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Context memory formation is a complex process that requires transcription in many subregions of the brain including the dorsal hippocampus and retrosplenial cortex. One critical gene necessary for memory formation is the circadian gene Period1 (Per1), which has been shown to function in the dorsal hippocampus to modulate spatial memory in addition to its well-documented role in regulating the diurnal clock within the suprachiasmatic nucleus (SCN). We recently found that alterations in Per1 expression in the dorsal hippocampus can modulate spatial memory formation, with reduced hippocampal Per1 impairing memory and overexpression of Per1 ameliorating age-related impairments in spatial memory. Whether Per1 similarly functions within other memory-relevant brain regions is currently unknown. Here, to test whether Per1 is a general mechanism that modulates memory across the brain, we tested the role of Per1 in the retrosplenial cortex (RSC), a brain region necessary for context memory formation. First, we demonstrate that context fear conditioning drives a transient increase in Per1 mRNA expression within the anterior RSC that peaks 60 m after training. Next, using HSV-CRISPRi-mediated knockdown of Per1, we show that reducing Per1 within the anterior RSC before context fear acquisition impairs memory in both male and female mice. In contrast, overexpressing Per1 with either HSV-CRISPRa or HSV-Per1 before context fear acquisition drives a sex-specific memory impairment; males show impaired context fear memory whereas females are not affected by Per1 overexpression. Finally, as Per1 levels are known to rhythmically oscillate across the day/night cycle, we tested the possibility that Per1 overexpression might have different effects on memory depending on the time of day. In contrast to the impairment in memory we observed during the daytime, Per1 overexpression has no effect on context fear memory during the night in either male or female mice. Together, our results indicate that Per1 modulates memory in the anterior retrosplenial cortex in addition to its documented role in regulating memory within the dorsal hippocampus, although this role may differ between males and females., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior., Competing Interests: The authors declare no competing interest.

Chemical communication via pheromones is an integral component in insect behavior, particularly for mate searching and reproduction. Aggregation pheromones, that attract conspecifics of both sexes, are particularly common and have been identified for hundreds of species. These pheromones are among the most ecologically selective pest suppression agents. In this study, we identified an activating effect of the aggregation pheromone of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenibroidae) on a highly conserved circadian clock gene (Tctimeless). Tribolium castaneum is one of the most damaging cosmopolitan pest of flour and other stored food products. Its male produced aggregation pheromone, 4,8-dimethyldecanal (DMD), attracts both conspecific males and females and is used for pest management via monitoring and mating disruption. The Tctimeless gene is an essential component for daily expression patterns of the circadian clock and plays vital roles in eclosion, egg production, and embryonic development. In this study, we demonstrate that constant exposure to the species-specific aggregation pheromone led to Tctimeless up-regulation and a different pattern of rhythmic locomotive behavior. We propose that changing the well-adapted "alarm clock", using DMD is liable to reduce fitness and can be highly useful for pest management., (© 2021. The Author(s).)

Circadian clock is involved in regulating most renal physiological functions, including water and electrolyte balance and blood pressure homeostasis, however, the role of circadian clock in renal pathophysiology remains largely unknown. Here we aimed to investigate the role of Bmal1, a core clock component, in the development of renal fibrosis, the hallmark of pathological features in many renal diseases. The inducible Bmal1 knockout mice (iKO) whose gene deletion occurred in adulthood were used in the study. Analysis of the urinary water, sodium and potassium excretion showed that the iKO mice exhibit abolished diurnal variations. In the model of renal fibrosis induced by unilateral ureteral obstruction, the iKO mice displayed significantly decreased tubulointerstitial fibrosis reflected by attenuated collagen deposition and mitigated expression of fibrotic markers α-SMA and fibronectin. The hedgehog pathway transcriptional effectors Gli1 and Gli2, which have been reported to be involved in the pathogenesis of renal fibrosis, were significantly decreased in the iKO mice. Mechanistically, ChIP assay and luciferase reporter assay revealed that BMAL1 bound to the promoter of and activate the transcription of Gli2, but not Gli1, suggesting that the involvement of Bmal1 in renal fibrosis was possibly mediated via Gli2-dependent mechanisms. Furthermore, treatment with TGF-β increased Bmal1 in cultured murine proximal tubular cells. Knockdown of Bmal1 abolished, while overexpression of Bmal1 increased, Gli2 and the expression of fibrosis-related genes. Collectively, these results revealed a prominent role of the core clock gene Bmal1 in tubulointerstitial fibrosis. Moreover, we identified Gli2 as a novel target of Bmal1, which may mediate the adverse effect of Bmal1 in obstructive nephropathy., (© 2021 Federation of American Societies for Experimental Biology.)

Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period ( per ) and timeless ( tim ) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity., (Copyright © 2021, American Association for the Advancement of Science.)

While circadian rhythms in physiology and behavior demonstrate remarkable day-to-day precision, they are also able to exhibit plasticity in a variety of parameters and under a variety of conditions. After-effects are one type of plasticity in which exposure to non-24-h light-dark cycles (T-cycles) will alter the animal's free-running rhythm in subsequent constant conditions. We use a mathematical model to explore whether the concept of synaptic plasticity can explain the observation of after-effects. In this model, the SCN is composed of a set of individual oscillators randomly selected from a normally distributed population. Each cell receives input from a defined set of oscillators, and the overall period of a cell is a weighted average of its own period and that of its inputs. The influence that an input has on its target's period is determined by the proximity of the input cell's period to the imposed T-cycle period, such that cells with periods near T will have greater influence. Such an arrangement is able to duplicate the phenomenon of after-effects, with relatively few inputs per cell (~4-5) being required. When the variability of periods between oscillators is low, the system is quite robust and results in minimal after-effects, while systems with greater between-cell variability exhibit greater magnitude after-effects. T-cycles that produce maximal after-effects have periods within ~2.5 to 3 h of the population period. Overall, this model demonstrates that synaptic plasticity in the SCN network could contribute to plasticity of the circadian period.

