Professor Malcolm von Schantz

Chronotype or diurnal preference is a questionnaire-based measure influenced both by circadian period and by the sleep homeostat. In order to further characterize the biological determinants of these measures, we used a hypothesis-free approach to investigate the association between the score of the morningness-eveningness questionnaire (MEQ) and the Munich chronotype questionnaire (MCTQ), as continuous variables, and volumetric measures of brain regions acquired by magnetic resonance imaging (MRI). Data were collected from the Baependi Heart Study cohort, based in a rural town in South-Eastern Brazil. MEQ and anatomical 1.5-T MRI scan data were available from 410 individuals, and MCTQ scores were available from a subset of 198 of them. The average MEQ (62.2±10.6) and MCTQ (average MSFsc 201±85 min) scores were suggestive of a previously reported strong general tendency towards morningness in this community. Setting the significance threshold at P>0.002 to account for multiple comparisons, we observed a significant association between lower MEQ score (eveningness) and greater volume of the left anterior occipital sulcus (β=-0.163, p=0.001) of the occipital lobe. No significant associations were observed for MCTQ. This may reflect the smaller dataset for MCTQ, and/or the fact that MEQ, which asks questions about preferred timings, is more trait-like than the MCTQ, which asks questions about actual timings. The association between MEQ and a brain region dedicated to visual information processing is suggestive of the increasingly recognized fluidity in the interaction between visual and non-visual photoreception and the circadian system, and the possibility that chronotype includes an element of masking.

Circadian rhythm disturbances are frequently described in psychiatric disorders such as major depressive disorder, bipolar disorder, and schizophrenia. Growing evidence suggests a biological connection between mental health and circadian rhythmicity, including the circadian influence on brain function and mood and the requirement for circadian entrainment by external factors, which is often impaired in mental illness. Mental (as well as physical) health is also adversely affected by circadian misalignment. The marked interindividual differences in this combined susceptibility, in addition to the phenotypic spectrum in traits related both to circadian rhythms and mental health, suggested the possibility of a shared genetic background and that circadian clock genes may also be candidate genes for psychiatric disorders. This hypothesis was further strengthened by observations in animal models where clock genes had been knocked out or mutated. The introduction of genome-wide association studies (GWAS) enabled hypothesis-free testing. GWAS analysis of chronotype confirmed the prominent role of circadian genes in these phenotypes and their extensive polygenicity. However, in GWAS on psychiatric traits, only one clock gene, ARNTL (BMAL1) was identified as one of the few loci differentiating bipolar disorder from schizophrenia, and macaque monkeys where the ARNTL gene has been knocked out display symptoms similar to schizophrenia. Another lesson from genomic analyses is that chronotype has an important genetic correlation with several psychiatric disorders and that this effect is unidirectional. We conclude that the effect of circadian disturbances on psychiatric disorders probably relates to modulation of rhythm parameters and extend beyond the core clock genes themselves.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature compensated circadian rhythms which, since RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on casein kinases in RBCs. As with nucleated cell types, broad spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Individual variability in diurnal preference or chronotype is commonly assessed with selfreport 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 build-up 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 analysing answers to MEQ items that compose these latent factors, it may be possible to extract further knowledge of factors that affect morningness-eveningness.

The association between obstructive sleep apnea (OSA) and increased cardiometabolic risk (CMR) has been well documented in higher-income countries. However, OSA and its association with CMR have not yet been investigated, based on objective measures, in Southern Africa. We measured polysomnography (PSG)-derived sleep characteristics, OSA prevalence and its association with cardiometabolic diseases in a rural, low-income, aging African-ancestry sample in South Africa. Seventy-five participants were recruited. BMI, hypertension, diabetes, dyslipidaemia, and HIV status were determined. A continuous CMR score was calculated using waist circumference (WC), random glucose, HDL-cholesterol, triglycerides, and mean arterial blood pressure. Sleep architecture, arousal index, and apnea-hypopnea index (AHI) for detection of OSA (AHI≥15) were assessed by home-based PSG. Associations between CMR score and age, sex, socio-economic status (SES), AHI and TST were investigated by multivariable analysis. In our sample (53 women, 66.1±10.7y, 12 HIV+), 60.7% were overweight/obese, 61.3% hypertensive and 29.3% had undiagnosed OSA. Being older (p=0.02), having a greater BMI (p=0.02) and higher WC (p<0.01) were associated with OSA. AHI severity (ß=0.011p=0.01) and being a woman (ß=0.369, p=0.01) were independently associated with a greater CMR score in SES- and age-adjusted analyses. In this aging South African community with obesity and hypertension, OSA prevalence is alarming and associated with CMR. We demonstrate feasibility of detecting OSA in a rural setting using PSG. Our results highlight the necessity for actively promoting health education and systematic screening and treatment of OSA in this population, to prevent future cardiovascular morbidity, especially among women.

