A dual-feedback loop model of the mammalian circadian clock for multi-input control of circadian phase.

The molecular circadian clock is driven by interlocked transcriptional-translational feedback loops, producing oscillations in the expressions of genes and proteins to coordinate the timing of biological processes throughout the body. Modeling this system gives insight into the underlying processes driving oscillations in an activator-repressor architecture and allows us to make predictions about how to manipulate these oscillations. The knockdown or upregulation of different cellular components using small molecules can disrupt these rhythms, causing a phase shift, and we aim to determine the dosing of such molecules with a model-based control strategy. Mathematical models allow us to predict the phase response of the circadian clock to these interventions and time them appropriately but only if the model has enough physiological detail to describe these responses while maintaining enough simplicity for online optimization. We build a control-relevant, physiologically-based model of the two main feedback loops of the mammalian molecular clock, which provides sufficient detail to consider multi-input control. Our model captures experimentally observed peak to trough ratios, relative abundances, and phase differences in the model species, and we independently validate this model by showing that the in silico model reproduces much of the behavior that is observed in vitro under genetic knockout conditions. Because our model produces valid phase responses, it can be used in a model predictive control algorithm to determine inputs to shift phase. Our model allows us to consider multi-input control through small molecules that act on both feedback loops, and we find that changes to the parameters of the negative feedback loop are much stronger inputs for shifting phase. The strongest inputs predicted by this model provide targets for new experimental small molecules and suggest that the function of the positive feedback loop is to stabilize the oscillations while linking the circadian system to other clock-controlled processes.

Chronopharmacology of simvastatin on hyperlipidaemia in high-fat diet-fed obese mice.

The chronopharmacology refers to the utilization of physiological circadian rhythms to optimize the administration time of drugs, thus increasing their efficacy and safety, or reducing adverse effects. Simvastatin is one of the most widely prescribed drugs for the treatment of hypercholesterolaemia, hyperlipidemia and coronary artery disease. There are conflicting statements regarding the timing of simvastatin administration, and convincing experimental evidence remains unavailable. Thus, we aimed to examine whether different administration times would influence the efficacy of simvastatin. High-fat diet-fed mice were treated with simvastatin at zeitgeber time 1 (ZT1) or ZT13, respectively, for nine weeks. Simvastatin showed robust anti-hypercholesterolaemia and anti-hyperlipidemia effects on these obese mice, regardless of administration time. However, simvastatin administrated at ZT13, compared to ZT1, was more functional for decreasing serum levels of total cholesterol, triglycerides, non-esterified free fatty acids and LDL cholesterol, as well as improving liver pathological characteristics. In terms of possible mechanisms, we found that simvastatin did not alter the expression of hepatic circadian clock gene in vivo, although it failed to change the period, phase and amplitude of oscillation patterns in Per2::Luc U2OS and Bmal1::Luc U2OS cells in vitro. In contrast, simvastatin regulated the expression of Hmgcr, Mdr1 and Slco2b1 in a circadian manner, which potentially contributed to the chronopharmacological function of the drug. Taken together, we provide solid evidence to suggest that different administration times affect the lipid-lowering effects of simvastatin.

ZEITLUPE facilitates the rhythmic movements of Nicotiana attenuata flowers.

Circadian organ movements are ubiquitous in plants. These rhythmic outputs are thought to be regulated by the circadian clock and auxin signalling, but the underlying mechanisms have not been clarified. Flowers of Nicotiana attenuata change their orientation during the daytime through a 140° arc to balance the need for pollinators and the protection of their reproductive organs. This rhythmic trait is under the control of the circadian clock and results from bending and re-straightening movements of the pedicel, stems that connect flowers to the inflorescence. Using an explant system that allowed pedicel growth and curvature responses to be characterized with high spatial and temporal resolution, we demonstrated that this movement is organ autonomous and mediated by auxin. Changes in the growth curvature of the pedicel are accompanied by an auxin gradient and dorsiventral asymmetry in auxin-dependent transcriptional responses; application of auxin transport inhibitors influenced the normal movements of this organ. Silencing the expression of the circadian clock component ZEITLUPE (ZTL) arrested changes in the growth curvature of the pedicel and altered auxin signalling and responses. IAA19-like, an Aux/IAA transcriptional repressor that is circadian regulated and differentially expressed between opposite tissues of the pedicel, and therefore possibly involved in the regulation of changes in organ curvature, physically interacted with ZTL. Together, these results are consistent with a direct link between the circadian clock and the auxin signalling pathway in the regulation of this rhythmic floral movement.

Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness.

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3 We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems.IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

Yin and Yang: Why did evolution implement and preserve the circadian rhythmicity?

Yin and Yang concept emphasizes the reciprocal and interrelated nature; neither is sufficient, both are needed to sustain the overall balance of the living system. Changing the balance, by implementing deficiency or excess of one of them, upsets the equilibrium (homeostasis) of the whole system. PURPOSE: In this opinion article intermittent exposure is presented as the stimulus for development and evolutionary conservation of circadian rhythm, an endogenous, entrainable oscillation of approximately 24 h, to counteract/balance the cells' natural tendency to attenuate their response during long-term exposure to different endogenous substances. RESULTS: The concept of Yin and Yang duality is an allegory on which the avoidance of attenuation of the cells' responses hypothesis is presented as an explanation for the circadian rhythmicity, which is integrated in all human cells, with the exception of stem and cancer cells. CONCLUSIONS: We hypothesize, that circadian rhythmicity has evolved, during evolution, into a mechanism that prevents disruption of the organism's negative-feedback-loop homeostasis.

The Snapdragon LATE ELONGATED HYPOCOTYL Plays A Dual Role in Activating Floral Growth and Scent Emission.

The plant circadian clock controls a large number of internal processes, including growth and metabolism. Scent emission displays a circadian pattern in many species such as the snapdragon. Here we show that knocking down LATE ELONGATED HYPOCOTYL in Antirrhinum majus affects growth and scent emission. In order to gain an understanding of the growth kinetics, we took a phenomic approach using in-house artificial vision systems, obtaining time-lapse videos. Wild type flowers showed a higher growth speed than knockdown plants. The maximal growth rate was decreased by 22% in plants with lower LHY expression. Floral volatiles were differentially affected as RNAi plants showed advanced emission of compounds synthesized from cinnamic acid and delayed emission of metabolites of benzoic acid. The monoterpenes myrcene and ocimene were delayed, whereas the sesquiterpene farnesene was advanced. Overall, transgenic lines showed an altered volatile emission pattern and displayed a modified scent profile. Our results show that AmLHY plays an important role in the quantitative and qualitative control of floral growth and scent emission.

Old and New Roles and Evolving Complexities of Cardiovascular Clocks.

The cardiovascular (CV) system has been established to be significantly influenced by the molecular components of circadian rhythm. Oscillations of circadian rhythm occur within the circulation to affect thrombosis and blood pressure and within CV tissues including arteries, heart, and kidney to control function. Physiologic and molecular oscillations of circadian rhythm have been well connected via global, tissue-specific, and transgenic reporter mouse models of key core clock signals such as Bmal1, Period, and Clock, which can produce both pathology and protection with their mutation. With different nuances of CV clock action continuing to emerge in studies of the cardiovascular system, new questions are raised in both new and old mouse model system observations that underscore the importance, complexity, and continued study of the circadian clock mechanism in cardiovascular disease.

Peripheral Circadian Oscillators.

