonis and Ruminiclostridium 5 were positively associated with each other and negatively associated with secondary and conjugated bile acids. Prebiotic diet, but not CDR, impacted beta diversity. Measures of alpha diversity evenness were decreased by CDR and prebiotic diet prevented that effect. Rats exposed to CDR while eating prebiotic, compared to control diet, more quickly realigned NREM sleep and core body temperature (ClockLab) diurnal rhythms to the altered light/dark cycle. Finally, both cholic acid and Ruminiclostridium 5 prior to CDR were associated with time to realign CBT rhythms to the new light/dark cycle after CDR; whereas both Ruminiclostridium 5 and taurocholic acid prior to CDR were associated with NREM sleep recovery after CDR. These results support our hypothesis and suggest that ingestion of prebiotic substrates is an effective strategy to increase the relative abundance of health promoting microbes, alter the fecal bile acid profile, and facilitate the recovery and realignment of sleep and diurnal rhythms after circadian disruption.

Affective behaviours and mental health are profoundly affected by disturbances in circadian rhythms. Casein kinase 1 epsilon (CSNK1E) is a core component of the circadian clock. Mice with tau or null mutation of this gene have shortened and lengthened circadian period respectively. Here, we examined anxiety-like, fear, and despair behaviours in both male and female mice of these two different mutants. Compared with wild-type mice, we found reductions in fear and anxiety-like behaviours in both mutant lines and in both sexes, with the tau mutants exhibiting the greatest phenotypic changes. However, the behavioural despair had distinct phenotypic patterns, with markedly less behavioural despair in female null mutants, but not in tau mutants of either sex. To determine whether abnormal light entrainment of tau mutants to 24-h light-dark cycles contributes to these phenotypic differences, we also examined these behaviours in tau mutants on a 20-h light-dark cycle close to their endogenous circadian period. The normalized entrainment restored more wild-type-like behaviours for fear and anxiety, but it induced behavioural despair in tau mutant females. These data show that both mutations of Csnk1e broadly affect fear and anxiety-like behaviours, while the effects on behavioural despair vary with genetics, photoperiod, and sex, suggesting that the mechanisms by which Csnk1e affects fear and anxiety-like behaviours may be similar, but distinct from those affecting behavioural despair. Our study also provides experimental evidence in support of the hypothesis of beneficial outcomes from properly entrained circadian rhythms in terms of the anxiety-like and fear behaviours.

Polyphasic sleep is the practice of distributing multiple short sleep episodes across the 24-hour day rather than having one major and possibly a minor ("nap") sleep episode each day. While the prevalence of polyphasic sleep is unknown, anecdotal reports suggest attempts to follow this practice are common, particularly among young adults. Polyphasic-sleep advocates claim to thrive on as little as 2 hours of total sleep per day. However, significant concerns have been raised that polyphasic sleep schedules can result in health and safety consequences. We reviewed the literature to identify the impact of polyphasic sleep schedules (excluding nap or siesta schedules) on health, safety, and performance outcomes. Of 40,672 potentially relevant publications, with 2,023 selected for full-text review, 22 relevant papers were retained. We found no evidence supporting benefits from following polyphasic sleep schedules. Based on the current evidence, the consensus opinion is that polyphasic sleep schedules, and the sleep deficiency inherent in those schedules, are associated with a variety of adverse physical health, mental health, and performance outcomes. Striving to adopt a schedule that significantly reduces the amount of sleep per 24 hours and/or fragments sleep into multiple episodes throughout the 24-hour day is not recommended.

Sleep disruption is a challenging and exceedingly common physiological state that contributes to a wide range of biochemical and molecular perturbations and has been linked to numerous adverse health outcomes. Modern society exerts significant pressure on the sleep/wake cycle via myriad factors, including exposure to electric light, psychological stressors, technological interconnection, jet travel, shift work, and widespread use of sleep-affecting compounds. Interestingly, recent research has identified a link between the microbiome and the regulation of sleep, suggesting that interventions targeting the microbiome may offer unique therapeutic approaches to challenges posed by sleep disruption. In this study, we test the hypothesis that administration of a prebiotic diet containing galactooligosaccharides (GOS) and polydextrose (PDX) in adult male rats improves sleep in response to repeated sleep disruption and during recovery sleep. We found that animals fed the GOS/PDX prebiotic diet for 4 weeks exhibit increased non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during 5 days of sleep disruption and increased total sleep time during 24 h of recovery from sleep disruption compared to animals fed a control diet, despite similar baseline sleep characteristics. Further, the GOS/PDX prebiotic diet led to significant changes in the fecal microbiome. Consistent with previous reports, the prebiotic diet increased the relative abundance of the species Parabacteroides distasonis, which positively correlated with sleep parameters during recovery sleep. Taken together, these findings suggest that the GOS/PDX prebiotic diet may offer an approach to improve resilience to the physiologic challenge of sleep disruption, in part through impacts on the microbiome.