Context Metabolic syndrome (MetS) is a complex condition comprising a ‘clustering’ of components representing cardiometabolic risk factors for heart disease and diabetes; its prevalence rate is high and consequences serious. Evidence suggests that light exposure patterns and misalignment of circadian rhythms might contribute to MetS etiology by impacting energy metabolism and glucose regulation. Objective We hypothesised that individuals with MetS would show disrupted circadian and sleep parameters alongside differences in light exposure profiles. We investigated this using data from a cohort study in Brazil. Methods Data from 103 individuals from the Baependi Heart Cohort Study aged between 50 and 70 were analysed. Motor activity and light exposure were measured using wrist-worn actigraphy devices. Cardiometabolic data were used to calculate the number of MetS components present in each participant, and participants grouped as MetS/non-MetS according to standard guidelines. Between-group comparisons were made for the actigraphy measures; additionally, correlation analyses were conducted. Results Motor activity and circadian profiles showed no differences between groups. However, the MetS group presented lower light exposure during the day and higher light exposure at night. Correlation analyses, including all participants, showed that greater daytime light exposure and greater light exposure difference between day and night were associated with reduced MetS risk (a lower number of MetS components). Also, the light exposure difference between day and night correlated with body mass index across all participants. Conclusions The observed results suggest a direct association between light exposure and MetS which appears to not be attributable to disruptions in circadian activity rhythm nor to sleep parameters. This link between light exposure patterns and MetS risk could inform possible prevention strategies.

Industrialisation greatly increased human night-time exposure to artificial light, which in animal models is a known cause of depressive phenotypes. Whilst many of these phenotypes are ‘direct’ effects of light on affect, an ‘indirect’ pathway via altered sleep-wake timing has been suggested. We have previously shown that the Period3 gene, which forms part of the biological clock, is associated with altered sleep-wake patterns in response to light. Here, we show that both wild-type and Per3-/- mice showed elevated levels of circulating corticosterone and increased hippocampal Bdnf expression after 3 weeks of exposure to dim light at night, but only mice deficient for the PERIOD3 protein (Per3-/-) exhibited a transient anhedonia-like phenotype, observed as reduced sucrose preference, in weeks 2-3 of dim light at night, whereas WT mice did not. Per3-/- mice also exhibited a significantly smaller delay in behavioural timing than WT mice during weeks 1, 2 and 4 of dim light at night exposure. When treated with imipramine, neither Per3-/- nor WT mice exhibited an anhedonia-like phenotype, and neither genotypes exhibited a delay in behavioural timing in responses to dLAN. While the association between both Per3-/- phenotypes remains unclear, both are alleviated by imipramine treatment during dim night-time light.

Circadian organization of the mammalian transcriptome is achieved by rhythmic recruitment of key modifiers of chromatin structure and transcriptional and translational processes. These rhythmic processes, together with posttranslational modification, constitute circadian oscillators in the brain and peripheral tissues, which drive rhythms in physiology and behavior, including the sleep-wake cycle. In humans, sleep is normally timed to occur during the biological night, when body temperature is low and melatonin is synthesized. Desynchrony of sleep-wake timing and other circadian rhythms, such as occurs in shift work and jet lag, is associated with disruption of rhythmicity in physiology and endocrinology. However, to what extent mistimed sleep affects the molecular regulators of circadian rhythmicity remains to be established. Here, we show that mistimed sleep leads to a reduction of rhythmic transcripts in the human blood transcriptome from 6.4% at baseline to 1.0% during forced desynchrony of sleep and centrally driven circadian rhythms. Transcripts affected are key regulators of gene expression, including those associated with chromatin modification (methylases and acetylases), transcription (RNA polymerase II), translation (ribosomal proteins, initiation, and elongation factors), temperature-regulated transcription (cold inducible RNA-binding proteins), and core clock genes including CLOCK and ARNTL (BMAL1). We also estimated the separate contribution of sleep and circadian rhythmicity and found that the sleep-wake cycle coordinates the timing of transcription and translation in particular. The data show that mistimed sleep affects molecular processes at the core of circadian rhythm generation and imply that appropriate timing of sleep contributes significantly to the overall temporal organization of the human transcriptome.

