CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational.

Complex societies precede moralizing gods throughout world history.

The origins of religion and of complex societies represent evolutionary puzzles1-8. The 'moralizing gods' hypothesis offers a solution to both puzzles by proposing that belief in morally concerned supernatural agents culturally evolved to facilitate cooperation among strangers in large-scale societies9-13. Although previous research has suggested an association between the presence of moralizing gods and social complexity3,6,7,9-18, the relationship between the two is disputed9-13,19-24, and attempts to establish causality have been hampered by limitations in the availability of detailed global longitudinal data. To overcome these limitations, here we systematically coded records from 414 societies that span the past 10,000 years from 30 regions around the world, using 51 measures of social complexity and 4 measures of supernatural enforcement of morality. Our analyses not only confirm the association between moralizing gods and social complexity, but also reveal that moralizing gods follow-rather than precede-large increases in social complexity. Contrary to previous predictions9,12,16,18, powerful moralizing 'big gods' and prosocial supernatural punishment tend to appear only after the emergence of 'megasocieties' with populations of more than around one million people. Moralizing gods are not a prerequisite for the evolution of social complexity, but they may help to sustain and expand complex multi-ethnic empires after they have become established. By contrast, rituals that facilitate the standardization of religious traditions across large populations25,26 generally precede the appearance of moralizing gods. This suggests that ritual practices were more important than the particular content of religious belief to the initial rise of social complexity.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Quantitative historical analysis uncovers a single dimension of complexity that structures global variation in human social organization.

Do human societies from around the world exhibit similarities in the way that they are structured, and show commonalities in the ways that they have evolved? These are long-standing questions that have proven difficult to answer. To test between competing hypotheses, we constructed a massive repository of historical and archaeological information known as "Seshat: Global History Databank." We systematically coded data on 414 societies from 30 regions around the world spanning the last 10,000 years. We were able to capture information on 51 variables reflecting nine characteristics of human societies, such as social scale, economy, features of governance, and information systems. Our analyses revealed that these different characteristics show strong relationships with each other and that a single principal component captures around three-quarters of the observed variation. Furthermore, we found that different characteristics of social complexity are highly predictable across different world regions. These results suggest that key aspects of social organization are functionally related and do indeed coevolve in predictable ways. Our findings highlight the power of the sciences and humanities working together to rigorously test hypotheses about general rules that may have shaped human history.

Peroxiredoxins are conserved markers of circadian rhythms.

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth's rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimized a bacterial CRISPR-hybrid system coupled with FACS, and identified novel high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allowed effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light.

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

Disentangling the evolutionary drivers of social complexity: A comprehensive test of hypotheses.

During the Holocene, the scale and complexity of human societies increased markedly. Generations of scholars have proposed different theories explaining this expansion, which range from broadly functionalist explanations, focusing on the provision of public goods, to conflict theories, emphasizing the role of class struggle or warfare. To quantitatively test these theories, we develop a general dynamical model based on the theoretical framework of cultural macroevolution. Using this model and Seshat: Global History Databank, we test 17 potential predictor variables proxying mechanisms suggested by major theories of sociopolitical complexity (and >100,000 combinations of these predictors). The best-supported model indicates a strong causal role played by a combination of increasing agricultural productivity and invention/adoption of military technologies (most notably, iron weapons and cavalry in the first millennium BCE).

A randomized trial of cash incentives for sexual behavior change among female sex workers in Dar es Salaam.

RATIONALE: Female sex workers (FSW) across the world are at high risk for HIV infection and much work is needed to scale up HIV prevention programs among this group. Conditional cash transfer (CCT) programs have been used successfully in recent years to encourage behavior change. We report the results of a CCT intervention among FSW in Tanzania. METHODS: We conducted a randomized controlled trial (N = 100) of a CCT intervention among FSW in Dar-es-Salaam, Tanzania in 2013. A respondent-driven sampling approach recruited women and randomized them into two groups based on the value of the cash incentive ($20 vs. $40 per visit). All women received testing for 2 curable sexually transmitted infections (STIs), trichomonas and syphilis, free treatment for those STIs and counseling. Women attended study visits at 0, 2 and 4 months and were tested for STIs and received counseling at each visit. Women testing negative for both STIs at the 2- and 4-month visits received a cash reward. RESULTS: Eighty-four women were retained in the study through all three visits. Participants reported significant reductions in the number of clients per week, and increases in the proportion of clients that they used condoms with over the course of the study. STI results showed decreases in prevalence from baseline to final study visit for syphilis and trichomonas. CONCLUSION: While this study was not powered to determine if the incentive resulted in statistically significant increases in condom use or decreases in STI prevalence, the results show the acceptability of the intervention, the feasibility of the recruitment methods, and the ability to retain FSW participants across multiple study visits. A follow-up randomized study with a larger number of participants is planned to test the efficacy of the intervention among high-risk populations of women engaging in transactional sex.

Revisiting Wasson's Soma: exploring the effects of preparation on the chemistry of Amanita muscaria.