Circadian rhythms are ~24-hour cycles of physiology and behavior that are synchronized to environmental cycles, such as the light-dark cycle. During the 20th century, most research focused on establishing the fundamental properties of circadian rhythms and discovering circadian pacemakers that were believed to reside in the nervous system of animals. During this time, studies that suggested the existence of circadian oscillators in peripheral organs in mammals were largely dismissed. The discovery of a single-locus circadian pacemaker in the nervous system of several animals affirmed the single-oscillator model of the circadian system. However, the discovery of the genes that constituted the molecular timekeeping system provided the tools for demonstrating the existence of bona fide circadian oscillators in nearly every peripheral tissue in animals, including rodents, in the late 1990s and early 2000s. These studies led to our current understanding that the circadian system in animals is a hierarchical multi-oscillatory network, composed of master pacemaker(s) in the brain and oscillators in peripheral organs. Further studies showed that altering the temporal relationship between these oscillators by simulating jet-lag and metabolic challenges in rodents caused adverse physiological outcomes. Herein we review the studies that led to our current understanding of the function and pathology of the hierarchical multi-oscillator circadian system.

CikA Modulates the Effect of KaiA on the Period of the Circadian Oscillation in KaiC Phosphorylation.

Cyanobacteria contain a circadian oscillator that can be reconstituted in vitro. In the reconstituted circadian oscillator, the phosphorylation state of KaiC oscillates with a circadian period, spending about 12 h in the phosphorylation phase and another 12 h in the dephosphorylation phase. Although some entrainment studies have been performed using the reconstituted oscillator, they were insufficient to fully explain entrainment mechanisms of the cyanobacterial circadian clock due to the lack of input pathway components in the in vitro oscillator reaction mixture. Here, we investigate how an input pathway component, CikA, affects the phosphorylation state of KaiC in vitro. In general, CikA affects the amplitude and period of the circadian oscillation of KaiC phosphorylation by competing with KaiA for the same binding site on KaiB. In the presence of CikA, KaiC switches from its dephosphorylation phase to its phosphorylation phase prematurely, due to an early release of KaiA from KaiB as a result of competitive binding between CikA and KaiA. This causes hyperphosphorylation of KaiC and lowers the amplitude of the circadian oscillation. The period of the KaiC phosphorylation oscillation is shortened by adding increased amounts of CikA. A constant period can be maintained as CikA is increased by proportionally decreasing the amount of KaiA. Our findings give insight into how to reconstitute the cyanobacterial circadian clock in vitro by the addition of an input pathway component, and explain how this affects circadian oscillations by directly interacting with the oscillator components.

Autonomic nerves and circadian control of renal function.

Cardiovascular and renal physiology follow strong circadian rhythms. For instance, renal excretion of solutes and water is higher during the active period compared to the inactive period, and blood pressure peaks early in the beginning of the active period of both diurnal and nocturnal animals. The control of these rhythms is largely dependent on the expression of clock genes both in the central nervous system and within peripheral organs themselves. Although it is understood that the central and peripheral clocks interact and communicate, few studies have explored the specific mechanism by which various organ systems within the body are coordinated to control physiological processes. The renal sympathetic nervous innervation has long been known to have profound effects on renal function, and because the sympathetic nervous system follows strong circadian rhythms, it is likely that autonomic control of the kidney plays an integral role in modulating renal circadian function. This review highlights studies that provide insight into this interaction, discusses areas lacking clarity, and suggests the potential for future work to explore the role of renal autonomics in areas such as blood pressure control and chronic kidney disease.

Integration of Melatonin Related Redox Homeostasis, Aging, and Circadian Rhythm.

Circadian rhythms (CRs) are intrinsic clocks organizing the behavior and physiology of organisms. These clocks are thought to have coevolved with cellular redox regulation. Metabolism, redox homeostasis, circadian clock, and diet offer insights into aging. Mitochondria play a pivotal role in redox homeostasis, CR, and aging. Melatonin is synthesized in mitochondria, is the key regulator of CRs, and shows substantial antioxidative effects. Melatonin levels tend to decrease significantly with advancing age. Recent reports showed that disruptions of CRs may render aging populations even more susceptible to age-related disorders. Recent and high-quality articles investigating CR, redox homeostasis, aging, and their relationship during aging process were included. Putting special emphasis on the possible effects of melatonin on redox homeostasis and mitochondrial dynamics, recent clinical evidence highlighting the importance of circadian mechanisms was utilized. A deeper understanding of the role of altered mitochondrial redox homeostasis in the pathogenesis of age-related disorders and its relationship with CR could offer novel therapeutic interventions. Chronotherapy, a therapeutic approach considering CR of organisms and best therapeutic times, could potentially reduce side effects and improve therapeutic efficiency. Redox homeostasis, energy metabolism, and CR are all intertwined.

