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CpG island prediction software, MethPrimer (www.urogene.org), identifies CpG islands in the 5' regulatory sequences of circadian clock genes, CLOCK, RORα, CRY1 and PER2. The regions from -2000 to +2000 were analysed, relative to the transcription start site of +1. In the linear scale depicted, 0 refers to 2000 bases upstream and 4000 refers to 2000 bases downstream of the transcription start site.

Supplementary figure legends

Correlations between gene expression fold changes (R values are shown)

OBJECTIVES: To investigate associations between self-reported sleep duration and cardiometabolic (CM) risk factors in African-origin adults residing in five countries spanning the epidemiologic transition. DESIGN: Cross-sectional SETTING AND PARTICIPANTS: Ghanaian (n=491), South African (n=503), Jamaican (n=508), Seychellois (n=501) and American (n=480) men and women. MEASUREMENTS: Self-reported sleep duration was obtained using questionnaires. Sex and sitestratified logistic regression analyses investigated relationships between sleep duration, individual CM risk factors and a binary CM risk variable (presence of ≥3 CM risk factors), adjusting for age, physical activity and education. RESULTS: Sleep duration distributions varied by cohort: 44.5%, 41.4%, 35.9%, 16.8% and 2.5% of American, Jamaican, Seychellois, Ghanaian and South African men reported

During lockdowns associated with the COVID-19 pandemic, individuals have experienced changes in sleep and lifestyle behaviours, and heightened depression and anxiety. However, the inter-relationship and relative strength of those behaviours on mental health outcomes is still unknown. Using structural equation modelling, we investigated how insomnia symptoms, sleep regularity, exercise intensity/frequency and sitting/screen-use (sedentary screen-use) interacted to predict depressive and anxiety-related symptoms before and during lockdown in 1048 South African adults (median age: 27y; n = 767 female; n = 473 students). We also controlled for the effects of sex and student status. Irrespective of lockdown, (a) more severe symptoms of insomnia and greater sedentary screen-use predicted greater symptoms of depression and anxiety and (b) the effects of sedentary screen-use on mental health outcomes were mediated by insomnia. The effects of physical activity on mental health outcomes, however, were only significant during lockdown. Low physical activity was associated with greater insomnia symptom severity, which in turn was associated with increased depressive and anxiety-related symptoms. Overall, relationships between the study variables and mental health outcomes were amplified during lockdown. The findings highlight the importance of maintaining physical activity and reducing sedentary screen-use to promote better sleep and mental health.

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 non-communicable 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

Diurnal preference, or chronotype, determined partly by genetics and modified by age, activity, and the environment, defines the time of day at which one feels at his/her best, when one feels sleepy, and when one would prefer to start his/her day. Chronotype affects the phase relationship of an individual's circadian clock with the environment such that morning types have earlier-phased circadian rhythms than evening types. The phases of circadian rhythms are synchronized to the environment on a daily basis, undergoing minor adjustments of phase each day. Light is the most potent time cue for phase-shifting circadian rhythms, but the timing and amount of solar irradiation vary dynamically with season, especially with increasing distance from the equator. There is evidence that chronotype is modified by seasonal change, most likely due to the changes in the light environment, but interindividual differences in photoperiod responsiveness mean that some people are more affected than others. Differences in circadian light sensitivity due to endogenous biological reasons and/or previous light history are responsible for the natural variation in photoperiod responsiveness. Modern lifestyles that include access to artificial light at night, temperature-controlled environments, and spending much less time outdoors offer a buffer to the environmental changes of the seasons and may contribute to humans becoming less responsive to seasons.