Study Objectives Sleep deprivation induces systemic inflammation that may contribute to stress vulnerability and other pathologies. We tested the hypothesis that immunization with heat-killed Mycobacterium vaccae NCTC 11659 (MV), an environmental bacterium with immunoregulatory and anti-inflammatory properties, prevents the negative impacts of 5 days of sleep disruption on stress-induced changes in sleep, behavior, and physiology in mice. Methods In a 2 × 2 × 2 experimental design, male C57BL/6N mice were given injections of either MV or vehicle on days –17, –10, and –3. On days 1–5, mice were exposed to intermittent sleep disruption, whereby sleep was disrupted for 20 h per day. Immediately following sleep disruption, mice were exposed to 1-h social defeat stress or novel cage (control) conditions. Object location memory (OLM) testing was conducted 24 h after social defeat, and tissues were collected 6 days later to measure inflammatory markers. Sleep was recorded using electroencephalography (EEG) and electromyography (EMG) throughout the experiment. Results In vehicle-treated mice, only the combination of sleep disruption followed by social defeat (double hit): (1) increased brief arousals and NREM beta (15–30 Hz) EEG power in sleep immediately post-social defeat compared to baseline; (2) induced an increase in the proportion of rapid-eye-movement (REM) sleep and number of state shifts for at least 5 days post-social defeat; and (3) induced hyperlocomotion and lack of habituation in the OLM task. Immunization with MV prevented most of these sleep and behavioral changes. Conclusions Immunization with MV ameliorates a stress-induced sleep and behavioral phenotype that shares features with human posttraumatic stress disorder.

Microbial transfer from the environment can influence a person’s health, but relevant studies often have confounding variables and short durations. Here, we used the unique environment of the International Space Station (ISS) to track movement of microbes between an astronaut’s commensal microbiomes and their environment. We identified several microbial taxa, includingSerratia proteamaculansandRickettsia australiswhich appear to have been transferred from the ISS to the commensal microbiomes of the astronaut. Strains were matched at the SNP and haplotype-level, and notably some strains persisted even after the astronaut’s return to Earth. Some transferred taxa correspond to secondary strains in the ISS environment, suggesting that transfer may be mediated by evolutionary selection. Finally, we show evidence that the T-Cell repertoire of the astronaut changes to become more specific to environmental taxa, suggesting that continual microbial and immune monitoring can help guide spaceflight mission planning, health monitoring, and habitat design.

Affective behaviors and mental health are profoundly affected by disturbances in circadian rhythms. Casein kinase 1 epsilon (CSNK1E) is an essential component of the core circadian clock. Mice with tau or null mutation of this gene have shortened and lengthened circadian period respectively. Here we examined anxiety-like, fear, and depressive-like behaviors in both male and female mice of these two different mutants. Compared with wild-type mice, we found reductions in fear and anxiety-like behaviors in both mutant lines and in both sexes, with the tau mutants exhibiting the greatest phenotypic changes. However, the depressive-like behaviors had distinct phenotypic patterns, with markedly less depressive-like behaviors in female null mutants, but not in tau mutants of either sex. To determine whether abnormal light entrainment of tau mutants to 24 hour light-dark cycles contributes to these phenotypic differences, we also examined these behaviors in tau mutants on a 20 hour light-dark cycle close to their endogenous circadian period. The normalized entrainment restored more wild-type-like behaviors for fear and anxiety, but it induced depressive-like behavior in tau mutant females. These data show that both mutations of Csnk1e broadly affect fear and anxiety-like behaviors, while the effects on depressive-like behavior vary with genetics, photoperiod, and sex, suggesting that the mechanisms by which Csnk1e affects fear and anxiety-like behaviors may be similar, but distinct from those affecting depressive-like behavior. Our study also provides experimental evidence in support of the hypothesis of beneficial outcomes from properly entrained circadian rhythms in terms of the anxiety-like and fear behaviors.

Because regular sleep disruption can increase vulnerability to stress-related psychiatric disorders, there is a need to explore novel countermeasures to increase stress resilience after inadequate sleep. In this study, we explored the impact of 5 days of intermittent sleep disruption on vulnerability to acute social defeat stress in mice, and investigated the ability of the environmental, immunomodulatory bacterium Mycobacterium vaccae NCTC 11659 (MV) to promote stress resilience in that context. We found that mice receiving sleep disruption plus acute stress developed sleep and behavioral phenotypes that had some features of human posttraumatic stress disorder (PTSD) including reduced NREM delta power and increased NREM beta power in post-stress sleep EEG, persistent increases in sleep fragmentation and the REM:Sleep ratio, and behavioral changes. Importantly, immunization with heat-killed MV prevented the development of this phenotype. These results support further research into novel, microbial-based countermeasures to improve health and increase resilience to sleep disruption.

Introduction Disturbed gut microbiome diversity has been associated with poor health outcomes and various disease states. We investigated the impact of combined sleep restriction (3h time in bed [TIB] sleep opportunities per day) and circadian misalignment (daytime sleep and nighttime wakefulness) on gut microbiome alpha diversity in healthy young individuals in a controlled laboratory setting. Methods Twenty healthy adults (8 female), mean age (±SD) 25.65(±4.2) completed a 39-day protocol consisting of two laboratory visits lasting 4 days each. Two weeks of ambulatory monitoring prior to laboratory visits confirmed ~8h habitual sleep duration per night. Participants consumed energy-balanced diets, identical within participants, 2 days before and during the laboratory visits. The laboratory visits consisted of sleep opportunities as follows: night 1 (8h TIB), night 2 (3h TIB), day 3 (3h TIB) and day 4 (3h TIB). Fecal microbiome samples were obtained at baseline between day 1 and 2, and during sleep and circadian disruption (between day 3 and 4). Alpha diversity measures were calculated using Pielou’s evenness, Faith’s phylogenetic diversity and number of observed OTUs. Results Linear mixed models with subject as a random factor and visit as a fixed factor were performed to assess whether any alpha diversity measures changed during sleep and circadian disruption compared to baseline. Alpha diversity did not change significantly between baseline and sleep and circadian disruption (all p > 0.57). Additionally, intraclass correlation coefficients (ICCs) were calculated at baseline and during sleep and circadian disruption to determine if alpha diversity measures showed trait-like stability at both time points. ICCs were substantial to almost perfect (ICC 0.64–0.84) at baseline and substantial (ICC 0.70–0.80) during sleep and circadian disruption. Conclusion Four days of combined sleep restriction and circadian misalignment does not appear to alter alpha diversity of gut microbiota species in healthy adults. Further, substantial to almost perfect intraclass correlation coefficients suggest alpha diversity of the human microbiome is stable during combined sleep and circadian perturbation and that examination at the level of microbiota community composition and functional outcomes are needed. Support (if any) Office of Naval Research MURI (N00014- 15-1-2809), NIH/NCATS (UL1TR002535), NIH T32 HL149646, CU Undergraduate Research Opportunities.