Drug addiction is a chronic and relapsing brain disorder, influenced by complex interactions between endogenous and exogenous factors. Per2, a circadian gene, plays a role in drug addiction. Previous studies using Per2-knockout mice have shown a role for Per2 in cocaine, morphine and alcohol addiction. In the present study, we investigated the role of Per2 in methamphetamine (METH) addiction using Per2-overexpression and knockout mice. We observed locomotor sensitization responses to METH administration, and rewarding effects using a conditioned place preference test. In addition, we measured expression levels of dopamine and dopamine-related genes (monoamine oxidase A, DA receptor 1, DA receptor 2, DA active transporter, tyrosine hydroxylase and cAMP response element-binding protein 1) in the striatum of the mice after repeated METH treatments, using qRT-PCR. Per2-overexpressed mice showed decreased locomotor sensitization and rewarding effects of METH compared to the wildtype mice, whereas the opposite was observed in Per2 knockout mice. Both types of transgenic mice showed altered expression levels of dopamine-related genes after repeated METH administration. Specifically, we observed lower dopamine levels in Per2-overexpressed mice and higher levels in Per2-knockout mice. Taken together, Per2 expression levels may influence the addictive effects of METH through the dopaminergic system in the striatum of mice., (© 2018 Society for the Study of Addiction.)

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls. Given the vital role of period 2 (Per2) in the core clock and the unrecognized role of Per2 in SCD, we transplanted the bone marrow (BM) of SCD mice to Per2 Luciferase mice, which revealed that Per2 expression was up-regulated in SCD mouse lung. Next, we transplanted the BM of SCD mice to period 1 ( Per1 ) /Per2 double deficient [ Per1/Per2 double knockout ( dKO )] and wild-type mice, respectively. We discovered that Per1/Per2 dKO mice transplanted with SCD BM (SCD → Per1/Per2 dKO ) displayed severe irradiation sensitivity and were more susceptible to an early death. Although we observed an increase of peripheral inflammatory cells, we did not detect differences in erythrocyte sickling. However, there was further lung damage due to elevated pulmonary congestion, inflammatory cell infiltration, iron overload, and secretion of IL-6 in lavage fluid. Overall, we demonstrate that Per1/Per2 is beneficial to counteract elevated systemic inflammation, lung tissue inflammation, and iron overload in SCD.-Adebiyi, M. G., Zhao, Z., Ye, Y., Manalo, J., Hong, Y., Lee, C. C., Xian, W., McKeon, F., Culp-Hill, R., D' Alessandro, A., Kellems, R. E., Yoo, S.-H., Han, L., Xia, Y. Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. Here we demonstrate that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. We observe a molecular synchronization defect explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx -mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. Our results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain., Competing Interests: Competing interestsThe authors declare no competing interests.

In mammals, the circadian rhythms are regulated by the central clock located in the hypothalamic suprachiasmatic nucleus (SCN), which is composed of heterogeneous neurons with various neurotransmitters. Among them an inhibitory neurotransmitter, γ-Amino-Butyric-Acid (GABA), is expressed in almost all SCN neurons, however, its role in the circadian physiology is still unclear. Here, we show that the SCN of fetal mice lacking vesicular GABA transporter (VGAT -/- ) or GABA synthesizing enzyme, glutamate decarboxylase (GAD65 -/- /67 -/- ), shows burst firings associated with large Ca 2+ spikes throughout 24 hours, which spread over the entire SCN slice in synchrony. By contrast, circadian PER2 rhythms in VGAT -/- and GAD65 -/- /67 -/- SCN remain intact. SCN-specific VGAT deletion in adult mice dampens circadian behavior rhythm. These findings indicate that GABA in the fetal SCN is necessary for refinement of the circadian firing rhythm and, possibly, for stabilizing the output signals, but not for circadian integration of multiple cellular oscillations., Competing Interests: Competing interestsThe authors declare no competing interests.

Environmental circadian disruption (ECD), characterized by repeated or long-term disruption in environmental timing cues which require the internal circadian clock to change its phase to resynchronize with the environment, is associated with numerous serious health issues in humans. While animal and isolated cell models exist to study the effects of destabilizing the relationship between the circadian system and the environment, neither approach provides an ideal solution. Here, we developed an in vitro model which incorporates both elements of a reductionist cellular model and disruption of the clock/environment relationship using temperature as an environmental cue, as occurs in vivo. Using this approach, we have demonstrated that some effects of in vivo ECD can be reproduced using only isolated peripheral oscillators. Specifically, we report exaggerated inflammatory responses to endotoxin following repeated environmental circadian disruption in explanted spleens. This effect requires a functional circadian clock but not the master brain clock, the suprachiasmatic nucleus (SCN). Further, we report that this is a result of cumulative, rather than acute, circadian disruption as has been previously observed in vivo. Finally, such effects appear to be tissue specific as it does not occur in lung, which is less sensitive to the temperature cycles employed to induce ECD. Taken together, the present study suggests that this model could be a valuable tool for dissecting the causes and effects of circadian disruption both in isolated components of physiological systems as well as the aggregated interactions of these systems that occur in vivo., Competing Interests: The authors have declared that no competing interests exist.

The floral perianth, comprising sepals and petals, conceals the sexual organs and attracts pollinators. The coordination of growth and scent emission is not fully understood. We have analyzed the effect of knocking down CHANEL ( PhCHL ), the ZEITLUPE ortholog in petunia ( PhCHL ) by hairpin RNAs. Plants with low PhCHL mRNA had overall decreased size. Growth evaluation using time lapse image analysis showed that early leaf movement was not affected by RNAi:PhCHL , but flower angle movement was modified, moving earlier during the day in knockdown plants than in wild types. Despite differences in stem length, growth rate was not significantly affected by loss of PhCHL . In contrast, petal growth displayed lower growth rate in RNAi:PhCHL . Decreased levels of PhCHL caused strongly modified scent profiles, including changes in composition and timing of emission resulting in volatile profiles highly divergent from the wild type. Our results show a role of PhCHL in controlling growth and development of vegetative and reproductive organs in petunia. The different effects of PhCHL on organ development indicate an organ-specific interpretation of the down regulation of PhCHL . Through the control of both timing and quantitative volatile emissions, PhCHL appears to be a major coordinator of scent profiles.