There is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study, the circadian clock was investigated in cervical and esophageal cancers, to determine whether it is disrupted in these cancer types. Oncomine datamining revealed downregulation of multiple members of the circadian clock gene family in cancer patient tissue compared with matched normal epithelium. Real-time RT-PCR analysis confirmed significant downregulation of CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBα, and RORα in esophageal tumor tissue. In cell line models, expression of several circadian clock genes was significantly decreased in transformed and cancer cells compared with noncancer controls, and protein levels were dysregulated. These effects were mediated, at least in part, by methylation, where CLOCK, CRY1, and RORα gene promoter regions were found to be methylated in cancer cells. Overexpression of CLOCK and PER2 in cancer cell lines inhibited cell proliferation and activation of RORα and REV-ERBα using agonists resulted in cancer cell death, while having a lesser effect on normal epithelial cells. Despite dysregulated circadian clock gene expression, cervical and esophageal cancer cells maintain functional circadian oscillations after Dexamethasone synchronization, as revealed using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are intact. Implications: This study is a first to describe dysregulated, yet oscillating, circadian clock gene expression in cervical and esophageal cancer cells, and knowledge of circadian clock functioning in these cancer types has the potential to inform chronotherapy approaches, where the timing of administration of chemotherapy is optimized on the basis of the circadian clock.

Introduction Sleep interacts with both infectious diseases and NCDs, but is a frequently neglected aspect of health and well-being. In South Africa, both NCD and HIV are highly prevalent, making the study of the association of sleep disorders with this double epidemic an imperative. Methods We investigated sleep habits and their interactions with HIV and NCDs in 5059 individuals 40 years and above from the study ‘Health and Aging in Africa: a Longitudinal Study of an INDEPTH community’ in Agincourt, South Africa. We collected sociodemographic data, anthropometric measurements, and blood pressure, and tested for glucose, haemoglobin and HIV. Major individual components of the Pittsburgh Sleep Quality index (PSQI) questionnaire were analysed, in addition to other questions on health and well-being. Sex comparisons were carried out using an independent t-test, a Mann-Whitney U test or a Fisher’s Exact test. The association of demographic variables and sleep parameters were measured using logistic regression. Results Self-reported sleep duration was 8.2±1.6h. Insufficient sleep associated with age, education, unemployment, and obesity. Restless sleep associated with age, education, unemployment, and being female and single table 1. Obesity associated with self-reported bedtime, wake time, insufficient sleep, snoring/gasping/periods of stopping breathing during sleep. Hypertension associated with shorter sleep duration, poor sleep quality, restless sleep, and sleep apnoea symptoms. Diabetes associated with bedtime, restless sleep, and snoring table 2. HIV+ individuals not on anti-retroviral therapy (ART) reported more nocturnal awakenings than those on ART (p=0.029) and HIV- individuals (p=0.024). Discussion These data provide a basis for further studies of the relationship between sleep and other risk factors. It also provides valuable data about sleep habits in Africa, with potential for future analysis of how it is affected by urbanisation and industrialisation. Finally, it offers a unique opportunity for a population-based comparison of the effects of treated and untreated HIV infection, increasingly unavailable elsewhere.

Studies have shown bidirectional relationships between short- or long-sleep duration and risk for obesity, non-communicable diseases, all-cause mortality and cardiovascular disease mortality. Increasing sleep duration may be an appropriate strategy to reduce cardiometabolic risk in short-sleeping individuals. The aim is to review the effects of sleep extension interventions on cardiometabolic risk in adults. The PubMed and Scopus databases were searched for relevant, English, peer-reviewed scientific publications (until August 2018). Seven studies that aimed to increase sleep duration in adults by any sleep extension intervention and described at least one cardiometabolic risk factor were included. These studies had a combined sample size of 138 participants who were either healthy (n = 14), healthy short-sleeping (n = 92), overweight short-sleeping (n = 10), or pre- or hypertensive short-sleeping (n = 22) individuals. The durations of the sleep extension interventions ranged from 3 days to 6 weeks, and all successfully increased total sleep time by between 21 and 177 min. Sleep extension was associated with improved direct and indirect measures of insulin sensitivity, decreased leptin and peptide tyrosine-tyrosine, and reductions in overall appetite, desire for sweet and salty foods, intake of daily free sugar, and percentage of daily caloric intake from protein. This review provides preliminary evidence for a role for sleep extension to improve cardiometabolic outcomes and directive towards future studies in the field of cardiometabolic health and sleep.