Over the past decade, a large body of literature has demonstrated that disruptions of the endogenous circadian clock, whether environmental or genetic, lead to metabolic dysfunctions that are associated with obesity, diabetes, and other metabolic disorders. The phrase, “It is not only what you eat and how much you eat, but also when you eat” sends a simple message about circadian timing and body weight regulation. Communicating this message to clinicians and patients, while also elucidating the neuroendocrine, molecular, and genetic mechanisms underlying this phrase is essential to embrace the growing knowledge of the circadian impact on metabolism as a part of healthy life style as well as to incorporate it into clinical practice for improvement of overall human health. In this review, we discuss findings from animal models, as well as epidemiological and clinical studies in humans, which collectively promote the awareness of the role of circadian clock in metabolic functions and dysfunctions.

It has been established in recent years that the gut microbiome plays a role in health and disease, potentially via alterations in metabolites that influence host physiology. Although sleep disruption and gut dysbiosis have been associated with many of the same diseases, studies investigating the gut microbiome in the context of sleep disruption have yielded inconsistent results, and have not assessed the fecal metabolome. We exposed mice to five days of sleep disruption followed by four days of ad libitum recovery sleep, and assessed the fecal microbiome and fecal metabolome at multiple timepoints using 16S rRNA gene amplicons and untargeted LC-MS/MS mass spectrometry. We found global shifts in both the microbiome and metabolome in the sleep-disrupted group on the second day of recovery sleep, when most sleep parameters had recovered to baseline levels. We observed an increase in the Firmicutes:Bacteroidetes ratio, along with decreases in the genus Lactobacillus, phylum Actinobacteria, and genus Bifidobacterium in sleep-disrupted mice compared to control mice. The latter two taxa remained low at the fourth day post-sleep disruption. We also identified multiple classes of fecal metabolites that were differentially abundant in sleep-disrupted mice, some of which are physiologically relevant and commonly influenced by the microbiome. This included bile acids, and inference of microbial functional gene content suggested reduced levels of the microbial bile salt hydrolase gene in sleep-disrupted mice. Overall, this study adds to the evidence base linking disrupted sleep to the gut microbiome and expands it to the fecal metabolome, identifying sleep disruption-sensitive bacterial taxa and classes of metabolites that may serve as therapeutic targets to improve health after poor sleep.

Study Objectives The present studies examine the effects of NMDAR activation by NYX-2925 diurnal rhythmicity of both sleep and wake as well as emotion. Methods Twenty-four-hour sleep EEG recordings were obtained in sleep-deprived and non-sleep-deprived rats. In addition, the day–night cycle of both activity and mood was measured using home cage ultrasonic-vocalization recordings. Results NYX-2925 significantly facilitated non-REM (NREM) sleep during the lights-on (sleep) period, and this effect persisted for 3 days following a single dose in sleep-deprived rats. Sleep-bout duration and REM latencies were increased without affecting total REM sleep, suggesting better sleep quality. In addition, delta power during wake was decreased, suggesting less drowsiness. NYX-2925 also rescued learning and memory deficits induced by sleep deprivation, measured using an NMDAR-dependent learning task. Additionally, NYX-2925 increased positive affect and decreased negative affect, primarily by facilitating the transitions from sleep to rough-and-tumble play and back to sleep. In contrast to NYX-2925, the NMDAR antagonist ketamine acutely (1–4 hours post-dosing) suppressed REM and non-REM sleep, increased delta power during wake, and blunted the amplitude of the sleep-wake activity rhythm. Discussion These data suggest that NYX-2925 could enhance behavioral plasticity via improved sleep quality as well as vigilance during wake. As such, the facilitation of sleep by NYX-2925 has the potential to both reduce symptom burden on neurological and psychiatric disorders as well as serve as a biomarker for drug effects through restoration of sleep architecture.