The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the PERIOD (PER) and TIMELESS (TIM) proteins repress the expression of their own gene by inhibiting the activity of the CLOCK (CLK) and CYCLE (CYC) transcription factors. A series of posttranslational modifications contribute to the oscillations of the PER and TIM proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. Here we report that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases TIM protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per 0 mutants, supporting a PER-dependent inhibition of tim mRNA deadenylation by POP2. We report a deadenylation mechanism that controls the oscillations of a core clock gene transcript., Competing Interests: The authors declare no conflict of interest.

Although emerging evidence indicates an important role of the circadian clock in modulating the diurnal oscillation of mammalian target of rapamycin complex 1 (mTORC1) signaling, the underlying molecular mechanism remains elusive. Here we show that Period2 (Per2), a core clock protein, functions as a scaffold protein to tether tuberous sclerosis complex 1 (Tsc1), Raptor, and mTOR together to specifically suppress the activity of mTORC1 complex. Due to the loss of its inhibition of mTORC1, Per2 deficiency significantly enhances protein synthesis and cell proliferation but reduces autophagy. Furthermore, we find that the glucagon-Creb/Crtc2 signaling cascade induces Per2 expression, which mediates the suppression of mTORC1 in mouse liver during fasting. Our study not only uncovers a novel role of Per2 in regulating the mTORC1 pathway, but also sheds new light on the mechanism of fasting inhibition on mTORC1 in the liver., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A plethora of physiological processes show stable and synchronized daily oscillations that are either driven or modulated by biological clocks. A circadian pacemaker located in the suprachiasmatic nucleus of the ventral hypothalamus coordinates 24-hour oscillations of central and peripheral physiology with the environment. The circadian clockwork involved in driving rhythmic physiology is composed of various clock genes that are interlocked via a complex feedback loop to generate precise yet plastic oscillations of ∼24 hours. This review focuses on the specific role of the core clockwork gene Period1 and its paralogs on intra-oscillator and extra-oscillator functions, including, but not limited to, hippocampus-dependent processes, cardiovascular function, appetite control, as well as glucose and lipid homeostasis. Alterations in Period gene function have been implicated in a wide range of physical and mental disorders. At the same time, a variety of conditions including metabolic disorders also impact clock gene expression, resulting in circadian disruptions, which in turn often exacerbates the disease state.

miRNAs are important regulators of diverse cellular processes including proliferation, apoptosis, and differentiation. In the context of bone marrow derived stromal cell and adipose derived stromal cell differentiation, miRNAs are established regulators of both differentiation or stemness depending on their target. Furthermore, miRNA dysregulation can play a key role in various disease states. Here we show that miR-181a is regulated in a circadian manner and is induced during both immortalized bone marrow derived stromal cell (iBMSC) as well as primary patient adipose derived stromal cell (PASC) adipogenesis. Enhanced expression of miR-181a in iBMSCs  and PASCs produced a robust increase in adipogenesis through the direct targeting of the circadian factor period circadian regulator 3 (PER3). Furthermore, we show that knocking down endogenous miR-181a expression in iBMSC has a profound inhibitory effect on iBMSC adipogenesis through its regulation of PER3. Additionally, we found that miR-181a regulates the circadian dependency of the adipogenesis master regulator PPARγ. Taken together, our data identify a previously unknown functional link between miR-181a and the circadian machinery in immortalized bone marrow stromal cells and adipose derived stromal cells highlighting its importance in iBMSC and ASC adipogenesis and circadian biology.

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

Throughout the evolutionary time, all organisms and species on Earth evolved with an adaptation to consistent oscillations of sunlight and darkness, now recognized as 'circadian rhythm.' Single-cellular to multisystem organisms use circadian biology to synchronize to the external environment and provide predictive adaptation to changes in cellular homeostasis. Dysregulation of circadian biology has been implicated in numerous prevalent human diseases, and subsequently targeting the circadian machinery may provide innovative preventative or treatment strategies. Discovery of 'peripheral circadian clocks' unleashed widespread investigations into the potential roles of clock biology in cellular, tissue, and organ function in healthy and diseased states. Particularly, oxygen-sensing pathways (e.g. hypoxia inducible factor, HIF1), are critical for adaptation to changes in oxygen availability in diseases such as myocardial ischemia. Recent investigations have identified a connection between the circadian rhythm protein Period 2 (PER2) and HIF1A that may elucidate an evolutionarily conserved cellular network that can be targeted to manipulate metabolic function in stressed conditions like hypoxia or ischemia. Understanding the link between circadian and hypoxia pathways may provide insights and subsequent innovative therapeutic strategies for patients with myocardial ischemia. This review addresses our current understanding of the connection between light-sensing pathways (PER2), and oxygen-sensing pathways (HIF1A), in the context of myocardial ischemia and lays the groundwork for future studies to take advantage of these two evolutionarily conserved pathways in the treatment of myocardial ischemia., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day-dependent manner. Normal progression of circadian rhythms is dependent on the daily cycling of core transcriptional factors that make up cell-autonomous molecular oscillators. In Drosophila , PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional-translational feedback mechanism to regulate the circadian transcriptome. Posttranslational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, casein kinase 1α (CK1α) has been identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. Using Drosophila as a model, we show that CK1α has an overall function of speeding up PER metabolism and is required to maintain the 24 h period of circadian rhythms. Our results indicate that CK1α collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, we observe that CK1α promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1α enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus. SIGNIFICANCE STATEMENT Circadian clocks are endogenous timers that integrate environmental signals to impose temporal control over organismal physiology over the 24 h day/night cycle. To maintain the 24 h period length of circadian clocks and to ensure that circadian rhythms are in synchrony with the external environment, key proteins that make up the molecular oscillator are extensively regulated by phosphorylation to ensure that they perform proper time-of-day-specific functions. Casein kinase 1α (CK1α) has previously been identified as a kinase that phosphorylates mammalian PERIOD (PER) proteins to promote their degradation, but the mechanism by which it modulates PER stability is unclear. In this study, we characterize the mechanisms by which CK1α interacts with DOUBLETIME (DBT) to achieve the overall function of speeding up PER metabolism and to ensure proper time-keeping., (Copyright © 2018 the authors 0270-6474/18/3810631-13$15.00/0.)

Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. Here, we show that mutations in the clock gene period result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. We further describe distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when we reintroduce a form of the period gene in glia, lateral neurons, and the fan-shaped body. Our results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: Delirium occurs in approximately 30% of critically ill patients, and the risk of dying during admission doubles in those patients. Molecular mechanisms causing delirium are largely unknown. However, critical illness and the ICU environment consistently disrupt circadian rhythms, and circadian disruptions are strongly associated with delirium. Exposure to benzodiazepines and constant light are suspected risk factors for the development of delirium. Thus, we tested the functional role of the circadian rhythm protein Period 2 (PER2) in different mouse models resembling delirium., Design: Animal study., Setting: University experimental laboratory., Subjects: Wildtype, Per2 mice., Interventions: Midazolam, lipopolysaccharide (lipopolysaccharide), constant light, nobiletin, or sham-treated animals., Measurements and Main Results: Midazolam significantly reduced the expression of PER2 in the suprachiasmatic nucleus and the hippocampus of wild-type mice. Behavioral tests following midazolam exposure revealed a robust phenotype including executive dysfunction and memory impairment suggestive of delirium. These findings indicated a critical role of hippocampal expressed PER2. Similar results were obtained in mice exposed to lipopolysaccharide or constant light. Subsequent studies in Per2 mice confirmed a functional role of PER2 in a midazolam-induced delirium-like phenotype. Using the small molecule nobiletin to enhance PER2 function, the cognitive deficits induced by midazolam or constant light were attenuated in wild-type mice., Conclusions: These experiments identify a novel role for PER2 during a midazolam- or constant light-induced delirium-like state, highlight the importance of hippocampal PER2 expression for cognitive function, and suggest the PER2 enhancer nobiletin as potential therapy in delirium-like conditions associated with circadian disruption.

Mammalian circadian rhythms are phase-adjusted and amplified by external cues such as light and food. While the light input pathway via the central clock, the suprachiasmatic nucleus, has been well defined, the mechanism of feeding-induced circadian resetting remains undefined, particularly in humans. Animal studies have indicated that insulin, a pancreatic hormone that is secreted rapidly in response to feeding, is an input factor for a few peripheral clocks, such as liver and adipose tissue. In this study, using plucked and cultured hair follicles as a representative human peripheral clock, we examined the effect of insulin on circadian characteristics of clock gene expression. Our results demonstrate that insulin phase-shifts or amplifies the clock gene expression rhythms of ex vivo cultured hair follicles in a phase-responsive manner. To reduce the possibility that differences in species, genetic or environmental background, and experimental methods affected experimental outcomes, we also treated surgically extracted whisker follicles of Period2::Luciferase (Per2 Luc ) mice with insulin and found that the effect of insulin on clock gene expression was reproducible. These results suggest the possibility that feeding-induced insulin resets peripheral circadian clocks in humans., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

In recent years, it has become increasingly apparent that antisense transcription plays an important role in the regulation of gene expression. The circadian clock is no exception: an antisense transcript of the mammalian core-clock gene PERIOD2 (PER2), which we shall refer to as Per2AS RNA, oscillates with a circadian period and a nearly 12 h phase shift from the peak expression of Per2 mRNA. In this paper, we ask whether Per2AS plays a regulatory role in the mammalian circadian clock by studying in silico the potential effects of interactions between Per2 and Per2AS RNAs on circadian rhythms. Based on the antiphasic expression pattern, we consider two hypotheses about how Per2 and Per2AS mutually interfere with each other's expression. In our pre-transcriptional model, the transcription of Per2AS RNA from the non-coding strand represses the transcription of Per2 mRNA from the coding strand and vice versa. In our post-transcriptional model, Per2 and Per2AS transcripts form a double-stranded RNA duplex, which is rapidly degraded. To study these two possible mechanisms, we have added terms describing our alternative hypotheses to a published mathematical model of the molecular regulatory network of the mammalian circadian clock. Our pre-transcriptional model predicts that transcriptional interference between Per2 and Per2AS can generate alternative modes of circadian oscillations, which we characterize in terms of the amplitude and phase of oscillation of core clock genes. In our post-transcriptional model, Per2/Per2AS duplex formation dampens the circadian rhythm. In a model that combines pre- and post-transcriptional controls, the period, amplitude and phase of circadian proteins exhibit non-monotonic dependencies on the rate of expression of Per2AS. All three models provide potential explanations of the observed antiphasic, circadian oscillations of Per2 and Per2AS RNAs. They make discordant predictions that can be tested experimentally in order to distinguish among these alternative hypotheses.

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates daily rhythms including sleep-wake, hormone release, and gene expression. The cells of the SCN must synchronize to each other to drive these circadian rhythms in the rest of the body. The ontogeny of circadian cycling and intercellular coupling in the SCN remains poorly understood. Recent in vitro studies have recorded circadian rhythms from the whole embryonic SCN. Here, we tracked the onset and precision of rhythms in PERIOD2 (PER2), a clock protein, within the SCN isolated from embryonic and postnatal mice of undetermined sex. We found that a few SCN cells developed circadian periodicity in PER2 by 14.5 d after mating (E14.5) with no evidence for daily cycling on E13.5. On E15.5, the fraction of competent oscillators increased dramatically corresponding with stabilization of their circadian periods. The cells of the SCN harvested at E15.5 expressed sustained, synchronous daily rhythms. By postnatal day 2 (P2), SCN oscillators displayed the daily, dorsal-ventral phase wave in clock gene expression typical of the adult SCN. Strikingly, vasoactive intestinal polypeptide (VIP), a neuropeptide critical for synchrony in the adult SCN, and its receptor, VPAC2R, reached detectable levels after birth and after the onset of circadian synchrony. Antagonists of GABA or VIP signaling or action potentials did not disrupt circadian synchrony in the E15.5 SCN. We conclude that endogenous daily rhythms in the fetal SCN begin with few noisy oscillators on E14.5, followed by widespread oscillations that rapidly synchronize on E15.5 by an unknown mechanism. SIGNIFICANCE STATEMENT We recorded the onset of PER2 circadian oscillations during embryonic development in the mouse SCN. When isolated at E13.5, the anlagen of the SCN expresses high, arrhythmic PER2. In contrast, a few cells show noisy circadian rhythms in the isolated E14.5 SCN and most show reliable, self-sustained, synchronized rhythms in the E15.5 SCN. Strikingly, this synchrony at E15.5 appears before expression of VIP or its receptor and persists in the presence of blockers of VIP, GABA or neuronal firing. Finally, the dorsal-ventral phase wave of PER2 typical of the adult SCN appears ∼P2, indicating that multiple signals may mediate circadian synchrony during the ontogeny of the SCN., (Copyright © 2018 the authors 0270-6474/18/381326-09$15.00/0.)

