Effect of 60 days of head down tilt bed rest on amplitude and phase of rhythms in physiology and sleep in men.

Twenty-four-hour rhythms in physiology and behaviour are shaped by circadian clocks, environmental rhythms, and feedback of behavioural rhythms onto physiology. In space, 24 h signals such as those associated with the light-dark cycle and changes in posture, are weaker, potentially reducing the robustness of rhythms. Head down tilt (HDT) bed rest is commonly used to simulate effects of microgravity but how HDT affects rhythms in physiology has not been extensively investigated. Here we report effects of -6° HDT during a 90-day protocol on 24 h rhythmicity in 20 men. During HDT, amplitude of light, motor activity, and wrist-temperature rhythms were reduced, evening melatonin was elevated, while cortisol was not affected during HDT, but was higher in the morning during recovery when compared to last session of HDT. During recovery from HDT, time in Slow-Wave Sleep increased. EEG activity in alpha and beta frequencies increased during NREM and REM sleep. These results highlight the profound effects of head-down-tilt-bed-rest on 24 h rhythmicity.

Extensive dynamic changes in the human transcriptome and its circadian organization during prolonged bed rest.

Physiological and molecular processes including the transcriptome change across the 24-h day, driven by molecular circadian clocks and behavioral and systemic factors. It is not known how the temporal organization of the human transcriptome responds to a long-lasting challenge. This may, however, provide insights into adaptation, disease, and recovery. We investigated the human 24-h time series transcriptome in 20 individuals during a 90-day constant bed rest protocol. We show that the protocol affected 91% of the transcriptome with 76% of the transcriptome still affected after 10 days of recovery. Dimensionality-reduction approaches revealed that many affected transcripts were associated with mRNA translation and immune function. The number, amplitude, and phase of rhythmic transcripts, including clock genes, varied significantly across the challenge. These findings of long-lasting changes in the temporal organization of the transcriptome have implications for understanding the mechanisms underlying health consequences of conditions such as microgravity and bed rest.

Non-traumatic hemorrhagic myelopathy in dogs.

BACKGROUND: Non-traumatic spinal cord hemorrhage (NTSH) is an uncommon cause of myelopathy in dogs. OBJECTIVES: Describe the clinical characteristics, concurrent medical conditions and underlying causes, magnetic resonance imaging (MRI) findings and outcome in dogs with NTSH. ANIMALS: Dogs diagnosed with NTSH using gradient echo T2-weighted (GRE) sequences with or without histopathological confirmation of hemorrhage were included. Dogs with a traumatic cause were excluded, including those with compressive intervertebral disc extrusion. METHODS: Retrospective descriptive study; the databases of 2 referral hospitals were searched between 2013 and 2021. RESULTS: Twenty-three dogs met inclusion criteria. The onset of signs was acute and progressive in 70% of cases; spinal hyperesthesia was variable (48%). Hemorrhage was identified in the thoracolumbar spinal segments in 65% of dogs. An underlying cause was identified in 65% of cases. Angiostrongylus vasorum represented 18% of the total cohort, followed by steroid-responsive meningitis arteritis (SRMA; 13%). Overall, 64% of dogs had a good or excellent outcome, regardless of cause; which was increased to 100% for SRMA, 75% for A. vasorum and 75% for idiopathic NTSH. Outcome was not associated with neurological severity. Recovery rate was 67% and 50% for nociception-intact and nociception-negative dogs, respectively. CONCLUSIONS: Larger prospective studies would be required to define prognostic factors for dogs with NTSH, but outcome appeared to be most influenced by the underlying cause, as opposed to neurological severity at presentation.

Mistimed sleep and waking activity in humans disrupts glucocorticoid signalling transcripts and SP1, but not plasma cortisol rhythms.