[The brain's biological clock].

Daily biological rhythms are controlled by a clock system, composed of a hierarchical multi-oscillator structure. Each cell in this system harbours a self-sustained autonomous molecular oscillator. Light adjusts the phase of the brain oscillator to the environmental light/dark cycle by intrinsically photosensitive retinal ganglion cells through their own photoreceptor, melanopsin, and by using the neuropeptide called pituitary adenylate cyclase-activating polypeptide as well as glutamate as neurotransmitters. The circadian synchronisation system is critical to health, and breakdown of the 24-hour temporal order could lead to pathological conditions.

A thermodynamically consistent model of the post-translational Kai circadian clock.

The principal pacemaker of the circadian clock of the cyanobacterium S. elongatus is a protein phosphorylation cycle consisting of three proteins, KaiA, KaiB and KaiC. KaiC forms a homohexamer, with each monomer consisting of two domains, CI and CII. Both domains can bind and hydrolyze ATP, but only the CII domain can be phosphorylated, at two residues, in a well-defined sequence. While this system has been studied extensively, how the clock is driven thermodynamically has remained elusive. Inspired by recent experimental observations and building on ideas from previous mathematical models, we present a new, thermodynamically consistent, statistical-mechanical model of the clock. At its heart are two main ideas: i) ATP hydrolysis in the CI domain provides the thermodynamic driving force for the clock, switching KaiC between an active conformational state in which its phosphorylation level tends to rise and an inactive one in which it tends to fall; ii) phosphorylation of the CII domain provides the timer for the hydrolysis in the CI domain. The model also naturally explains how KaiA, by acting as a nucleotide exchange factor, can stimulate phosphorylation of KaiC, and how the differential affinity of KaiA for the different KaiC phosphoforms generates the characteristic temporal order of KaiC phosphorylation. As the phosphorylation level in the CII domain rises, the release of ADP from CI slows down, making the inactive conformational state of KaiC more stable. In the inactive state, KaiC binds KaiB, which not only stabilizes this state further, but also leads to the sequestration of KaiA, and hence to KaiC dephosphorylation. Using a dedicated kinetic Monte Carlo algorithm, which makes it possible to efficiently simulate this system consisting of more than a billion reactions, we show that the model can describe a wealth of experimental data.

Structural and biophysical methods to analyze clock function and mechanism.

Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttranslational oscillator. X-ray crystallography and solution NMR were deployed to gain an understanding of the three-dimensional structures of the three proteins KaiA, KaiB, and KaiC that make up the inner timer in cyanobacteria. A hybrid structural biology approach including crystallography, electron microscopy, and solution scattering has shed light on the shapes of binary and ternary Kai protein complexes. Structural studies of the cyanobacterial oscillator demonstrate both the strengths and the limitations of the divide-and-conquer strategy. Thus, investigations of complexes involving domains and/or peptides have afforded valuable information into Kai protein interactions. However, high-resolution structural data are still needed at the level of complexes between the 360-kDa KaiC hexamer that forms the heart of the clock and its KaiA and KaiB partners.

Historia de los terrenos del Hospital Clínico y la Facultad de Medicina de la Universidad de Chile / History of the University of Chile Faculty of Medicine and clinical hospital location

The history of the location of the University of Chile Faculty of Medicine North Campus is derived from a farm of Pedro de Valdivia founder of the city of Santiago de la Nueva Extremadura and governor of the “Reyno de Chile”. This work narrates succinctly the history of this particular location from the Spanish Conquest period to present days.