Introduction Individual differences in cognition during sleep restriction and circadian misalignment have been shown to be trait-like. Here we explored the consistency of individual differences in cardiovascular responses to the cold pressor test (CPT) measured by changes in the distal-proximal skin temperature gradient (DPG) using the contralateral hand to that immersed in the ice water bath, which reflects a reflex cutaneous vasoconstriction. Methods Eighteen healthy participants (8 females) mean (±SD) age 25.28 (±4.3), underwent two identical in-laboratory combined sleep restriction and circadian misalignment protocols preceded by an 8h baseline in-laboratory sleep opportunity. The participants were given a 3h sleep opportunity on night 2 and a 3h sleep opportunity on days 3 and 4 followed by recovery sleep. The CPT occurred the morning after the baseline sleep opportunity and the morning before recovery sleep. Participants maintained a seated posture beginning 30 min prior to the CPT and skin temperature was assessed starting 15 min before until 45 min after the CPT. DPG (proximal=subclavicular; distal=hand palmar) data were averaged into 3 min bins. Changes in DPG from pre-CPT and 5 min post-CPT were assessed as the primary outcome using mixed-model ANOVAs. Intra-class correlation coefficients (ICC) were calculated to measure consistency of individual differences for DPG responses. Results Mixed-model ANOVA revealed significant effects of time and combined sleep restriction and circadian misalignment on DPG during the CPT (both p Conclusion Findings support that combined sleep restriction and circadian misalignment is associated with sympathetic activation and that individual differences in the DPG response to cold pressor stress are consistent. Support (if any) Office of Naval Research MURI grant N00014-15-1-2809, NIH/NCATS Colorado CTSA Grant UL1TR002535

Background Electroencephalogram (EEG) and electromyogram (EMG) recordings are often used in rodents to study sleep architecture and sleep-associated neural activity. These recordings must be scored to designate what sleep/wake state the animal is in at each time point. Manual sleep-scoring is very time-consuming, so machine-learning classifier algorithms have been used to automate scoring. New method Instead of using single classifiers, we implement a multiple classifier system. The multiple classifier is built from six base classifiers: decision tree, k-nearest neighbors, naive Bayes, support vector machine, neural net, and linear discriminant analysis. Decision tree and k-nearest neighbors were improved into ensemble classifiers by using bagging and random subspace. Confidence scores from each classifier were combined to determine the final classification. Ambiguous epochs can be rejected and left for a human to classify. Results Support vector machine was the most accurate base classifier, and had error rate of 0.054. The multiple classifier system reduced the error rate to 0.049, which was not significantly different from a second human scorer. When 10% of epochs were rejected, the remaining epochs’ error rate dropped to 0.018. Comparison with existing method(s) Compared with the most accurate single classifier (support vector machine), the multiple classifier reduced errors by 9.4%. The multiple classifier surpassed the accuracy of a second human scorer after rejecting only 2% of epochs. Conclusions Multiple classifier systems are an effective way to increase automated sleep scoring accuracy. Improvements in autoscoring will allow sleep researchers to increase sample sizes and recording lengths, opening new experimental possibilities.

Disruption of circadian rhythms results in metabolic dysfunction. Casein kinase 1 epsilon (CK1ε) is a canonical circadian clock gene. Null and tau mutations in CK1ε show distinct effects on circadian period. To investigate the role of CK1ε in body weight regulation under both regular chow (RC) and high fat (HF) diet conditions, we examined body weight on both RC and HF diets in CK1ε-/- and CK1εtau/tau mice on a standard 24 hr light-dark (LD) cycle. Given the abnormal entrainment of CK1εtau/tau mice on a 24 hr LD cycle, a separate set of CK1εtau/tau mice were tested under both diet conditions on a 20 hr LD cycle, which more closely matches their endogenous period length. On the RC diet, both CK1ε-/- and CK1εtau/tau mutants on a 24 hr LD cycle and CK1εtau/tau mice on a 20 hr LD cycle exhibited significantly lower body weights, despite similar overall food intake and activity levels. On the HF diet, CK1εtau/tau mice on a 20 hr LD cycle were protected against the development of HF diet-induced excess weight gain. These results provide additional evidence supporting a link between circadian rhythms and energy regulation at the genetic level, particularly highlighting CK1ε involved in the integration of circadian biology and metabolic physiology.

Chrononutrition is an emerging discipline that builds on the intimate relation between endogenous circadian (24-h) rhythms and metabolism. Circadian regulation of metabolic function can be observed from the level of intracellular biochemistry to whole-organism physiology and even postprandial responses. Recent work has elucidated the metabolic roles of circadian clocks in key metabolic tissues, including liver, pancreas, white adipose, and skeletal muscle. For example, tissue-specific clock disruption in a single peripheral organ can cause obesity or disruption of whole-organism glucose homeostasis. This review explains mechanistic insights gained from transgenic animal studies and how these data are being translated into the study of human genetics and physiology. The principles of chrononutrition have already been demonstrated to improve human weight loss and are likely to benefit the health of individuals with metabolic disease, as well as of the general population.

Study Objectives Determine stability of individual differences in executive function, cognitive processing speed, selective visual attention, and maintenance of wakefulness during simulated sustained operations with combined sleep restriction and circadian misalignment. Methods Twenty healthy adults (eight female), aged 25.7 (±4.2 SD), body mass index (BMI) 22.3 (±2.1) kg/m2 completed an 18-day protocol twice. Participants maintained habitual self-selected 8-hour sleep schedules for 2 weeks at home prior to a 4-day laboratory visit that included one sleep opportunity per day: 8 hours on night 1, 3 hours on night 2, and 3 hours on mornings 3 and 4. After 3 days of unscheduled sleep at home, participants repeated the entire protocol. Stability and task dependency of individual differences in performance were quantified by intra-class correlation coefficients (ICC) and Kendall’s Tau, respectively. Results Performance on Stroop, Visual Search, and the Maintenance of Wakefulness Test were highly consistent within individuals during combined sleep restriction and circadian misalignment. Individual differences were trait-like as indicated by ICCs (0.54–0.96) classified according to standard criteria as moderate to almost perfect. Individual differences on other performance tasks commonly reported in sleep studies showed fair to almost perfect ICCs (0.22–0.94). Kendall’s rank correlations showed that individual vulnerability to sleep restriction and circadian misalignment varied by task and by metric within a task. Conclusions Consistent vulnerability of higher-order cognition and maintenance of wakefulness to combined sleep restriction and circadian misalignment has implications for the development of precision countermeasure strategies for workers performing safety-critical tasks, e.g. military, police, health care workers and emergency responders.