Cortisol is a robust circadian signal that synchronises peripheral circadian clocks with the central clock in the suprachiasmatic nucleus via glucocorticoid receptors that regulate peripheral gene expression. Misalignment of the cortisol rhythm with the sleep-wake cycle, as occurs in shift work, is associated with negative health outcomes, but underlying molecular mechanisms remain largely unknown. We experimentally induced misalignment between the sleep-wake cycle and melatonin and cortisol rhythms in humans and measured time series blood transcriptomics while participants slept in-phase and out-of-phase with the central clock. The cortisol rhythm remained unchanged, but many glucocorticoid signalling transcripts were disrupted by mistimed sleep. To investigate which factors drive this dissociation between cortisol and its signalling pathways, we conducted bioinformatic and temporal coherence analyses. We found that glucocorticoid signalling transcripts affected by mistimed sleep were enriched for binding sites for the transcription factor SP1. Furthermore, changes in the timing of the rhythms of SP1 transcripts, a major regulator of transcription, and changes in the timing of rhythms in transcripts of the glucocorticoid signalling pathways were closely associated. Associations between the rhythmic changes in factors that affect SP1 expression and its activity, such as STAT3, EP300, HSP90AA1, and MAPK1, were also observed. We conclude that plasma cortisol rhythms incompletely reflect the impact of mistimed sleep on glucocorticoid signalling pathways and that sleep-wake driven changes in SP1 may mediate disruption of these pathways. These results aid understanding of mechanisms by which mistimed sleep affects health.

Diurnal and circadian rhythmicity of the human blood transcriptome overlaps with organ- and tissue-specific expression of a non-human primate.

BACKGROUND: Twenty-four-hour rhythmicity in mammalian tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker and light-dark cycles. At the physiological level, the neural and endocrine systems synchronise gene expression in peripheral tissues and organs to the 24-h-day cycle, and disruption of such regulation has been shown to lead to pathological conditions. Thus, monitoring rhythmicity in tissues/organs holds promise for circadian medicine; however, most tissues and organs are not easily accessible in humans and alternative approaches to quantify circadian rhythmicity are needed. We investigated the overlap between rhythmic transcripts in human blood and transcripts shown to be rhythmic in 64 tissues/organs of the baboon, how these rhythms are aligned with light-dark cycles and each other, and whether timing of tissue-specific rhythmicity can be predicted from a blood sample. RESULTS: We compared rhythmicity in transcriptomic time series collected from humans and baboons using set logic, circular cross-correlation, circular clustering, functional enrichment analyses, and least squares regression. Of the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue and most of these genes were associated with basic processes such as transcription and protein homeostasis. In total, 109 (17%) of the 652 overlapping rhythmic genes were reported as rhythmic in only one baboon tissue or organ and several of these genes have tissue/organ-specific functions. The timing of human and baboon rhythmic transcripts displayed prominent 'night' and 'day' clusters, with genes in the dark cluster associated with translation. Alignment between baboon rhythmic transcriptomes and the overlapping human blood transcriptome was significantly closer when light onset, rather than midpoint of light, or end of light period, was used as phase reference point. The timing of overlapping human and baboon rhythmic transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood. CONCLUSIONS: The human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs in baboon. The rhythmic sets vary across tissues/organs, but the timing of most rhythmic genes is similar in human blood and baboon tissues/organs. These results have implications for development of blood transcriptome-based biomarkers for circadian rhythmicity in tissues and organs.

What are we measuring with the morningness-eveningness questionnaire? Exploratory factor analysis across four samples from two countries.

Individual variability in diurnal preference or chronotype is commonly assessed with self-report scales such as the widely used morningness-eveningness questionnaire (MEQ). We sought to investigate the MEQ's internal consistency by applying exploratory factor analysis (EFA) to determine the number of underlying latent factors in four different adult samples, two each from the United Kingdom and Brazil (total N = 3,457). We focused on factors that were apparent in all samples, irrespective of particular sociocultural diversity and geographical characteristics, so as to show a common core reproducible structure across samples. Results showed a three-factor solution with acceptable to good model fit indexes in all studied populations. Twelve of the 19 MEQ items in the three-correlated factor solution loaded onto the same factors across the four samples. This shows that the scale measures three distinguishable, yet correlated constructs: (1) items related to how people feel in the morning, which we termed efficiency of dissipation of sleep pressure (recovery process) (items 1, 3, 4, 5, 7, 9, 13, and 19); (2) items related to how people feel before sleep, which we called sensitivity to buildup of sleep pressure (items 2, 10, and 12); and (3) peak time of cognitive arousal (item 11). Although the third factor was not regarded as consistent since only one item was common among all samples, it might represent subjective amplitude. These results suggested that the latent constructs of the MEQ reflect dissociable homeostatic processes in addition to a less consistent propensity for cognitive arousal at different times of the day. By analyzing answers to MEQ items that compose these latent factors, it may be possible to extract further knowledge of factors that affect morningness-eveningness.