Later chronotype (i.e. evening preference) and later timing of sleep have been associated with greater morbidity, including higher rates of metabolic dysfunction and cardiovascular disease. However, no one has examined whether chronotype is associated with mortality risk to date. Our objective was to test the hypothesis that being an evening type is associated with increased mortality in a large cohort study, the UK Biobank. Our analysis included 433,268 adults aged 38-73 at the time of enrolment and an average 6.5- year follow-up. The primary exposure was chronotype, as assessed through a single self-reported question defining participants as definite morning types, moderate morning types, moderate evening types, or definite evening types. The primary outcomes were all-cause mortality and mortality due to cardiovascular disease (CVD). Prevalent disease was also compared among the chronotype groups. Analyses were adjusted for age, sex, ethnicity, smoking, body mass index, sleep duration, socioeconomic status and comorbidities. Greater eveningness, particularly being a definite evening type, was significantly associated with a higher prevalence of all comorbidities. Comparing definite evening type to definite morning type, the associations were strongest for psychological disorders (OR 1.94, 95% CI 1.86 to 2.02, p=<.001), followed by diabetes (OR 1.30, 95% CI 1.24 to 1.36, p=<.001), neurological disorders (OR 1.25, 95% CI 1.20 to 1.30, p=<.001), gastrointestinal/ abdominal disorders (OR 1.23, 95% CI 1.19 to 1.27, p=<.001), and respiratory disorders (OR 1.22, 95% CI 1.18 to 1.26, p=<.001). The total number of deaths was 10,534, out of which 2,127 were due to CVD. Greater eveningness, based on chronotype as an ordinal variable, was associated with a small increased risk of all-cause mortality (HR 1.02, 95% CI 1.004 to 1.05, p=.017) and CVD mortality (HR 1.04, 95% CI 1.00 to 1.09, p=.06). Compared to definite morning types, definite evening types had significantly increased risk of all-cause mortality (HR 1.10, 95% CI 1.02 to 1.18, p=.012). This first report of increased mortality in evening types is consistent with previous reports of increased levels of cardiometabolic risk factors in this group. Mortality risk in evening types may be due to behavioural, psychological, and physiological risk factors, many of which may be attributable to chronic misalignment between internal physiological timing and externally imposed timing of work and social activities. These findings suggest the need for researching possible interventions aimed at either modifying circadian rhythms in individuals or at allowing evening types greater working hour flexibility.

Our own species has a diurnal activity pattern and an average circadian period of 24.2 hours. Exact determination of circadian period requires expensive and intrusive protocols, and investigators are therefore using chronotype questionnaires as a proxy quantitative measure. Both measures show a normal distribution suggestive of a polygenic trait. The genetic components of the 24-hour feedback loop that generates circadian rhythms within our cells have been mapped in detail, identifying a number of candidate genes which have been investigated for genetic polymorphisms relating to the phenotypic variance. Key in this mechanism is the inhibitory complex containing period and cryptochrome proteins and interacting protein kinases and ubiquitin ligases, and the stability of this complex is recognized as the major determinant of circadian periodicity. The identification of the causative mutations in familial circadian rhythms sleep disorders has shed additional light into this mechanism. Mutations in the negative feedback protein-encoding genes PER2 and CRY2 as well as the CSNK1D gene encoding casein kinase I delta have been shown to cause advanced sleep phase disorder, and a mutation in the CRY1 gene delayed sleep phase disorder. The candidate gene approach has also yielded a number of genetic associations with chronotype as determined by questionnaires. More recently, genome-wide association studies (GWAS) of chronotype have both confirmed associations with the candidate clock gene PER2 and identified a serious of novel genes associated with variability in circadian rhythmicity, which have yet to be explored. Whilst considerable progress has thus been made with mapping the phenotypic diversity in human circadian rhythms and the genomic variability that causes it, studies to date have been mostly focused on individuals of European descent, and there is a strong need for research on other populations.

The well-established negative health outcomes of sleep deprivation, and the suggestion that availability of electricity may enable later bed times without compensating sleep extension in the morning, have stimulated interest in studying communities whose sleep pattern may resemble a preindustrial state. Here, we describe sleep and activity in two neighbouring communities, one urban (Milange) and one rural (Tengua), in a region of Mozambique where urbanisation is an ongoing process. The two communities differ in the amount and timing of daily activity and of light exposure, with later bedtimes (≈1 h) associated with more evening and less daytime light exposure seen in the town of Milange. In contrast to previous reports comparing communities with and without electricity, sleep duration did not differ between Milange (7.28 h) and Tengua (7.23 h). Notably, calculated sleep quality was significantly poorer in rural Tengua than in Milange, and poor sleep quality was associated with a number of attributes more characteristic of rural areas, including more intense physical labour and less comfortable sleeping arrangements. Thus, whilst our data support the hypothesis that access to electricity delays sleep timing, the higher sleep quality in the urban population also suggests that some aspects of industrialisation are beneficial to sleep.