Proteas are woody perennial plants that develop large, complex inflorescences which are popular as cut flowers. Four paralogous genes with sequence similarity to the meristem identity gene LEAFY (LFY) of Arabidopsis thaliana were identified in the Protea cultivar ‘Carnival’ (Protea compacta x Protea neriifolia). The deduced amino acid sequences of Protea LFY (ProLFY) paralogues displayed highly conserved N- and C-terminal regions comparable to that of other LFY proteins. Paralogous sequences shared 97–99% similarity at nucleotide and deduced amino acid levels. To determine if these ProLFY paralogues are involved in floral transition and development, three paralogues were heterologously expressed in A. thaliana Col-0 wild-type and lfy-2 mutant plants. These three paralogues were chosen as they had the greatest sequence dissimilarities which may suggest a different function. Expression of the ProLFY-WL4 paralogue rescued the low fertility of the lfy-2 mutant by restoring seed yield to wild-type levels, and resulted in the conversion of ‘leaf-like’ inflorescences to flowers with a wild-type appearance. ProLFY-WL4 expression levels during ‘Carnival’ meristem development confirmed a role for this paralogue in inflorescence development. The other two paralogues used in heterologous A. thaliana expression studies, ProLFY-L5 and ProLFY-D1, did not rescue fertility or the abnormal floral phenotype of lfy-2. However, transgenic plants developed either smaller leaves when expressing 35S::ProLFY-L5, or larger leaves when expressing 35S::ProLFY-D1, compared to the non-transgenic lfy-2 plants. Together these results suggest that ProLFY paralogues may be involved in both floral and vegetative development of Protea.

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 pre-industrial 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.

There is a perception that time spent asleep is time wasted. Anecdotally one hears of people sleeping no more than 3–4 h per night with no apparent ill effects. As tempting as a short sleep regime sounds, we know that sleep is critical for survival. In 1983 Rechtschaffen and colleagues showed that rats developed pathology and died within 14 to 21 days of total sleep deprivation. 1 Data accumulated over the past 40 years from prospective cohort studies indicate higher all-cause mortality rates among people who sleep either less than 6 h or more than 9 h per night. 2 We also know that sleep loss affects neurobehavioural performance, metabolism and obesity, and psychological health. 3 (Full text available online at www.medpharm.tandfonline.com/oemd ) Journal of Endocrinology, Metabolism and Diabetes of South Africa 2016; DOI: 10.1080/16089677.2016.1150574

The new strategy to initiate the induction of bone formation is to carve smart, self-inducing geometric cues assembled within biomimetic medical devices. These are endowed with the striking prerogative of differentiating myoblastic and/or pericytic stem cells into osteoblastic-like cells attached to the morphogenetic concavities; osteoblastic-like cells secrete osteogenic gene products of the TGF-beta supergene family, further differentiating invading stem cells into osteoblastic-like cells, and initiating bone formation by induction as a secondary response.

Objectives: Four adult non-human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor-b2 (hTGF-b2) together with muscle-derived stem cells. Materials and methods: The hTGF-b2 was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein1, bone morphogenetic protein-3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Results: Heterotopic intramuscular implantation of 5 and 25 lg hTGF-b2 combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. Conclusions :I nPapio ursinus, in marked contrast to rodents and lagomorphs, hTGF-b2 induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF-b2. Induction of bone formation by hTGF-b2 in the nonhuman primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF-b2; the bone induction cascade initiated by mammalian TGF-b proteins in Papio ursinus needs to be re-evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.

Xerophyta humilis is a monocotyledonous resurrection plant found in arid and semi-arid summer rainfall areas of Southern Africa, which undergoes desiccation to survive periods of extreme drought. In order for X.humilis to thrive in their natural habitat, correct timing of the floral transition, coincident with wet periods of sufficient duration, is essential. In this study, the environmental cues involved in the regulation of the floral transition in X.humilis were analysed. No single parameter tested was sufficient to induce flowering, but it was found that flowering was promoted by a combination of a cool period experienced while plants were hydrated, followed by transfer to long-day photoperiods of relatively high light intensity. Plants retained competence to flower if desiccated during exposure to cold, but no flowering occurred if dried prior to this exposure. These data suggest that exposure to cold temperature facilitates vernalisation and subsequent exposure to high light and long days are inductive for floral initiation in X. humilis.