Alzheimer's disease genetic risk and sleep phenotypes in healthy young men: association with more slow waves and daytime sleepiness.

STUDY OBJECTIVES: Sleep disturbances and genetic variants have been identified as risk factors for Alzheimer's disease (AD). Our goal was to assess whether genome-wide polygenic risk scores (PRS) for AD associate with sleep phenotypes in young adults, decades before typical AD symptom onset. METHODS: We computed whole-genome PRS for AD and extensively phenotyped sleep under different sleep conditions, including baseline sleep, recovery sleep following sleep deprivation, and extended sleep opportunity, in a carefully selected homogenous sample of 363 healthy young men (22.1 years ± 2.7) devoid of sleep and cognitive disorders. RESULTS: AD PRS was associated with more slow-wave energy, that is, the cumulated power in the 0.5-4 Hz EEG band, a marker of sleep need, during habitual sleep and following sleep loss, and potentially with larger slow-wave sleep rebound following sleep deprivation. Furthermore, higher AD PRS was correlated with higher habitual daytime sleepiness. CONCLUSIONS: These results imply that sleep features may be associated with AD liability in young adults, when current AD biomarkers are typically negative, and support the notion that quantifying sleep alterations may be useful in assessing the risk for developing AD.

A PERIOD3 variable number tandem repeat polymorphism modulates melatonin treatment response in delayed sleep-wake phase disorder.

We examined whether a polymorphism of the PERIOD3 gene (PER3; rs57875989) modulated the sleep-promoting effects of melatonin in Delayed Sleep-Wake Phase Disorder (DSWPD). One hundred and four individuals (53 males; 29.4 ±10.0 years) with DSWPD and a delayed dim light melatonin onset (DLMO) collected buccal swabs for genotyping (PER34/4 n = 43; PER3 5 allele [heterozygous and homozygous] n = 60). Participants were randomised to placebo or 0.5 mg melatonin taken 1 hour before desired bedtime (or ~1.45 hours before DLMO), with sleep attempted at desired bedtime (4 weeks; 5-7 nights/week). We assessed sleep (diary and actigraphy), Pittsburgh Sleep Quality Index (PSQI), Insomnia Severity Index (ISI), Patient-Reported Outcomes Measurement Information System (PROMIS: Sleep Disturbance, Sleep-Related Impairment), Sheehan Disability Scale (SDS) and Patient- and Clinician-Global Improvement (PGI-C, CGI-C). Melatonin treatment response on actigraphic sleep onset time did not differ between genotypes. For PER34/4 carriers, self-reported sleep onset time was advanced by a larger amount and sleep onset latency (SOL) was shorter in melatonin-treated patients compared to those receiving placebo (P = .008), while actigraphic sleep efficiency in the first third of the sleep episode (SE T1) did not differ. For PER3 5 carriers, actigraphic SOL and SE T1 showed a larger improvement with melatonin (P < .001). Melatonin improved ISI (P = .005), PROMIS sleep disturbance (P < .001) and sleep-related impairment (P = .017), SDS (P = .019), PGI-C (P = .028) and CGI-C (P = .016) in PER34/4 individuals only. Melatonin did not advance circadian phase. Overall, PER34/4 DSWPD patients have a greater response to melatonin treatment. PER3 genotyping may therefore improve DSWPD patient outcomes.

A Topological Cluster of Differentially Regulated Genes in Mice Lacking PER3.