Significant questions remain unanswered regarding the genetic versus environmental contributions to racial/ethnic differences in sleep and circadian rhythms. We addressed this question by investigating the association between diurnal preference, using the Morningness-Eveningness questionnaire (MEQ), and genetic ancestry within the Baependi Heart Study cohort, a highly admixed Brazilian population based in a rural town. Analysis was performed using measures of ancestry, using the Admixture program, and MEQ from 1,453 individuals. We found an association between the degree of Amerindian (but not European of African) ancestry and morningness, equating to 0.16 units for each additional percent of Amerindian ancestry, after adjustment for age, sex, education, and residential zone. To our knowledge, this is the first published report identifying an association between genetic ancestry and MEQ, and above all, the first one based on ancestral contributions within individuals living in the same community. This previously unknown ancestral dimension of diurnal preference suggests a stratification between racial/ethnic groups in an as yet unknown number of genetic polymorphisms.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Sleep complaints and irregular sleep patterns, such as curtailed sleep during workdays and longer and later sleep during weekends, are common. It is often implied that differences in circadian period and in entrained phase contribute to these patterns, but few data are available. We assessed parameters of the circadian rhythm of melatonin at baseline and in a forced desynchrony protocol in 35 participants (18 women) with no sleep disorders. Circadian period varied between 23 h 50 min and 24 h 31 min, and correlated positively (n = 31, rs = 0.43, P = 0.017) with the timing of the melatonin rhythm relative to habitual bedtime. The phase of the melatonin rhythm correlated with the Insomnia Severity Index (n = 35, rs = 0.47, P = 0.004). Self-reported time in bed during free days also correlated with the timing of the melatonin rhythm (n = 35, rs = 0.43, P = 0.01) as well as with the circadian period (n = 31, rs = 0.47, P = 0.007), such that individuals with a more delayed melatonin rhythm or a longer circadian period reported longer sleep during the weekend. The increase in time in bed during the free days correlated positively with circadian period (n = 31, rs = 0.54, P = 0.002). Polysomnographically assessed latency to persistent sleep (n = 34, rs = 0.48, P = 0.004) correlated with the timing of the melatonin rhythm when participants were sleeping at their habitual bedtimes in the laboratory. This correlation was significantly stronger in women than in men (Z = 2.38, P = 0.017). The findings show that individual differences in circadian period and phase of the melatonin rhythm associate with differences in sleep, and suggest that individuals with a long circadian period may be at risk of developing sleep problems.

Circadian rhythms and sleep are two separate but intimately related processes. Circadian rhythms are generated through the precisely controlled, cyclic expression of a number of genes designated clock genes. Genetic variability in these genes has been associated with a number of phenotypic differences in circadian, but also in sleep parameters both in mouse models and in humans. Diurnal preferences, as determined by the self-reported Horne-Östberg questionnaire, has been associated with polymorphisms in the human genes CLOCK, PER1, PER2, and PER3. Circadian rhythms sleep disorders have also been associated with mutations and polymorphisms in clock genes, with the advanced type cosegrating in an autosomal dominant inheritance pattern with mutations in the genes PER2 and CSNK1D, and the delayed type associating without discernible Mendelian inheritance with polymorphisms in CLOCK and PER3. Several mouse models of clock gene null alleles have been demonstrated to have affected sleep homeostasis. Recent findings have shown that the variable number tandem polymorphism in PER3, previously linked to diurnal preference, has profound effects on sleep homeostasis and cognitive performance following sleep loss, confirming the close association between the processes of circadian rhythms and sleep on the genetic level.

In spite of suspected circadian differences between different ancestral groups, most human studies have used individuals of European descent. This also applies to three recent genome-wide association studies (GWAS), which pinpointed a number of chronotype loci. We investigated the distribution of these hits in different 1000 Genomes populations. We found six out of the 41 alleles previously identified by GWAS in European participants (in the genes RGS16, PER2, AK5, and between the genes APH1A and CA14) to be absent from some non-European population groups. This highlights the need for ancestral diversity in circadian research, and may reflect differences affecting the phenotype of individuals of East Asian ancestry.