Robustness in biology is the stability of phenotype under diverse genetic and/or environmental perturbations. The circadian clock has remarkable stability of period and phase that-unlike other biological oscillators-is maintained over a wide range of conditions. Here, we show that the high fidelity of the circadian system stems from robust degradation of the clock protein PERIOD. We show that PERIOD degradation is regulated by a balance between ubiquitination and deubiquitination, and that disruption of this balance can destabilize the clock. In mice with a loss-of-function mutation of the E3 ligase gene β-Trcp2, the balance of PERIOD degradation is perturbed and the clock becomes dramatically unstable, presenting a unique behavioral phenotype unlike other circadian mutant animal models. We believe that our data provide a molecular explanation for how circadian phases, such as wake-sleep onset times, can become unstable in humans, and we present a unique mouse model to study human circadian disorders with unstable circadian rhythm phases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Period 2 (PER2) is an important factor in daily oscillations called circadian rhythms, which are emerging as one of the most important regulatory networks, responsible for homeostasis and transcriptional regulation of a number of genes. Our work shows that PER2 could act as a co-activator of the constitutive androstane receptor (CAR), a key nuclear receptor (NR) that regulates the metabolism of endobiotics and xenobiotics. Bioinformatic analysis shows that PER2 and CAR possess structural elements that could enable them to interact which was confirmed experimentally by CoIP experiment. Co-transfection of mouse hepatocarcinoma cells with plasmids overexpressing Per2 and Car increases expression of Bmal1, a potential CAR target gene, more than transfections with Car only. This is the first report indicating the interaction of PER2 and CAR.

Rationale: Previous studies have shown that repeated exposure to drugs of abuse is associated with changes in clock genes expression and that mice strains with various mutations in clock genes show alterations in drug-induced behaviors., Objective: The objective of this study is to characterize the role of the clock gene mPer1 in the development of morphine-induced behaviors and a possible link to histone deacetylase (HDAC) activity., Methods: In Per1 Brdm1 null mutant mice and wild-type (WT) littermates, we examined whether there were any differences in the development of morphine antinociception, tolerance to antinociception, withdrawal, sensitization to locomotion, and conditioned place preference (CPP)., Results: Per1 Brdm1 mutant mice did not show any difference in morphine antinociception, tolerance development, nor in physical withdrawal signs precipitated by naloxone administration compared to WT. However, morphine-induced locomotor sensitization and CPP were significantly impaired in Per1 Brdm1 mutant mice. Because a very similar dissociation between tolerance and dependence vs. sensitization and CPP was recently observed after the co-administration of morphine and the HDAC inhibitor sodium butyrate (NaBut), we studied a possible link between mPer1 and HDAC activity. As opposed to WT controls, Per1 Brdm1 mutant mice showed significantly enhanced striatal global HDAC activity within the striatum when exposed to a locomotor-sensitizing morphine administration regimen. Furthermore, the administration of the HDAC inhibitor NaBut restored the ability of morphine to promote locomotor sensitization and reward in Per1 Brdm1 mutant mice., Conclusions: Our results reveal that although the mPer1 gene does not alter morphine-induced antinociception nor withdrawal, it plays a prominent role in the development of morphine-induced behavioral sensitization and reward via inhibitory modulation of striatal HDAC activity. These data suggest that PER1 inhibits deacetylation to promote drug-induced neuroplastic changes.

Environmental disruption of molecular clocks promoted liver carcinogenesis and accelerated cancer progression in rodents. We investigated the specific role of clock gene Period 2 (Per2) for liver carcinogenesis and clock-controlled cellular proliferation, genomic instability and inflammation. We assessed liver histopathology, and determined molecular and physiology circadian patterns in mice on chronic diethylnitrosamine (DEN) exposure according to constitutive Per2 mutation. First, we found that Per2m/m liver displayed profound alterations in proliferation gene expression, including c-Myc derepression, phase-advanced Wee1, and arrhythmic Ccnb1 and K-ras mRNA expressions, as well as deregulated inflammation, through arrhythmic liver IL-6 protein concentration, in the absence of any DEN exposure. These changes could then make Per2m/m mice more prone to subsequently develop liver cancers on DEN. Indeed, primary liver cancers were nearly fourfold as frequent in Per2m/m mice as compared to wild-type (WT), 4 months after DEN exposure. The liver molecular clock was severely disrupted throughout the whole carcinogenesis process, including the initiation stage, i.e. within the initial 17 days on DEN. Per2m/m further exhibited increased c-Myc and Ccnb1 mean 24h expressions, lack of P53 response, and arrhythmic ATM, Wee1 and Ccnb1 expressions. DEN-induced tumor related inflammation was further promoted through increased protein concentrations of liver IL-6 and TNF-α as compared to WT during carcinogenesis initiation. Per2 mutation severely deregulated liver gene or protein expressions related to three cancer hallmarks, including uncontrolled proliferation, genomic instability, and tumor promoting inflammation, and accelerated liver carcinogenesis several-fold. Clock gene Per2 acted here as a liver tumor suppressor from initiation to progression.