INTRODUCTION To date, 559 humans have been flown into space, but long-duration (>300 days) missions are rare (n = 8 total). Long-duration missions that will take humans to Mars and beyond are planned by public and private entities for the 2020s and 2030s; therefore, comprehensive studies are needed now to assess the impact of long-duration spaceflight on the human body, brain, and overall physiology. The space environment is made harsh and challenging by multiple factors, including confinement, isolation, and exposure to environmental stressors such as microgravity, radiation, and noise. The selection of one of a pair of monozygotic (identical) twin astronauts for NASA’s first 1-year mission enabled us to compare the impact of the spaceflight environment on one twin to the simultaneous impact of the Earth environment on a genetically matched subject. RATIONALE The known impacts of the spaceflight environment on human health and performance, physiology, and cellular and molecular processes are numerous and include bone density loss, effects on cognitive performance, microbial shifts, and alterations in gene regulation. However, previous studies collected very limited data, did not integrate simultaneous effects on multiple systems and data types in the same subject, or were restricted to 6-month missions. Measurement of the same variables in an astronaut on a year-long mission and in his Earth-bound twin indicated the biological measures that might be used to determine the effects of spaceflight. Presented here is an integrated longitudinal, multidimensional description of the effects of a 340-day mission onboard the International Space Station. RESULTS Physiological, telomeric, transcriptomic, epigenetic, proteomic, metabolomic, immune, microbiomic, cardiovascular, vision-related, and cognitive data were collected over 25 months. Some biological functions were not significantly affected by spaceflight, including the immune response (T cell receptor repertoire) to the first test of a vaccination in flight. However, significant changes in multiple data types were observed in association with the spaceflight period; the majority of these eventually returned to a preflight state within the time period of the study. These included changes in telomere length, gene regulation measured in both epigenetic and transcriptional data, gut microbiome composition, body weight, carotid artery dimensions, subfoveal choroidal thickness and peripapillary total retinal thickness, and serum metabolites. In addition, some factors were significantly affected by the stress of returning to Earth, including inflammation cytokines and immune response gene networks, as well as cognitive performance. For a few measures, persistent changes were observed even after 6 months on Earth, including some genes’ expression levels, increased DNA damage from chromosomal inversions, increased numbers of short telomeres, and attenuated cognitive function. CONCLUSION Given that the majority of the biological and human health variables remained stable, or returned to baseline, after a 340-day space mission, these data suggest that human health can be mostly sustained over this duration of spaceflight. The persistence of the molecular changes (e.g., gene expression) and the extrapolation of the identified risk factors for longer missions (>1 year) remain estimates and should be demonstrated with these measures in future astronauts. Finally, changes described in this study highlight pathways and mechanisms that may be vulnerable to spaceflight and may require safeguards for longer space missions; thus, they serve as a guide for targeted countermeasures or monitoring during future missions.

Background Insomnia is increasingly recognized to be comorbid with one or more medical conditions. This study used an online research platform to characterize insomnia across different mental and physical conditions. Methods A custom cross-sectional survey was fielded online to 31,208 users of the patient community PatientsLikeMe. The survey queried members on National Sleep Foundation-defined insomnia risk ( waking up feeling unrefreshed , difficulty falling asleep , waking in the middle of the night , or waking too early ). Results Complete results were obtained from 5256 patients with 11 comorbid conditions. Seventy-six percent of US-based respondents were at risk for insomnia. Patients who reported difficulty falling asleep were found to have nearly twice the odds of self-reporting insomnia (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.5–2.1) when compared to those who do not have difficulty falling asleep, whereas those who reported waking during the night or waking up unrefreshed were no more likely (OR: 1.025 and 1.032, respectively) to report that they suffered from insomnia than those who did not experience these issues. Although insomnia was self-reported as severe or very severe across most conditions, few respondents had actually been diagnosed with insomnia by a physician. After adjustment for age and gender, there was an independent and strong effect of primary condition severity on insomnia risk, and those with severe epilepsy (0.93), depressive disorders (0.92), and fibromyalgia (0.92) occupied the highest risk probabilities. Conclusions The high rate of severity and frequency of insomnia across a multitude of mental and physical conditions reveals an opportunity for better disease management through enhanced insomnia awareness.

Children with atopic dermatitis (AD) experience significant sleep disruption, and clinically, the disease is noted to worsen in a circadian manner at night. Epidemiologic findings highlight many negative consequences of AD, such as impaired linear growth, which is uniquely related to disturbed sleep. Clinical guidelines currently recommend assessing sleep in patients with AD as a crucial parameter of disease control with appropriate treatment. In this review we describe our current understanding of the roles of sleep cycles and circadian rhythms in the nighttime exacerbation of AD (nocturnal eczema). We present a schematic to explain the mechanism of nocturnal eczema. Treatment options for sleep disturbance and future directions for research are discussed in the context of AD.

Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA-like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild-type (WT) mice were maintained on a constant 12 hour:12 hour LD cycle (12:12 LD) or exposed to weekly 12 hour phase shifts. Alternatively, male circadian mutant mice (ClockΔ19 or Csnk1etau mutants) were compared with age-matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix-degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ-ERK-RUNX2/NFκB and β-catenin signaling pathways, stimulation of MMP-13 and ADAMTS-5, as well as suppression of the anabolic mediators SOX9 and TIMP-3 in articular chondrocytes of phase-shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA-like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA-like pathological changes in the mouse knee joint.

Background/Objectives Mounting evidence supports a link between circadian disruption and metabolic disease. Humans with circadian disruption (e.g., night-shift workers) have an increased risk of obesity and cardiometabolic diseases compared to the non-disrupted population. However, it is unclear if the obesity and obesity-related disorders associated with circadian disruption respond to therapeutic treatments as well as individuals with other types of obesity. Subjects/Methods Here, we test the effectiveness of the commonly used bariatric surgical procedure, Vertical Sleeve Gastrectomy (VSG) in mouse models of genetic and environmental circadian disruption. Results VSG led to a reduction in body weight and fat mass in both ClockΔ19 mutant and constant-light mouse models (P < .05), resulting in an overall metabolic improvement independent of circadian disruption. Interestingly, the decrease in body weight occurred without altering diurnal feeding or activity patterns (P > .05). Within circadian-disrupted models, VSG also led to improved glucose tolerance and lipid handling (P < .05). Conclusions Together these data demonstrate that VSG is an effective treatment for the obesity associated with circadian disruption, and that the potent effects of bariatric surgery are orthogonal to circadian biology. However, since the effects of bariatric surgery are independent of circadian disruption, VSG cannot be considered a cure for circadian disruption. These data have important implications for circadian-disrupted obese patients. Moreover, these results reveal new information about the metabolic pathways governing the effects of bariatric surgery as well as of circadian disruption.

The discovery of the molecular mechanisms underlying the circadian clock, which functions in virtually every cell throughout the body to coordinate biological processes to anticipate and better adapt to daily rhythmic changes in the environment, is one of the major biomedical breakthroughs in the 20(th) century. Twenty years after this breakthrough, the biomedical community is now at a new frontier to incorporate the circadian clock mechanisms into many areas of biomedical research, as studies continue to reveal an important role of the circadian clock in a wide range of biological functions and diseases. A forefront of this exciting area is the research of interactions between the clock and energy metabolism. In this review, we summarize animal and human studies linking disruptions of the circadian clock, either environmental or genetic, to metabolic dysfunctions associated with obesity, diabetes, and other metabolic disorders. We also discuss how these advances in circadian biology may pave the way to revolutionize clinical practice in the era of precision medicine.

We identified gene networks linking sleep and affect, highlighting those oppositely perturbed by sleep loss and depression., To understand the transcriptomic organization underlying sleep and affective function, we studied a population of (C57BL/6J × 129S1/SvImJ) F2 mice by measuring 283 affective and sleep phenotypes and profiling gene expression across four brain regions. We identified converging molecular bases for sleep and affective phenotypes at both the single-gene and gene-network levels. Using publicly available transcriptomic datasets collected from sleep-deprived mice and patients with major depressive disorder (MDD), we identified three cortical gene networks altered by the sleep/wake state and depression. The network-level actions of sleep loss and depression were opposite to each other, providing a mechanistic basis for the sleep disruptions commonly observed in depression, as well as the reported acute antidepressant effects of sleep deprivation. We highlight one particular network composed of circadian rhythm regulators and neuronal activity–dependent immediate-early genes. The key upstream driver of this network, Arc, may act as a nexus linking sleep and depression. Our data provide mechanistic insights into the role of sleep in affective function and MDD.

Recent advances in the understanding of the molecular, genetic, neural, and physiologic basis for the generation and organization of circadian clocks in mammals have revealed profound bidirectional interactions between the circadian clock system and pathways critical for the regulation of metabolism and energy balance. The discovery that mice harboring a mutation in the core circadian gene circadian locomotor output cycles kaput (Clock) develop obesity and evidence of the metabolic syndrome represented a seminal moment for the field, clearly establishing a link between circadian rhythms, energy balance, and metabolism at the genetic level. Subsequent studies have characterized in great detail the depth and magnitude of the circadian clock’s crucial role in regulating body weight and other metabolic processes. Dietary nutrients have been shown to influence circadian rhythms at both molecular and behavioral levels; and many nuclear hormone receptors, which bind nutrients as well as other circulating ligands, have been observed to exhibit robust circadian rhythms of expression in peripheral metabolic tissues. Furthermore, the daily timing of food intake has itself been shown to affect body weight regulation in mammals, likely through, at least in part, regulation of the temporal expression patterns of metabolic genes. Taken together, these and other related findings have transformed our understanding of the important role of time, on a 24-h scale, in the complex physiologic processes of energy balance and coordinated regulation of metabolism. This research has implications for human metabolic disease and may provide unique and novel insights into the development of new therapeutic strategies to control and combat the epidemic of obesity.