Purpose Cardiovascular disease (CVD) is a major challenge to global health. The same epidemiological transition scenario is replayed as countries develop, but with variations based on environment, culture and ethnic mixture. The Baependi Heart Study was set up in 2005 to develop a longitudinal family-based cohort study that reflects on some of the genetic and lifestyle-related peculiarities of the Brazilian populations, in order to evaluate genetic and environmental influences on CVD risk factor traits. Participants Probands were recruited in Baependi, a small rural town in the state of Minas Gerais, Brazil, following by first-degree and then increasingly more distant relatives. The first follow-up wave took place in 2010, and the second in 2016. At baseline, the study evaluated 1691 individuals across 95 families. Cross-sectional data have been collected for 2239 participants. Findings to date Environmental and lifestyle factors and measures relevant to cardiovascular health have been reported. Having expanded beyond cardiovascular health outcomes, the phenotype datasets now include genetics, biochemistry, anthropometry, mental health, sleep and circadian rhythms. Many of these have yielded heritability estimates, and a shared genetic background of anxiety and depression has recently been published. In spite of universal access to electricity, the population has been found to be strongly shifted towards morningness compared with metropolitan areas. Future plans A new follow-up, marking 10 years of the study, is ongoing in 2016, in which data are collected as in 2010 (with the exception of the neuropsychiatric protocol). In addition to this, a novel questionnaire package collecting information about intelligence, personality and spirituality is being planned. The data set on circadian rhythms and sleep will be amended through additional questionnaires, actimetry, home sleep EEG recording and dim light melatonin onset (DLMO) analysis. Finally, the anthropometric measures will be expanded by adding three-dimensional facial photography, voice recording and anatomical brain MRI.

As part of the Health and Aging in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI), we investigated sleep habits and their interactions with HIV or noncommunicable diseases (NCDs) in 5059 participants (median age: 61, interquartile range: 52—71, 54% females). Self-reported sleep duration was 8.2±1.6h, and bed and rise times were 20:48±1:15 and 05:31±1:05 respectively. Ratings of insufficient sleep were associated with older age, lack of formal education, unemployment, and obesity (p<0.05). Ratings of restless sleep were associated with being older, female, having more education, being unemployed, and single. Hypertension was associated with shorter self-reported sleep duration, poor sleep quality, restless sleep, and periods of stopping breathing during the night (p<0.05). HIV positive individuals not on antiretroviral treatment (ART) reported more nocturnal awakenings than those on ART (p=0.029) and HIV negative individuals (p=0.024), suggesting a negative net effect of untreated infection, but not of ART, on sleep quality. In this cohort, shorter, poor-quality sleep was associated with hypertension, but average self-reported sleep duration was longer than reported in other regions globally. It remains to be determined whether this is particular to this cohort, South Africa in general, or low- to middle-income countries undergoing transition.

Study Objectives: To screen the PER3 promoter for polymorphisms and investigate the phenotypic associations of these polymorphisms with diurnal preference, delayed sleep phase disorder/syndrome (DSPD/DSPS), and their effects on reporter gene expression. Design: Interspecific comparison was used to define the approximate extent of the PER3 promoter as the region between the transcriptional start site and nucleotide position −874. This region was screened in DNA pools using PCR and direct sequencing, which was also used to screen DNA from individual participants. The different promoter alleles were cloned into a luciferase expression vector and a deletion library created. Promoter activation was measured by chemiluminescence. Setting: N/A Patients or Participants: DNA samples were obtained from volunteers with defined diurnal preference (3 x 80, selected from a pool of 1,590), and DSPD patients (n = 23). Interventions: N/A Measurements and Results: We verified three single nucleotide polymorphisms (G −320T, C −319A, G −294A), and found a novel variable number tandem repeat (VNTR) polymorphism (−318 1/2 VNTR). The −320T and −319A alleles occurred more frequently in DSPD compared to morning (P = 0.042 for each) or evening types (P = 0.006 and 0.033). The allele combination TA2G was more prevalent in DSPD compared to morning (P = 0.033) or evening types (P = 0.002). Luciferase expression driven by the TA2G combination was greater than for the more common GC2A (P < 0.05) and the rarer TA1G (P < 0.001) combinations. Deletion reporter constructs identified two enhancer regions (−703 to −605, and −283 to −80). Conclusions: Polymorphisms in the PER3 promoter could affect its expression, leading to potential differences in the observed functions of PER3.