Aged animals exhibit diminished circadian rhythms, and both aging and circadian disruption sensitize neuroinflammatory responses. Microglia-the innate immune cell of the central nervous system-possess endogenous timekeeping mechanisms that regulate immune responses. Here, we explored whether aging is associated with disrupted diurnal rhythms in microglia and neuroinflammatory processes. First, hippocampal microglia isolated from young rats (4 months F344XBN) rhythmically expressed circadian clock genes, whereas microglia isolated from the hippocampus of aged rats (25 months) had aberrant Per1 and Per2 rhythms. Unstimulated microglia from young rats exhibited robust rhythms of TNFα and IL-1β mRNA expression, whereas those from aged rats had flattened and tonically elevated cytokine expression. Similarly, microglial activation markers were diurnally regulated in the hippocampus of young but not aged rats and diurnal differences in responsiveness to both ex vivo and in vivo inflammatory challenges were abolished in aged rats. Corticosterone is an entraining signal for extra-suprachiasmatic nucleus circadian rhythms. Here, corticosterone stimulation elicited similar Per1 induction in aged and young microglia. Overall, these results indicate that aging dysregulates circadian regulation of neuroinflammatory functions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The various clock genes in normal cells, through their interaction, establish a number of positive and negative feedback loops that compose a network structure. These genes play an important role in regulating normal physiological activities. The expression of clock gene PER1 is decreased in many types of cancer. PER1 is highly correlated with the initiation and progression of cancer by regulating numerous downstream genes. However, it is still unclear whether the lower expression of PER1 in cancer can influence the expression of other clock genes in the clock gene network. In this study, we used short hairpin RNA interference to knock down PER1 effectively in SCC15 human oral squamous cell carcinoma cells. These cancer cells later were subcutaneously injected into the back of nude mice. We discovered that after PER1 knockdown, apoptosis was decreased and cell proliferation and in vivo tumor formation were enhanced. Quantitative real-time PCR result indicated that in vitro and in vivo cancer cells after PER1 knockdown, PER2, DEC1, DEC2, CRY1, CRY2 and NPAS2 were significantly down-regulated at the mRNA level, while PER3, TIM, RORα and REV-ERBα were significantly up-regulated. It prompts that the role of PER1 in carcinogenesis is exerted not only by regulating downstream genes, but also through the synergistic dysregulation of many other clock genes in the clock gene network.

In mammals, the circadian system is composed of a principal circadian oscillator located in the suprachiasmatic nucleus (SCN) and a number of subordinate oscillators in extra-SCN brain regions and peripheral tissues/organs. However, how the time-keeping functions of this multiple oscillator circuit are affected by aberrant lighting environments remains largely unknown. In the present study, we investigated the effects of chronic light exposure in the middle of the night on the circadian system by comparing the mice housed in a 12:4:4:4-h L:DLD condition with the controls in 12:12-h L:D condition. Daily rhythms in locomotor activity were analyzed and the expression patterns of protein products of clock genes Period1 and Period2 (PER1 and PER2) were examined in the SCN and extra-SCN brain regions, including the dorsal striatum, hippocampus, paraventricular nucleus (PVN), and basolateral amygdala (BLA). Following 2 weeks of housing in the L:DLD condition, animals showed disturbed daily rhythms in locomotor activity and lacked daily rhythms of PER1 and PER2 in the SCN. In the extra-SCN brain regions, the PER1 and PER2 rhythms were affected in a region-specific pattern, such that they were relatively undisturbed in the striatum and hippocampus, phase-shifted in the BLA, and abolished in the PVN. In addition, mice in the L:DLD condition showed increased anxiety-like behaviors and reduced brain-derived neurotropic factor messenger RNA expression in the hippocampus, amygdala, and medial prefrontal cortex, which are brain regions that are involved in emotional regulation. These results indicate that nighttime light exposure leads to circadian disturbances not only by abolishing the circadian rhythms in the SCN but also by inducing misalignment among brain oscillators and negatively affects emotional processing. These observations serve to identify risks associated with decisions regarding lifestyle in our modern society., (© 2016 The Author(s).)

Background & Aims: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small non-coding RNAs that trigger mRNA translation inhibition. We aimed to evaluate the role of Per1 and related miRNAs in cholangiocarcinoma growth., Methods: The expression of clock genes was evaluated in human cholangiocarcinoma tissue arrays and cholangiocarcinoma lines. The rhythmic expression of clock genes was evaluated in cholangiocarcinoma cells and H69 (non-malignant cholangiocytes) by qPCR. We measured cell proliferation, cell cycle and apoptosis in Mz-ChA-1 cells after Per1 overexpression. We examined tumor growth in vivo after injection of Per1 overexpressing cells. We verified miRNAs that targets Per1. The circadian rhythm of miR-34a was evaluated in cholangiocarcinoma and H69 cells. We evaluated cell proliferation, apoptosis and invasion after inhibition of miR-34a in vitro, and the potential molecular mechanisms by mRNA profiling after overexpression of Per1., Results: Expression of Per1 was decreased in cholangiocarcinoma. The circadian rhythm of Per1 expression was lost in cholangiocarcinoma cells. Decreased cell proliferation, lower G2/M arrest, and enhanced apoptosis were shown in Per1 overexpressing cells. An in vivo study revealed decreased tumor growth, decreased proliferation, angiogenesis and metastasis after overexpressing Per1. Per1 was verified as a target of miR-34a. miR-34a was rhythmically expressed in cholangiocarcinoma cells and H69. The inhibition of miR-34a decreased proliferation, migration and invasion in cholangiocarcinoma cells. mRNA profiling has shown that overexpression of Per1 inhibits cell growth through regulation of multiple cancer-related pathways, such as cell cycle, cell growth and apoptosis pathways., Conclusions: Disruption of circadian rhythms of clock genes contribute to the malignant phenotypes of human cholangiocarcinoma., Lay Summary: The current study is about how biological clock and its regulators affect the bile duct tumor growth. The disruption of biological clock has a negative impact in different cancers. Per1 is a gene that is involved in maintaining the biological clock and show 24h oscillation. Reduced levels of Per1 and disruption of 24h circadian rhythm was found in bile duct cancer cells. Therefore, a genetic modified bile duct cancer cells was created. It has a higher level of Per1 expression and partially recovered circadian rhythm. Those genetic modified cells also displayed slower cell growth or higher rate of cell death. We also used mice model that lack of immune system to show that our genetic modified bile duct cells form smaller tumor. In addition, we tried to see how Per1 is communicating with other genes in regarding of controlling the tumor growth. We found Per1 is regulated by microRNA-34a, a small non-coding RNA that directly binds to genes and inhibit gene expression. Decreased level of miR-34a has also significantly reduced tumor growth through controlling the cell growth and cell death balance. Therefore bile duct cancer patients may be treated with miR-34a inhibitor or Per1 stimulator in the future., (Published by Elsevier B.V.)