STUDY OBJECTIVES: Efforts to identify the genetic basis of mammalian sleep have included quantitative trait locus (QTL) mapping and gene targeting of known core circadian clock genes. We combined three different genetic approaches to identify and test a positional candidate sleep gene - the circadian gene casein kinase 1 epsilon (Csnk1e), which is located in a QTL we identified for rapid eye movement (REM) sleep on chromosome 15. MEASUREMENTS AND RESULTS: Using electroencephalographic (EEG) and electromyographic (EMG) recordings, baseline sleep was examined in a 12-h light:12-h dark (LD 12:12) cycle in mice of seven genotypes, including Csnk1e(tau/tau) and Csnk1e(-/-) mutant mice, Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice, and their respective wild-type littermate control mice. Additionally, Csnk1e(tau/tau) and wild-type mice were examined in constant darkness (DD). Csnk1e(tau/tau) mutant mice and both Csnk1e (B6.D2) and Csnk1e (D2.B6) congenic mice showed significantly higher proportion of sleep time spent in REM sleep during the dark period than wild-type controls - the original phenotype for which the QTL on chromosome 15 was identified. This phenotype persisted in Csnk1e(tau/tau) mice while under free-running DD conditions. Other sleep phenotypes observed in Csnk1e(tau/tau) mice and congenics included a decreased number of bouts of nonrapid eye movement (NREM) sleep and an increased average NREM sleep bout duration. CONCLUSIONS: These results demonstrate a role for Csnk1e in regulating not only the timing of sleep, but also the REM sleep amount and NREM sleep architecture, and support Csnk1e as a causal gene in the sleep QTL on chromosome 15.

Osteoarthritis (OA) is characterized by a slow and progressive deterioration of articular cartilage. OA likely arises from a combination of systemic (genetics, age, environmental factors) and local factors (abnormal joint loading, overuse or trauma) working in concert to create a condition with definable morphologic and clinical characteristics. Several risk factors for OA have been previously identified, including genetic predisposition, obesity, diabetes, hypertension, hyperuricemia, previous trauma, and aging (1). However, due in large part to an inability to control for confounding factors, the underlying pathogenesis and causative factors responsible for initiation and progression of the disease remain largely unknown. There is a growing body of evidence correlating progression of OA with an upregulation of inflammatory processes (2). Oxidative stress elicited by reactive oxygen species (ROS) further disturbs cartilage homeostasis and promotes catabolism via induction of cell death, breakdown of matrix proteoglycans (PGs), upregulation of latent matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) synthesis, and oxidation of intracellular and extracellular molecules (3). Thus, environmental factors that promote oxidative stress and inflammatory states could potentially act as a risk factor for OA. Alcohol consumption could be one potential risk factor because: [1] chronic alcohol consumption, highly common in Western and industrial societies, generates reactive oxygen species (ROS), leading to systemic and tissue oxidative stress in human and rodents, and [2] alcohol is capable of inducing pro-inflammatory states in multiple organs such as liver, heart, central nervous system, and pancreas (4, 5). Several studies have previously attempted to elucidate a relationship between alcohol consumption and inflammatory arthritis such as rheumatoid arthritis, with conflicting results (6, 7). However, despite recent evidence demonstrating the importance of oxidative stress and pro-inflammatory states in the development and progression of degenerative joint disease, the impact of alcohol consumption on OA has not yet been studied. The findings of the present study suggest that chronic alcohol exposure may increase susceptibility to the development and/or progression of OA. Using a validated in vivo model of chronic alcohol treatment, we show that chronic alcohol consumption increases PG loss in both knee and shoulder joints of mice, stimulates multiple inflammatory, catabolic, and anti-anabolic mediators involved in cartilage. In our experimental protocol, young adult male (aged 7–9 weeks) C57BL/6 mice were provided ad libitum access to an alcohol-diet (i.e., the Nanji diet), containing 4.5% (v/v) ethanol (29% ethanol-derived calories) or an isocaloric alcohol-free control diet for 8 weeks (n=14–16 per group). All animal protocols and practices were reviewed and approved in advance by the Institutional Animal Care and Use Committees at Rush University and Northwestern University. Following eight weeks of alcohol-containing diet or the control diet, mice were euthanized and joint sections were collected, fixed, paraffin-embedded, and stained with Safranin-O to assess cartilage structure and matrix proteoglycan (PG) content. The alcohol diet is a slight modification of the well-validated Leiber-DiCarli diet in which the fat source comes from fish oil and consumption of this diet in BL6 mice has been shown to produce blood alcohol levels that are in the low to moderate level (8). Our group, and others, have successfully used this alcohol diet to induce a variety of alcohol pathologies including colon cancer, intestinal hyperpermeability, endotoxemia, and liver pathology in rodents (8–10). Serum alcohol level in the alcohol-fed mice at the time of euthanasia was ~3mg/dL and these mice did not show any overt behavioral abnormalities during our experimental protocol. Histological examination of knee joints of control diet-fed mice demonstrated normal architecture of articular cartilage with intense Safranin-O staining. In contrast, knee joints of alcohol-fed mice displayed OA-like characteristics, with increases in PG loss represented by Safranin-O staining and mild fibrillation (Figure 1A). These results were quantified using an OARSI semi-quantitative scoring system. Alcohol-fed mice scored significantly higher (1.3±0.67) than control mice (0.3±0.27; P < 0.05), indicating more severe arthritic changes in knee joints of alcohol-fed mice compared to control (Figure 1A, lower panel). Similar results were found in shoulder joints (Figure 1B), as Safranin-O staining revealed a decrease in gross PG content and an irregular cartilage surface in shoulder joints of alcohol-fed mice compared to the control group. We applied the OARSI scoring to quantify pathological changes in shoulder, and found significantly higher OARSI scores for alcohol-fed mice (0.75±0.28) compared to control mice (0.12±0.25; P < 0.05) (Figure 1B, lower panel). Interestingly, the intervertebral discs showed no pathological changes in alcohol-fed mice compared to control mice (Figure 1C). Figure 1 Chronic ethanol consumption induces pathological changes in articular cartilage Our results demonstrate a pathologic role of alcohol on specific catabolic and anti-anabolic mediators in knee joints (Supplemental Figure 1) that may increase susceptibility of OA induction. In our study, phospho protein kinase C δ (pPKCδ), pNF-κB, and pERK1/2 were significantly increased in knee joints of alcohol-fed compared to control mice (Supplemental Figure 1A, P < 0.05), suggesting that chronic alcohol consumption stimulates these catabolic signaling pathways, which may result in subsequent production of cartilage destructive enzymes. Hypertrophic marker RUNX2, as well as a key cartilage destructive enzymes matrix metalloprotease- 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), were significantly increased in knee joints of alcohol-fed mice compared to the control mice (Supplemental Figure 1B; P < 0.05). In addition to induction of catabolic effects, we observed that chronic alcohol consumption resulted in a striking reduction of anabolic and anti-inflammatory mediators in articular chondrocytes as assessed by immunohistochemistry (Supplemental Figure 1C, P < 0.05). These molecules include tissue inhibitor of metalloproteinase-3 (TIMP-3), SOX-9, high-mobility group protein-2 (HMGB2) and suppressor of cytokine signaling-2 (SOCS-2), molecules that were significantly decreased in OA cartilage and associated with both cartilage protection and repair. These findings clearly demonstrate that chronic alcohol consumption augments catabolic signaling pathways and suppresses anabolic, reparative, and anti-inflammatory activity in mouse knee articular chondrocytes. We also performed micro-computed tomography (uCT) to determine whether any pathological changes in bone pathology after chronic alcohol treatment were evident. However, we did not find significant bone pathology in the knee-joints of alcohol-fed mice from our experimental protocol (data not shown). To our knowledge, these findings provide the first evidence that chronic alcohol consumption may be a novel risk factor for the development of OA. The pathology associated with chronic alcohol consumption is multi-factorial, with diverse consequences in different cell types through direct toxic effects as well as indirect effects of specific metabolites, oxidative stress, immunologic/inflammatory processes. It is well-established that alcohol consumption causes endotoxemia that is thought to contribute to alcohol liver damage (11). Indeed, we have shown that alcohol consumption promotes intestinal hyperpermeability and endotoxemia in rodents including BL6 mice (8) phenomenon that are also observed in human alcoholics (12). Future studies are necessary to evaluate the contribution of intestinal barrier dysfunction and endotoxemia in our studies. There are, however, several limitations of this study that need to be mentioned. First, we have addressed the effects of chronic alcohol consumption in an in vivo mouse model of the knee joint, which makes it difficult to generalize results across species to human joint tissue. Clinical studies are necessary to confirm our data to associate chronic alcoholism and joint pathology. Second, the current study reports the potent catabolic activity of chronic alcohol in cartilage homeostasis via PKCδ, and MAPK signaling, but a detailed understanding of the specific cell signaling pathways and molecular mechanisms underlying these findings remains to be elucidated. Finally, although these results have significant potential clinical relevance, several aspects of the underlying mechanisms remain unknown. Further studies will facilitate a better understanding of the multiple, complex effects stimulated by alcohol in joint tissue.