Light affects the circadian axis in at least two ways. It can cause the acute suppression of pineal melatonin synthesis, and/or a phase-shift of the circadian oscillator. As recent evidence has suggested that extraocular light exposure may cause phase-shifts of the circadian clock, we have investigated whether suppression of melatonin can be induced by the same type of light exposure. In the first study subjects’ eyes were exposed to white light (2250 lux for 30 min) via a fibre optic cable. As expected, suppression of nighttime plasma melatonin levels (61 ± 6%) was observed. In the second study, light of the same quality but higher intensity (14,000 or 67,500 lux for 180 mins) was delivered in the same manner to the popliteal region behind the subjects’ knees, whilst shielding their eyes. No suppression of plasma melatonin levels (4 ± 7%) was detected in any of the subjects. Thus, extraocular photoreception, if it exists in mammals, does not affect the suprachiasmatic nuclei-pineal pathway.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. By using electrophysiological and pharmacological approaches, we show that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Sleep is modulated by several factors, including sex, age, and chronotype. It has been hypothesised that contemporary urban populations are under pressure towards shorter sleep duration and poorer sleep quality. Baependi is a small town in Brazil that provides a window of opportunity to study the influence of sleep patterns in a highly admixed rural population with a conservative lifestyle. We evaluated sleep characteristics, excessive daytime sleepiness, and chronotype using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale and Morningness-Eveningness Questionnaire questionnaires, respectively. The sample consisted of 1,334 subjects from the Baependi Heart study (41.5% male; age: 46.5±16.2 y, range: 18—89 years). Average self-reported sleep duration was 07:07±01:31 (bedtime 22:32±01:27, wake up time: 06:17±01:25 hh:min), sleep quality score was 4.9+3.2, chronotype was 63.6±10.8 and daytime sleepiness was 7.4±4.8. Despite a shift towards morningness in the population, chronotype remained associated with reported actual sleep timing. Age and sex modulated the ontogeny of sleep and chronotype, increasing age was associated with earlier sleep time and shorter sleep duration. Women slept longer and later, and reported poorer sleep quality than men (p<0.0001). This study provides indirect evidence in support of the hypothesis that sleep timing was earlier prior to full urbanisation.

Studying communities at different stages of urbanisation and industrialisation can teach us how timing and intensity of light affects the circadian clock under real-life conditions. We have previously described a strong tendency towards morningness in the Baependi Heart Study, located in a small rural town in Brazil. Here, we tested the hypothesis that this morningness tendency is associated with early circadian phase based on objective measurements (as determined by dim light melatonin onset, DLMO, and activity) and light exposure. We also analysed how well the previously collected chronotype questionnaire data was able to predict these DLMO values. The average DLMO observed in 73 participants (40 female) was 20:03±01:21, SD, with an earlier average onset in men (19:38±01:16) than in women (20:24±01:21; p≤0.01). However, men presented larger phase angle between DLMO and sleep onset time as measured by actigraphy (4.11 hours vs 3.16 hours; p≤0.01). Correlational analysis indicated associations between light exposure, activity rhythms, and DLMO, such that early DLMO was observed in participants with higher exposure to light, higher activity and earlier light exposure. The strongest significant predictor of DLMO was morningness-eveningness questionnaire (MEQ) (beta=-0.35, p≤0.05), followed by age(beta=-0.47, p≤0.01). Sex, light exposure, and variables derived from the Munich Chronotype Questionnaire were not significant predictors. Our observations demonstrate that both early sleep patterns and earlier circadian phase have been retained in this small rural town in spite of availability of electrification, in contrast to metropolitan post-industrial areas.

Individual variability in word generation is a product of genetic and environmental influences. The genetic effects on semantic verbal fluency were estimated in 1,735 participants from the Brazilian Baependi Heart Study. The numbers of exemplars produced in 60 s were broken down into time quartiles because of the involvement of different cognitive processes — predominantly automatic at the beginning, controlled/executive at the end. Heritability in the unadjusted model for the 60-s measure was 0.32. The best-fit model contained age, sex, years of schooling, and time of day as covariates, giving a heritability of 0.21. Schooling had the highest moderating effect. The highest heritability (0.17) was observed in the first quartile, decreasing to 0.09, 0.12, and 0.0003 in the following ones. Heritability for average production starting point (intercept) was 0.18, indicating genetic influences for automatic cognitive processes. Production decay (slope), indicative of controlled processes, was not significant. The genetic influence on different quartiles of the semantic verbal fluency test could potentially be exploited in clinical practice and genome-wide association studies.