Previous studies have shown that the aberrant expression of period circadian clock 2 (Per2) is closely related to the occurrence and development of cancers, but the specific mechanism remains unclear. In the present study, we used shRNA to downregulate Per2 in oral squamous cell carcinoma (OSCC) Tca8113 cells, and then detected the alterations in cell cycle, cell proliferation and apoptosis by flow cytometric analysis and mRNA expression alterations in all the important genes in the cyclin/cyclin-dependent protein kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) cell cycle network by RT-qPCR. We found that in the Tca8113 cells, after Per2 downregulation, the mRNA expression levels of cyclin A2, B1 and D1, CDK4, CDK6 and E2F1 were significantly increased (P<0.05), the mRNA expression levels of p53, p16 and p21 were significantly decreased (P<0.05), cell proliferation was significantly higher (P<0.05), apoptosis was significantly lower (P<0.05) and the number of cells in the G1/G0 phase was significantly decreased (P<0.05). The present study proves that in OSCC, clock gene Per2 plays an important role in cell cycle progression and the balance of cell proliferation and apoptosis by regulation of the cyclin/CDK/CKI cell cycle network. Further research on Per2 may provide a new effective molecular target for cancer treatments.

Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied MS-based quantitative proteomics on isolated mitochondria from mice killed throughout the day and identified extensive oscillations in the mitochondrial proteome. Remarkably, the majority of cycling mitochondrial proteins peaked during the early light phase. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or on a high-fat diet. We propose that PERIOD proteins optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby, serve as a rheostat for mitochondrial nutrient utilization.

Organismal homeostasis relies on coherent interactions among tissues, specifically between brain-driven functions and peripheral metabolic organs. Hypothalamic circuits compute metabolic information to optimize energetic resources, but the role of the circadian clock in these pathways remains unclear. We have generated mice with targeted ablation of the core-clock gene Bmal1 within Sf1-neurons of the ventromedial hypothalamus (VMH). While this mutation does not affect the central clock in the suprachiasmatic nucleus (SCN), the VMH clock controls cyclic thermogenesis in brown adipose tissue (BAT), a tissue that governs energy balance by dissipating chemical energy as heat. VMH-driven control is exerted through increased adrenergic signaling within the sympathetic nervous system, without affecting the BAT's endogenous clock. Moreover, we show that the VMH circadian clock computes light and feeding inputs to modulate basal energy expenditure. Thus, we reveal a previously unsuspected circuit where an SCN-independent, hypothalamic circadian clock controls BAT function, energy expenditure, and thermogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (∼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length.

The circadian (∼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood. We found that a light- and circadian clock-regulated MAPK/MNK pathway led to phosphorylation of the cap-binding protein eIF4E in the mouse suprachiasmatic nucleus of the hypothalamus, the locus of the master circadian clock in mammals. Phosphorylation of eIF4E specifically promoted translation of Period 1 (Per1) and Period 2 (Per2) mRNAs and increased the abundance of basal and inducible PER proteins, which facilitated circadian clock resetting and precise timekeeping. Together, these results highlight a critical role for light-regulated translational control in the physiology of the circadian clock.

Beyond their established role in the mammalian circadian clock, recent studies have confirmed that the circadian genes have been implicated in tumor onset and progression. Currently, the biological effects of circadian genes on osteosarcoma cells' proliferation and migration are not well understood. Period2 (Per2) is one of the core circadian genes that act as master regulators of development and is frequently dysregulated in several cancers. However, the effects of human Per2 (hPer2) on the biological behavior of osteosarcoma cells are rarely reported. In the present study, to address the expression of hPer2 in osteosarcoma cells, the pEGFP-N1-hPer2 eukaryotic expression vector was constructed and transfected into cultured MG63 cells using Lipofectamine™ 2000. The overexpression of hPer2 in MG63 cells was verified by qRT-PCR and Western blotting, respectively. Finally, we investigated the effects of hPer2 protein overexpression on MG63 cells' viability, cycle, apoptosis, and invasive ability. In conclusion, the recombinant pEGFP-N1-hPer2 plasmid had been constructed successfully and expressed effectively in MG63 cells. Furthermore, results also showed that the viability, proliferation, and invasive abilities were suppressed, and the apoptosis was enhanced in MG63 cells. This preliminary study provides ground work for further research on the roles of circadian gene hPer2 in osteosarcoma cells MG63 and would offer promise for the development of novel therapeutic strategies in the treatment of osteosarcoma.

Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss., (© The Author(s).)

Rhythmic physiology is central to retinal function and survival and adapts vision to daily light intensity changes. Mammalian retina rhythmically releases melatonin when cultured under constant conditions, and the occurrence of clock gene [e.g., Period (Per)] expression has been shown for most cellular layers. However, contribution of the distinct layers to genesis of circadian rhythms within the retina is still debated. To characterize their endogenous oscillatory capacity and their communication at the whole-tissue level, we used a vibratome-based method to isolate individual or paired retina cellular layers from the mPer2(Luc) mouse and Per1-luciferase (Per1-Luc) rat, and real-time recorded bioluminescence. We report that each layer of the mouse retina harbors a self-sustained oscillator whose period is significantly longer (∼ 26 hours) than in whole-retina explants (∼ 22.9 hours), indicating that the period is correlated with the degree of coupling. Accordingly, the maximal period (∼ 29 hours) is reached upon complete enzymatic dissociation of the retina. By using pharmacological approaches, we demonstrate that connection between retina oscillators involves gap junctions but only minor contribution from the main retina neurochemicals. Taken together with results from Per1-Luc rats, these data show that mammalian retina consists of a network of layer-specific oscillators whose period is determined by their connectivity., (© FASEB.)