Polymorphisms in the human circadian clock gene PERIOD3 (PER3) are associated with a wide variety of phenotypes such as diurnal preference, delayed sleep phase disorder, sleep homeostasis, cognitive performance, bipolar disorder, type 2 diabetes, cardiac regulation, cancer, light sensitivity, hormone and cytokine secretion, and addiction. However, the molecular mechanisms underlying these phenotypic associations remain unknown. Per3 knockout mice (Per3-/- ) have phenotypes related to activity, sleep homeostasis, anhedonia, metabolism, and behavioral responses to light. Using a protocol that induces behavioral differences in response to light in wild type and Per3-/- mice, we compared genome-wide expression in the eye and hypothalamus in the two genotypes. Differentially expressed transcripts were related to inflammation, taste, olfactory and melatonin receptors, lipid metabolism, cell cycle, ubiquitination, and hormones, as well as receptors and channels related to sleep regulation. Differentially expressed transcripts in both tissues co-localized with Per3 on an ∼8Mbp region of distal chromosome 4. The most down-regulated transcript is Prdm16, which is involved in adipocyte differentiation and may mediate altered body mass accumulation in Per3-/- mice. eQTL analysis with BXD mouse strains showed that the expression of some of these transcripts and also others co-localized at distal chromosome 4, is correlated with brain tissue expression levels of Per3 with a highly significant linkage to genetic variation in that region. These data identify a cluster of transcripts on mouse distal chromosome 4 that are co-regulated with Per3 and whose expression levels correlate with those of Per3. This locus lies within a topologically associating domain island that contains many genes with functional links to several of the diverse non-circadian phenotypes associated with polymorphisms in human PER3.

Circadian regulation in human white adipose tissue revealed by transcriptome and metabolic network analysis.

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled 'constant routine' conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

Identifying and validating blood mRNA biomarkers for acute and chronic insufficient sleep in humans: a machine learning approach.

Acute and chronic insufficient sleep are associated with adverse health outcomes and risk of accidents. There is therefore a need for biomarkers to monitor sleep debt status. None are currently available. We applied elastic net and ridge regression to transcriptome samples collected in 36 healthy young adults during acute total sleep deprivation and following 1 week of either chronic insufficient (<6 hr) or sufficient sleep (~8.6 hr) to identify panels of mRNA biomarkers of sleep debt status. The size of identified panels ranged from 9 to 74 biomarkers. Panel performance, assessed by leave-one-subject-out cross-validation and independent validation, varied between sleep debt conditions. Using between-subject assessments based on one blood sample, the accuracy of classifying "acute sleep loss" was 92%, but only 57% for classifying "chronic sleep insufficiency." A reasonable accuracy for classifying "chronic sleep insufficiency" could only be achieved by a within-subject comparison of blood samples. Biomarkers for sleep debt status showed little overlap with previously identified biomarkers for circadian phase. Biomarkers for acute and chronic sleep loss also showed little overlap but were associated with common functions related to the cellular stress response, such as heat shock protein activity, the unfolded protein response, protein ubiquitination and endoplasmic reticulum-associated protein degradation, and apoptosis. This characteristic response of whole blood to sleep loss can further aid our understanding of how sleep insufficiencies negatively affect health. Further development of these novel biomarkers for research and clinical practice requires validation in other protocols and age groups.

Phenotyping of PER3 variants reveals widespread effects on circadian preference, sleep regulation, and health.

Period3 (Per3) is one of the most robustly rhythmic genes in humans and animals. It plays a significant role in temporal organisation in peripheral tissues. The effects of PER3 variants on many phenotypes have been investigated in targeted and genome-wide studies. PER3 variants, especially the human variable number tandem repeat (VNTR), associate with diurnal preference, mental disorders, non-visual responses to light, brain and cognitive responses to sleep loss/circadian misalignment. Introducing the VNTR into mice alters responses to sleep loss and expression of sleep homeostasis-related genes. Several studies were limited in size and some findings were not replicated. Nevertheless, the data indicate a significant contribution of PER3 to sleep and circadian phenotypes and diseases, which may be connected by common pathways. Thus, PER3-dependent altered light sensitivity could relate to high retinal PER3 expression and may contribute to altered brain response to light, diurnal preference and seasonal mood. Altered cognitive responses during sleep loss/circadian misalignment and changes to slow wave sleep may relate to changes in wake/activity-dependent patterns of hypothalamic gene expression involved in sleep homeostasis and neural network plasticity. Comprehensive characterisation of effects of clock gene variants may provide new insights into the role of circadian processes in health and disease.