In 1968 R. Gordon Wasson first proposed his groundbreaking theory identifying Soma, the hallucinogenic sacrament of the Vedas, as the Amanita muscaria mushroom. While Wasson's theory has garnered acclaim, it is not without its faults. One omission in Wasson's theory is his failure to explain how pressing and filtering Soma, as described in the Rig Veda, supports his theory of Soma's identity. Several critics have reasoned that such preparation should be unnecessary if equivalent results can be obtained by consuming the raw plant, as is done with other psychoactive mushrooms. In order to address these specific criticisms over 600 anecdotal accounts of Amanita muscaria inebriation were collected and analyzed to determine the impact of preparation on Amanita muscaria's effects. The findings of this study demonstrated that the effects of Amanita muscaria were related to the type of preparation employed, and that its toxic effects were considerably reduced by preparations that paralleled those described for Soma in the Rig Veda. While unlikely to end debate over the identity of Soma, this study's findings help to solidify the foundation of Wasson's theory, and also to demonstrate the importance of preparation in understanding and uncovering the true identity of Soma.

Transcriptome analysis of grain development in hexaploid wheat.

BACKGROUND: Hexaploid wheat is one of the most important cereal crops for human nutrition. Molecular understanding of the biology of the developing grain will assist the improvement of yield and quality traits for different environments. High quality transcriptomics is a powerful method to increase this understanding. RESULTS: The transcriptome of developing caryopses from hexaploid wheat (Triticum aestivum, cv. Hereward) was determined using Affymetrix wheat GeneChip oligonucleotide arrays which have probes for 55,052 transcripts. Of these, 14,550 showed significant differential regulation in the period between 6 and 42 days after anthesis (daa). Large changes in transcript abundance were observed which were categorised into distinct phases of differentiation (6-10 daa), grain fill (12-21 daa) and desiccation/maturation (28-42 daa) and were associated with specific tissues and processes. A similar experiment on developing caryopses grown with dry and/or hot environmental treatments was also analysed, using the profiles established in the first experiment to show that most environmental treatment effects on transcription were due to acceleration of development, but that a few transcripts were specifically affected. Transcript abundance profiles in both experiments for nine selected known and putative wheat transcription factors were independently confirmed by real time RT-PCR. These expression profiles confirm or extend our knowledge of the roles of the known transcription factors and suggest roles for the unknown ones. CONCLUSION: This transcriptome data will provide a valuable resource for molecular studies on wheat grain. It has been demonstrated how it can be used to distinguish general developmental shifts from specific effects of treatments on gene expression and to diagnose the probable tissue specificity and role of transcription factors.

Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations.

AIMS: Circadian rhythms permeate all levels of biology to temporally regulate cell and whole-body physiology, although the cell-autonomous mechanism that confers ∼24-h periodicity is incompletely understood. Reports describing circadian oscillations of over-oxidized peroxiredoxin abundance have suggested that redox signaling plays an important role in the timekeeping mechanism. Here, we tested the functional contribution that redox state and primary metabolism make to mammalian cellular timekeeping. RESULTS: We found a circadian rhythm in flux through primary glucose metabolic pathways, indicating rhythmic NAD(P)H production. Using pharmacological and genetic perturbations, however, we found that timekeeping was insensitive to changes in glycolytic flux, whereas oxidative pentose phosphate pathway (PPP) inhibition and other chronic redox stressors primarily affected circadian gene expression amplitude, not periodicity. Finally, acute changes in redox state decreased PER2 protein stability, phase dependently, to alter the subsequent phase of oscillation. INNOVATION: Circadian rhythms in primary cellular metabolism and redox state have been proposed to play a role in the cellular timekeeping mechanism. We present experimental data testing that hypothesis. CONCLUSION: Circadian flux through primary metabolism is cell autonomous, driving rhythmic NAD(P)+ redox cofactor turnover and maintaining a redox balance that is permissive for circadian gene expression cycles. Redox homeostasis and PPP flux, but not glycolysis, are necessary to maintain clock amplitude, but neither redox nor glucose metabolism determines circadian period. Furthermore, cellular rhythms are sensitive to acute changes in redox balance, at least partly through regulation of PER protein. Redox and metabolic state are, thus, both inputs and outputs, but not state variables, of cellular circadian timekeeping. Antioxid. Redox Signal. 28, 507-520.

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing.

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse's active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

Comparison of hypertension healthcare outcomes among older people in the USA and England.