The Mesembryanthemoideae and Ruschioideae subfamilies are a major component of the Greater Cape Floristic Region in southern Africa. The Ruschioideae show an astonishing diversity of leaf shape and growth forms. Although 1,585 species are recognised within the morphologically diverse Ruschioideae, these species show minimal variation in plastid DNA sequence. We have investigated whether changes in selected leaf development transcription factors underpin the recent, rapid diversification of this large group of succulent plants. Degenerate primers designed to conserved regions of Asymmetric Leaves1/Rough Sheath 2/Phantastica (ARP) and the Class III HD-ZIP family of genes, were used to amplify sequences corresponding to these genes from several species within the Mesembryanthemoideae and Ruschioideae subfamilies. Two members of the Class III HD-ZIP family were identified in both the Mesembryanthemoideae and Ruschioideae, and were derived from an ancient gene duplication event that preceded the divergence of gymnosperms and angiosperms. While a single ARP orthologue was identified in the Mesembryanthemoideae, two paralogues, ARPa and ARPb, were identified in the Ruschioideae subfamily. ARPa was present in all species of Ruschioideae analysed in this study. ARPb has been lost from the Apatesieae and Dorotheantheae tribes, which form an early evolutionary branch from the Ruschieae tribe, as well as from selected species within the Ruschieae. The recent duplication and subsequent selected gene loss of the ARP transcription factor correlates with the rapid diversification of plant forms in the Ruschioideae.

Summary The circadian clock, an internal time‐keeping mechanism, allows plants to anticipate regular changes in the environment, such as light and dark, and biotic challenges such as pathogens and herbivores. Here, we demonstrate that the plant circadian clock influences susceptibility to the necrotrophic fungal pathogen, Botrytis cinerea. Arabidopsis plants show differential susceptibility to B. cinerea depending on the time of day of inoculation. Decreased susceptibility after inoculation at dawn compared with night persists under constant light conditions and is disrupted in dysfunctional clock mutants, demonstrating the role of the plant clock in driving time‐of‐day susceptibility to B. cinerea. The decreased susceptibility to B. cinerea following inoculation at subjective dawn was associated with faster transcriptional reprogramming of the defence response with gating of infection‐responsive genes apparent. Direct target genes of core clock regulators were enriched among the transcription factors that responded more rapidly to infection at subjective dawn than subjective night, suggesting an influence of the clock on the defence‐signalling network. In addition, jasmonate signalling plays a crucial role in the rhythmic susceptibility of Arabidopsis to B. cinerea with the enhanced susceptibility to this pathogen at subjective night lost in a jaz6 mutant., Significance Statement The circadian clock impacts plant metabolism and responses to the environment, including host susceptibility to pathogens. Here we demonstrate that Arabidopsis is less susceptible to fungal inoculation at dawn, because the defence transcriptional network is more rapidly activated. Genes differentially expressed at dawn and night were highly enriched for known direct targets of core clock transcription factors, suggesting direct links between the clock and the defence regulatory network.

The importance of light with respect to the outcome of plant-pathogen interactions is becoming increasingly evident: light affects both the host response and the virulence of some pathogens. The response of plants to environmental signals and stresses is modulated by the circadian clock, and it is apparent that this may include immune responses. Photo and temporal regulation of immune responses may allow plants to anticipate and react more effectively to particular pathogen infections. These aspects of regulation are sometimes overlooked when designing experiments to understand plant-pathogen interactions, complicating the interpretation of the outcomes and the direct comparisons of studies. We review recent key findings in these areas and discuss the implications for experimental design and analyses.