Prediction of Streptococcus uberis clinical mastitis risk using Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) in dairy herds.

The purpose of this study was to evaluate whether the risk of Streptococcus uberis clinical mastitis at cow level could be predicted from the historical presence of specific strains of S. uberis on dairy farms. Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to identify S. uberis isolates potentially capable of contagious transmission. Data were available from 10,652 cows from 52 English and Welsh dairy farms over a 14 month period, and 521 isolates of S. uberis from clinical mastitis cases were available for analysis. As well as the temporal herd history of clinical mastitis associated with particular S. uberis strains, other exposure variables included cow parity, stage of lactation, milk yield, and somatic cell count. Observations were structured longitudinally as repeated weekly measures through the study period for each cow. Data were analyzed in a Bayesian framework using multilevel logistic regression models. Similarity of mass spectral profiles between isolates of S. uberis from consecutive clinical cases of mastitis in herds was used to indicate potential for contagious phenotypic characteristics. Cross validation showed that new isolates with these characteristics could be identified with an accuracy of 90% based on bacterial protein mass spectral characteristics alone. The cow-level risk in any week of these S. uberis clinical mastitis cases increased with the presence of the same specific strains of S. uberis in other cows in the herd during the previous 2 weeks. The final statistical model indicated there would be a 2-3 fold increase in the risk of S. uberis clinical mastitis associated with particular strains if these occurred in the herd 1 and 2 weeks previously. The results suggest that specific strains of S. uberis may be involved with contagious transmission, and predictions based on their occurrence could be used as an early warning surveillance system to enhance the control of S. uberis mastitis.

Meal Timing Regulates the Human Circadian System.

Circadian rhythms, metabolism, and nutrition are intimately linked [1, 2], although effects of meal timing on the human circadian system are poorly understood. We investigated the effect of a 5-hr delay in meals on markers of the human master clock and multiple peripheral circadian rhythms. Ten healthy young men undertook a 13-day laboratory protocol. Three meals (breakfast, lunch, dinner) were given at 5-hr intervals, beginning either 0.5 (early) or 5.5 (late) hr after wake. Participants were acclimated to early meals and then switched to late meals for 6 days. After each meal schedule, participants' circadian rhythms were measured in a 37-hr constant routine that removes sleep and environmental rhythms while replacing meals with hourly isocaloric snacks. Meal timing did not alter actigraphic sleep parameters before circadian rhythm measurement. In constant routines, meal timing did not affect rhythms of subjective hunger and sleepiness, master clock markers (plasma melatonin and cortisol), plasma triglycerides, or clock gene expression in whole blood. Following late meals, however, plasma glucose rhythms were delayed by 5.69 ± 1.29 hr (p < 0.001), and average glucose concentration decreased by 0.27 ± 0.05 mM (p < 0.001). In adipose tissue, PER2 mRNA rhythms were delayed by 0.97 ± 0.29 hr (p < 0.01), indicating that human molecular clocks may be regulated by feeding time and could underpin plasma glucose changes. Timed meals therefore play a role in synchronizing peripheral circadian rhythms in humans and may have particular relevance for patients with circadian rhythm disorders, shift workers, and transmeridian travelers.

Blood transcriptome based biomarkers for human circadian phase.

Diagnosis and treatment of circadian rhythm sleep-wake disorders both require assessment of circadian phase of the brain's circadian pacemaker. The gold-standard univariate method is based on collection of a 24-hr time series of plasma melatonin, a suprachiasmatic nucleus-driven pineal hormone. We developed and validated a multivariate whole-blood mRNA-based predictor of melatonin phase which requires few samples. Transcriptome data were collected under normal, sleep-deprivation and abnormal sleep-timing conditions to assess robustness of the predictor. Partial least square regression (PLSR), applied to the transcriptome, identified a set of 100 biomarkers primarily related to glucocorticoid signaling and immune function. Validation showed that PLSR-based predictors outperform published blood-derived circadian phase predictors. When given one sample as input, the R2 of predicted vs observed phase was 0.74, whereas for two samples taken 12 hr apart, R2 was 0.90. This blood transcriptome-based model enables assessment of circadian phase from a few samples.