BACKGROUND: The USA and England have very different health systems. Comparing hypertension care outcomes in each country enables an evaluation of the effectiveness of each system. METHOD: The English Longitudinal Study of Ageing and the Health and Retirement Survey are used to compare the prevalence of controlled, uncontrolled and undiagnosed hypertension within the hypertensive population (diagnosed or measured within the survey data used) aged 50 years and above in the USA and in England. RESULTS: Controlled hypertension is more prevalent within the hypertensive population in the USA (age 50-64: 0.53 (0.50 to 0.57) and age 65+: 0.51 (0.49 to 0.53)) than in England (age 50-64: 0.45 (0.42 to 0.48) and age 65+: 0.42 (0.40 to 0.45)). This difference is driven by lower undiagnosed hypertension in the USA (age 50-64: 0.18 (0.15-0.21) and age 65+: 0.13 (0.12 to 0.14)) relative to England (age 50-64: 0.26 (0.24 to 0.29) and age 65+: 0.22 (0.20 to 0.24)). The prevalence of uncontrolled hypertension within the hypertensive population is very similar in the USA (age 50-64: 0.29 (0.26 to 0.32) and age 65+: 0.36 (0.34 to 0.38)) and England (age 50-64: 0.29 (0.26 to 0.32) and age 65+: 0.36 (0.34 to 0.39)). Hypertension care outcomes are comparable across US insurance categories. In both countries, undiagnosed hypertension is positively correlated with wealth (ages 50-64). Uncontrolled hypertension declines with rising wealth in the USA. CONCLUSIONS: Different diagnostic practices are likely to drive the cross-country differences in undiagnosed hypertension. US government health systems perform at least as well as private healthcare and are more equitable in the distribution of care outcomes. Higher undiagnosed hypertension among the affluent may reflect less frequent medical contact.

The Pentose Phosphate Pathway Regulates the Circadian Clock.

The circadian clock is a ubiquitous timekeeping system that organizes the behavior and physiology of organisms over the day and night. Current models rely on transcriptional networks that coordinate circadian gene expression of thousands of transcripts. However, recent studies have uncovered phylogenetically conserved redox rhythms that can occur independently of transcriptional cycles. Here we identify the pentose phosphate pathway (PPP), a critical source of the redox cofactor NADPH, as an important regulator of redox and transcriptional oscillations. Our results show that genetic and pharmacological inhibition of the PPP prolongs the period of circadian rhythms in human cells, mouse tissues, and fruit flies. These metabolic manipulations also cause a remodeling of circadian gene expression programs that involves the circadian transcription factors BMAL1 and CLOCK, and the redox-sensitive transcription factor NRF2. Thus, the PPP regulates circadian rhythms via NADPH metabolism, suggesting a pivotal role for NADPH availability in circadian timekeeping.

Metabolic Cycles in Yeast Share Features Conserved among Circadian Rhythms.

Cell-autonomous circadian rhythms allow organisms to temporally orchestrate their internal state to anticipate and/or resonate with the external environment. Although ∼24-hr periodicity is observed across aerobic eukaryotes, the central mechanism has been hard to dissect because few simple models exist, and known clock proteins are not conserved across phylogenetic kingdoms. In contrast, contributions to circadian rhythmicity made by a handful of post-translational mechanisms, such as phosphorylation of clock proteins by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), appear conserved among phyla. These kinases have many other essential cellular functions and are better conserved in their contribution to timekeeping than any of the clock proteins they phosphorylate. Rhythmic oscillations in cellular redox state are another universal feature of circadian timekeeping, e.g., over-oxidation cycles of abundant peroxiredoxin proteins. Here, we use comparative chronobiology to distinguish fundamental clock mechanisms from species and/or tissue-specific adaptations and thereby identify features shared between circadian rhythms in mammalian cells and non-circadian temperature-compensated respiratory oscillations in budding yeast. We find that both types of oscillations are coupled with the cell division cycle, exhibit period determination by CK1 and GSK3, and have peroxiredoxin over-oxidation cycles. We also explore how peroxiredoxins contribute to YROs. Our data point to common mechanisms underlying both YROs and circadian rhythms and suggest two interpretations: either certain biochemical systems are simply permissive for cellular oscillations (with frequencies from hours to days) or this commonality arose via divergence from an ancestral cellular clock.

Circadian redox and metabolic oscillations in mammalian systems.

SIGNIFICANCE: A substantial proportion of mammalian physiology is organized around the day/night cycle, being regulated by the co-ordinated action of numerous cell-autonomous circadian oscillators throughout the body. Disruption of internal timekeeping, by genetic or environmental perturbation, leads to metabolic dysregulation, whereas changes in metabolism affect timekeeping. RECENT ADVANCES: While gene expression cycles are essential for the temporal coordination of normal physiology, it has become clear that rhythms in metabolism and redox balance are cell-intrinsic phenomena, which may regulate gene expression cycles reciprocally, but persist in their absence. For example, a circadian rhythm in peroxiredoxin oxidation was recently observed in isolated human erythrocytes, fibroblast cell lines in vitro, and mouse liver in vivo. CRITICAL ISSUES: Mammalian timekeeping is a cellular phenomenon. While we understand many of the cellular systems that contribute to this biological oscillation's fidelity and robustness, a comprehensive mechanistic understanding remains elusive. Moreover, the formerly clear distinction between "core clock components" and rhythmic cellular outputs is blurred since several outputs, for example, redox balance, can feed back to regulate timekeeping. As with any cyclical system, establishing causality becomes problematic. FUTURE DIRECTIONS: A detailed molecular understanding of the temporal crosstalk between cellular systems, and the coincidence detection mechanisms that allow a cell to discriminate clock-relevant from irrelevant stimuli, will be essential as we move toward an integrated model of how this daily biological oscillation works. Such knowledge will highlight new avenues by which the functional consequences of circadian timekeeping can be explored in the context of human health and disease.