We have shown that alcohol increases Caco-2 intestinal epithelial cell monolayer permeability in vitro by inducing the expression of redox-sensitive circadian clock proteins CLOCK and PER2 and that these proteins are necessary for alcohol-induced hyperpermeability. We hypothesized that alcohol metabolism by intestinal Cytochrome P450 isoform 2E1 (CYP2E1) could alter circadian gene expression ( Clock and Per2), resulting in alcohol-induced hyperpermeability. In vitro Caco-2 intestinal epithelial cells were exposed to alcohol, and CYP2E1 protein, activity, and mRNA were measured. CYP2E1 expression was knocked down via siRNA and alcohol-induced hyperpermeability, and CLOCK and PER2 protein expression were measured. Caco-2 cells were also treated with alcohol or H2O2 with or without N-acetylcysteine (NAC) anti-oxidant, and CLOCK and PER2 proteins were measured at 4 or 2 h. In vivo Cyp2e1 protein and mRNA were also measured in colon tissue from alcohol-fed mice. Alcohol increased CYP2E1 protein by 93% and enzyme activity by 69% in intestinal cells in vitro. Alcohol feeding also increased mouse colonic Cyp2e1 protein by 73%. mRNA levels of Cyp2e1 were not changed by alcohol in vitro or in mouse intestine. siRNA knockdown of CYP2E1 in Caco-2 cells prevented alcohol-induced hyperpermeability and induction of CLOCK and PER2 proteins. Alcohol-induced and H2O2-induced increases in intestinal cell CLOCK and PER2 were significantly inhibited by treatment with NAC. We concluded that our data support a novel role for intestinal CYP2E1 in alcohol-induced intestinal hyperpermeability via a mechanism involving CYP2E1-dependent induction of oxidative stress and upregulation of circadian clock proteins CLOCK and PER2.