Humans have evolved in a rhythmic environment and display daily (circadian) rhythms in physiology, metabolism and behaviour that are in synchrony with the solar day. Modern lifestyles have compromised the exposure to bright light during the day and dark nights, resulting in the desynchronisation of endogenously generated circadian rhythms from the external environment and loss of coordination between rhythms within the body. This has detrimental effects on physical and mental health, due to the misregulation and uncoupling of important cellular and physiological processes. Long-term shift workers who are exposed to bright light at night experience the greatest disruption of their circadian rhythms. Studies have shown an association between exposure to light at night, circadian rhythm disruption and an increased risk of cancer. Previous reviews have explored the relevance of light and melatonin in cancer, but here we explore the correlation of circadian rhythm disruption and cancer in terms of molecular mechanisms affecting circadian gene expression and melatonin secretion.

The spontaneous induction of bone formation in heterotopic rectus abdominis and orthotopic calvarial sites by coral-derived biomimetic matrices of different chemical compositions was investigated in a long-term study in the non-human primate Papio ursinus. Coral-derived calcium carbonate constructs were converted to hydroxyapatite by hydrothermal exchange. Limited conversion produced hydroxyapatite/calcium carbonate (HA/CC) constructs of 5% and 13% hydroxyapatite. Rods of 20 mm in length and 7 mm in diameter were implanted in heterotopic rectus abdominis sites; discs 25 mm in diameter were implanted in orthotopic calvarial defects of six adult non-human primates P. ursinus. Heterotopic samples also included fully converted hydroxyapatite replicas sintered at 1100 °C. To further enhance spontaneous osteoinductive activity, fully converted hydroxyapatite replicas were coated with the synthetic peptide P15 known to increase the adhesion of fibroblasts to anorganic bovine mineral. Bone induction was assessed at 60, 90 and 365 days by histological examination, alkaline phosphatase and osteocalcin expression, as well as by the expression of BMP-7, GDF-10 and collagen type IV mRNAs. Induction of bone occurred in the concavities of the matrices at all time points. At 365 days, bone marrow was evident in the P15-coated and uncoated implants. Resorption of partially converted calcium carbonate/hydroxyapatite was apparent, as well as remodeling of the newly formed bone. Northern blot analyses of samples from heterotopic specimens showed high levels of expression of BMP-7 and collagen type IV mRNA in all specimen types at 60 days, correlating with the induction of the osteoblastic phenotype in invading fibrovascular cells. Orthotopic specimens showed prominent bone formation across the different implanted constructs. The concavities of the matrices biomimetize the remodeling cycle of the osteonic primate cortico-cancellous bone and promote the ripple-like cascade of the induction of bone formation. This study demonstrates for the first time that partially converted HA/CC constructs also induce spontaneous differentiation of bone, albeit only seen one year post-implantation.

Daylength, or photoperiod, is perceived as a seasonal signal for the control of flowering of many plants. The measurement of daylength is thought to be mediated through the interaction of phototransduction pathways with a circadian rhythm, so that flowering is induced (in long-day plants) or repressed (in short-day plants) when light coincides with a sensitive phase of the circadian cycle. To test this hypothesis in the facultative long-day plant, Arabidopsis thaliana , we used varying, non-24-hr light/dark cycles to alter the timing of circadian rhythms of gene expression relative to dawn and dusk. Effects on circadian rhythms were correlated with those on flowering times. We show that conditions that displaced subjective night events, such as expression of the flowering time regulator CONSTANS into the light portion of the cycle, were perceived as longer days. This work demonstrates that the perception of daylength in Arabidopsis relies on adjustments of the phase angle of circadian rhythms relative to the light/dark cycle, rather than on the measurement of the absolute duration of light and darkness.

Molecular models have been described for the circadian clocks of representatives of several different taxa. Much of the work on the plant circadian system has been carried out using the thale cress,Arabidopsis thaliana, as a model. We discuss the roles of genes implicated in the plant circadian system, with special emphasis onArabidopsis. Plants have an endogenous clock that regulates many aspects of circadian and photoperiodic behaviour. Despite the discovery of components that resemble those involved in the clocks of animals or fungi, no coherent model of the plant clock has yet been proposed. In this review, we aim to provide an overview of studies of theArabidopsiscircadian system. We shall compare these with results from different taxa and discuss them in the context of what is known about clocks in other organisms.

The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.