In recent years, strong evidence has emerged suggesting that insufficient duration, quality, and/or timing of sleep are associated with cardiovascular disease (CVD), and various mechanisms for this association have been proposed. Such associations may be related to endophenotypic features of the sleep homeostat and the circadian oscillator, or may be state-like effects of the environment. Here, we review recent literature on sleep, circadian rhythms and CVD with a specific emphasis on differences between racial/ethnic groups. We discuss the reported differences, mainly between individuals of European and African descent, in parameters related to sleep (architecture, duration, quality) and circadian rhythms (period length and phase shifting). We further review racial/ethnic differences in cardiovascular disease and its risk factors, and develop the hypothesis that racial/ethnic health disparities may, to a greater or smaller degree, relate to differences in parameters related to sleep and circadian rhythms. When humans left Africa some 100,000 years ago, some genetic differences between different races/ethnicities were acquired. These genetic differences have been proposed as a possible predictor of CVD disparities, but concomitant differences in culture and lifestyle between different groups may equally explain CVD disparities. We discuss the evidence for genetic and environmental causes of these differences in sleep and circadian rhythms, and their usefulness as health intervention targets.

Sleep complaints and irregular sleep patterns, such as curtailed sleep during workdays and longer and later sleep during weekends are common. It is often implied that differences in circadian period and in entrained phase contribute to these patterns but few data are available. We assessed parameters of the circadian rhythm of melatonin at baseline and in a forced desynchrony protocol in 35 participants (18 women) with no sleep disorders. Intrinsic circadian period varied between 23h50min and 24h31min and correlated positively (n=31, rs=0.43, P=0.017) with the timing of the melatonin rhythm relative to habitual bedtime. This phase of the melatonin rhythm correlated with the insomnia severity score (n=35, rs=0.47, P=0.004). Self-reported time in bed (TIB) during free days also correlated with the timing of the melatonin rhythm (n=35, rs=0.43, P=0.01) as well as with circadian period (n=31, rs=0.47, P=0.007) such that individuals with a more delayed melatonin rhythm or a longer circadian period reported longer sleep during the weekend. The increase in TIB during the free days correlated positively with circadian period (n=31, rs=0.54, P=0.002). Polysomnographically-assessed latency to persistent sleep (n=34, rs=0.48, P=0.004) correlated with the timing of the melatonin rhythm when participants were sleeping at their habitual bedtimes in the laboratory. This correlation was significantly stronger in women than in men (Z=2.38, P=0.017). The findings show that individual differences in period and phase of the circadian melatonin rhythm associate with differences in sleep and imply that individuals with a long circadian period are at risk of developing sleep problems.

Cognitive performance deteriorates during extended wakefulness and circadian phase misalignment, and some individuals are more affected than others. Whether performance is affected similarly across cognitive domains, or whether cognitive processes involving Executive Functions are more sensitive to sleep and circadian misalignment than Alertness and Sustained Attention, is a matter of debate.

Insufficient sleep and circadian rhythm disruption are associated with negative health outcomes, including obesity, cardiovascular disease, and cognitive impairment, but the mechanisms involved remain largely unexplored. Twenty-six participants were exposed to 1 wk of insufficient sleep (sleep-restriction condition 5.70 h, SEM = 0.03 sleep per 24 h) and 1 wk of sufficient sleep (control condition 8.50 h sleep, SEM = 0.11). Immediately following each condition, 10 whole-blood RNA samples were collected from each participant, while controlling for the effects of light, activity, and food, during a period of total sleep deprivation. Transcriptome analysis revealed that 711 genes were up- or down-regulated by insufficient sleep. Insufficient sleep also reduced the number of genes with a circadian expression profile from 1,855 to 1,481, reduced the circadian amplitude of these genes, and led to an increase in the number of genes that responded to subsequent total sleep deprivation from 122 to 856. Genes affected by insufficient sleep were associated with circadian rhythms (PER1, PER2, PER3, CRY2, CLOCK, NR1D1, NR1D2, RORA, DEC1, CSNK1E), sleep homeostasis (IL6, STAT3, KCNV2, CAMK2D), oxidative stress (PRDX2, PRDX5), and metabolism (SLC2A3, SLC2A5, GHRL, ABCA1). Biological processes affected included chromatin modification, gene-expression regulation, macromolecular metabolism, and inflammatory, immune and stress responses. Thus, insufficient sleep affects the human blood transcriptome, disrupts its circadian regulation, and intensifies the effects of acute total sleep deprivation. The identified biological processes may be involved with the negative effects of sleep loss on health, and highlight the interrelatedness of sleep homeostasis, circadian rhythmicity, and metabolism.