Impacts of shift work on sleep and circadian rhythms.

Shift work comprises work schedules that extend beyond the typical "nine-to-five" workday, wherein schedules often comprise early work start, compressed work weeks with 12-hour shifts, and night work. According to recent American and European surveys, between 15 and 30% of adult workers are engaged in some type of shift work, with 19% of the European population reportedly working at least 2 hours between 22:00 and 05:00. The 2005 International Classification of Sleep Disorders estimates that a shift work sleep disorder can be found in 2-5% of workers. This disorder is characterized by excessive sleepiness and/or sleep disruption for at least one month in relation with the atypical work schedule. Individual tolerance to shift work remains a complex problem that is affected by the number of consecutive work hours and shifts, the rest periods, and the predictability of work schedules. Sleepiness usually occurs during night shifts and is maximal at the end of the night. Impaired vigilance and performance occur around times of increased sleepiness and can seriously compromise workers' health and safety. Indeed, workers suffering from a shift work sleep-wake disorder can fall asleep involuntarily at work or while driving back home after a night shift. Working on atypical shifts has important socioeconomic impacts as it leads to an increased risk of accidents, workers' impairment and danger to public safety, especially at night. The aim of the present review is to review the circadian and sleep-wake disturbances associated with shift work as well as their medical impacts.

Circadian rhythms - from genes to physiology and disease.

Most physiological processes in our body oscillate in a daily fashion. These include cerebral activity (sleep-wake cycles), metabolism and energy homeostasis, heart rate, blood pressure, body temperature, renal activity, and hormone as well as cytokine secretion. The daily rhythms in behaviour and physiology are not just acute responses to timing cues provided by the environment, but are driven by an endogenous circadian timing system. A central pacemaker in the suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, coordinates all overt rhythms in our body through neuronal and humoral outputs. The SCN consists of two tiny clusters of ~100,000 neurones in humans, each harbouring a self-sustained, cell-autonomous molecular oscillator. Research conducted during the past years has shown, however, that virtually all of our thirty-five trillion body cells possess their own clocks and that these are indistinguishable from those operative in SCN neurones. Here we give an overview on the molecular and cellular architecture of the mammalian circadian timing system and provide some thoughts on its medical and social impact.

Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.

For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.

The role of the circadian clock in animal models of mood disorders.

An association between circadian clock function and mood regulation is well established and has been proposed as a factor in the development of mood disorders. Patients with depression or mania suffer disturbed sleep-wake cycles and altered rhythms in daily activities. Environmentally disrupted circadian rhythms increase the risk of mood disorders in the general population. However, proof that a disturbance of circadian rhythms is causally involved in the development of psychiatric disorders remains elusive. Using clock gene mutants, manipulations of sleep-wake and light-dark cycles, and brain lesions affecting clock function, animal models have been developed to investigate whether circadian rhythm disruptions alter mood. In this review, selected animal models are examined to address the issue of causality between circadian rhythms and affective behavior.

[Clock and molecular genetics in Drosophila]. / Horloge et génétique moléculaire chez la drosophile.

Most living organisms possess a circadian clock (24 h period) which allows them to adapt to environmental conditions. Numerous studies in Drosophila allowed to discover various key clock genes, such as period and timeless. The powerful tools of drosophila genetics have shown that the molecular clock relies on negative feedback loops that generate oscillations of the clock genes mRNA. A delay between the accumulation of mRNAs and proteins is required for the feedback loop. It is generated by post-translational modifications as phosphorylations and ubiquitinations, which control protein stability and determine the period of their oscillations. Clock cells are present in brain as well as in multiple peripheric tissues where they run autonomously. The synchronisation of clock cells by light relies on cryptochrome in both brain and peripheral tissues. In the brain, synchronisation also involves the eye photoreceptors. The clock that drives sleep-wake rhythms is controlled by different groups of neurons in the brain. Each group has a distinct function in the generation of the behavioral rhythm and this function is modulated by environmental conditions.