Cardiometabolic risk factors influence white matter hyperintensity (WMH) development: in metabolic syndrome (MetS), higher WMH load is often reported but the relationships between specific cardiometabolic variables, WMH load and cognitive performance are uncertain. We investigated these in a Brazilian sample (aged 50-85) with (N=61) and without (N=103) MetS. Stepwise regression models identified effects of cardiometabolic and demographic variables on WMH load (from FLAIR MRI) and verbal recall performance. WMH volume was greater in MetS, but verbal recall performance was not impaired. Age showed the strongest relationship with WMH load. Across all participants, systolic blood pressure (SBP) and fasting blood glucose were also contributors, and WMH volume was negatively associated with verbal recall performance. In non-MetS, higher HbA1c, SBP, and number of MetS components were linked to poorer recall performance while higher triglyceride levels appeared to be protective. In MetS only, these relationships were absent but education exerted a strongly protective effect on recall performance. Thus, results support MetS as a construct: the clustering of cardiometabolic variables in MetS alters their individual relationships with cognition; instead, MetS is characterised by a greater reliance on cognitive reserve mechanisms. In non-MetS, strategies to control HbA1c and SBP should be prioritised as these have the largest impact on cognition.

Study Objectives: Individual sleep timing differs and is governed partly by circadian oscillators, which may be assessed by hormonal markers, or by clock gene expression. Clock gene expression oscillates in peripheral tissues, including leukocytes. The study objective was to determine whether the endogenous phase of these rhythms, assessed in the absence of the sleep-wake and light-dark cycle, correlates with habitual sleep-wake timing. Design: Observational, cross-sectional. Setting: Home environment and Clinical Research Center. Participants: 24 healthy subjects aged 25.0 ± 3.5 (SD) years. Measurements: Actigraphy and sleep diaries were used to characterize sleep timing. Circadian rhythm phase and amplitude of plasma melatonin, cortisol, and BMAL1, PER2, and PER3 expression were assessed during a constant routine. Results: Circadian oscillations were more robust for PER3 than for BMAL1 or PER2. Average peak timings were 6:05 for PER3, 8:06 for PER2, 15:06 for BMAL1, 4:20 for melatonin, and 10:49 for cortisol. Individual sleep-wake timing correlated with the phases of melatonin and cortisol. Individual PER3 rhythms correlated significantly with sleep-wake timing and the timing of melatonin and cortisol, but those of PER2 and BMAL1 did not reach significance. The correlation between sleep timing and PER3 expression was stronger in individuals homozygous for the variant of the PER3 polymorphism that is associated with morningness. Conclusions: Individual phase differences in PER3 expression during a constant routine correlate with sleep timing during entrainment. PER3 expression in leukocytes represents a useful molecular marker of the circadian processes governing sleep-wake timing.

In humans, little is known about the entrainment of peripheral clocks by environmental cues or the circadian transcriptome of peripheral tissues. Meal timing entrains peripheral clocks rhythms of rodents but the effect of this on the human circadian system is unknown. It was hypothesised that meal timing would phase shift peripheral clock rhythms, but not master clock markers. Also hypothesised was that the transcriptome of subcutaneous adipose tissue would be under circadian regulation. Healthy male participants underwent two, separate clinical trials; one gave a food pulse containing 50% of the daily energetic need in one meal during a 4-hour ultradian light/dark cycle; another gave three isocaloric meals at 5-hourly intervals beginning at 0.5 then 5.5 hours after wake under a fixed light/dark cycle. All circadian rhythms were assessed before and after interventions, under constant routine conditions. Master clock marker, melatonin, was not significantly phase shifted by meal timing, as hypothesised. Plasma glucose and leptin rhythms showed large phase shifts in response to meal timing. Plasma triglycerides were minimally phase shifted by food pulse, but not by a change to meal schedule. A 5-hour delay in three isocaloric meals caused approximately a 1-hour delay in clock gene expression in serial adipose biopsies (PER2, PER3) but no shift in expression in whole blood (PER3, REVERB-β). Subcutaneous adipose biopsies taken under controlled conditions revealed that 1% of the transcriptome was circadian, with bimodal distribution of morning and evening peak times. Gene ontology enrichment analysis identified evening peaking probes as primarily involved in lipid metabolism. Morning peaking probes were involved in circadian rhythms and transcriptional regulation. These results demonstrate for the first time that meal timing differentially affects some peripheral, but not central, components of the human circadian system and that key metabolic processes are under circadian variation in the human adipose tissue transcriptome.