As a sensory micro-organ, pancreatic β-cells continually respond to nutritional signals and neuroendocrine input from other glucoregulatory organs. This sensory ability is essential for normal β-cell function and systemic glucose homeostasis. Period circadian protein (Per)-aryl hydrocarbon receptor nuclear translocator protein (Arnt)-single-minded protein (Sim) (PAS) domain proteins have a conserved role as sensory proteins, critical in adaptation to changes in voltage, oxygen potential, and xenobiotics. Within β-cells, PAS domain proteins such as hypoxia inducible factor 1α (Hif1α), Arnt, PAS kinase, Bmal1, and Clock respond to disparate stimuli, but act in concert to maintain proper β-cell function. Elucidating the function of these factors in islets offers a unique insight into the sensing capacity of β-cells, the consequences of impaired sensory function, and the potential to develop novel therapeutic targets for preserving β-cell function in diabetes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The period circadian clock 2 (per2) gene plays an important role in modulating the circadian rhythm in the central nervous system. Its protein product, PER2, is mainly expressed in the suprachiasmatic nucleus (SCN) and limbic system, including the central nucleus of the amygdala (CeA), the bed nucleus of the stria terminalis (BNST) and the hippocampus. PER2 rhythmic expression regulates hypothalamus‑pituitary‑adrenal (HPA) axis excitability and circadian rhythm via integration of optical signals and corticotropin‑releasing factor (CRF) stress‑related neurotransmitters, resulting in circadian rhythmicity in target organs. Moreover, glucocorticoids and glucocorticoid receptors exert strong negative feedback to the HPA axis and certain regions of the limbic system, modulating rhythmic per2 expression in peripheral organs. To date, the mechanism of action of PER2 in the limbic system and the HPA axis remains unclear, yet the per2 gene is considered valuable in clinical research for the study of metabolic syndromes, functional gastrointestinal disorders and certain liver diseases. In this review, we summarize the biological effects of the per2 gene and its protein product, PER2, in the limbic system, its involvement in regulation of the HPA axis by the limbic system and the resulting effects on the biological rhythm of target organs, and its clinical significance.

The mammalian circadian system synchronizes daily timing of activity and rest with the environmental light-dark cycle. Although the underlying molecular oscillatory mechanism is well studied, factors that influence phenotypic plasticity in daily activity patterns (temporal niche switching, chronotype) are presently unknown. Molecular evidence suggests that metabolism may influence the circadian molecular clock, but evidence at the level of the organism is lacking. Here we show that a metabolic challenge by cold and hunger induces diurnality in otherwise nocturnal mice. Lowering ambient temperature changes the phase of circadian light-dark entrainment in mice by increasing daytime and decreasing nighttime activity. This effect is further enhanced by simulated food shortage, which identifies metabolic balance as the underlying common factor influencing circadian organization. Clock gene expression analysis shows that the underlying neuronal mechanism is downstream from or parallel to the main circadian pacemaker (the hypothalamic suprachiasmatic nucleus) and that the behavioral phenotype is accompanied by phase adjustment of peripheral tissues. These findings indicate that nocturnal mammals can display considerable plasticity in circadian organization and may adopt a diurnal phenotype when energetically challenged. Our previously defined circadian thermoenergetics hypothesis proposes that such circadian plasticity, which naturally occurs in nocturnal mammals, reflects adaptive maintenance of energy balance. Quantification of energy expenditure shows that diurnality under natural conditions reduces thermoregulatory costs in small burrowing mammals like mice. Metabolic feedback on circadian organization thus provides functional benefits by reducing energy expenditure. Our findings may help to clarify relationships between sleep-wake patterns and metabolic phenotypes in humans.

The circadian clock plays an integral role in the regulation of physiological processes, including the regulation of blood pressure. However, deregulation of the clock can lead to pathophysiological states including hypertension. Recent work has implicated the circadian clock genes in the regulation of processes in the heart, kidney, vasculature, and the metabolic organs, which are all critical in the regulation of the blood pressure. The goal of this review is to provide an introduction and general overview into the role of circadian clock genes in the regulation of blood pressure with a focus on their deregulation in the etiology of hypertension. This review will focus on the core circadian clock genes CLOCK, BMAL1, Per, and Cry.

Circadian clocks in the peripheral tissues of mice are known to be entrained by pulse stimuli such as restricted feeding, novel wheel running, and several other agents. However, there are no reports on high temperature pulse-mediated entrainment on the phase-shift of peripheral clocks in vivo. Here we show that temperature treatment of mice for two days at 41°C, instead of 37°C, for 1-2 h during the inactive period, using a temperature controlled water bath stimulated phase-advance of peripheral clocks in the kidney, liver, and submandibular gland of PER2::LUCIFERASE mice. On the other hand, treatment for 2 days at 35°C ambient room temperature for 2 h did not cause a phase-advance. Maintenance of mice at 41°C in a water bath, sustained the core body temperature at 40-41°C. However, the use of 37°C water bath or the 35°C ambient room temperature elevated the core body temperature to 38.5°C, suggesting that at least a core body temperature of 40-41°C is necessary to cause phase-advance under light-dark cycle conditions. The temperature pulse stimulation at 41°C, instead of 37°C water bath for 2 h led to the elevated expression of Per1 and Hsp70 in the peripheral tissue of mice. In summary, the present study demonstrates that transient high temperature pulse using water bath during daytime causes phase-advance in mouse peripheral clocks in vivo. The present results suggest that hot water bath may affect the phase of peripheral clocks.

It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

The mammalian circadian clock is built on a molecular feedback loop in which the Period (PER) proteins, acting in a large, poorly understood complex, repress Clock-Bmal1, the transcription factor driving their expression. We found that mouse PER complexes include the histone methyltransferase HP1γ-Suv39h. PER proteins recruited HP1γ-Suv39h to the Per1 and Per2 promoters, and HP1γ-Suv39h proved important for circadian di- and trimethylation of histone H3 Lys9 (H3K9) at the Per1 promoter, feedback repression and clock function. HP1γ-Suv39h was recruited to the Per1 and Per2 promoters ~4 h after recruitment of HDAC1, a PER-associated protein previously implicated in clock function and H3K9 deacetylation at the Per1 promoter. PER complexes containing HDAC1 or HP1γ-Suv39h appeared to be physically separable. Circadian clock negative feedback by the PER complex thus involves dynamic, ordered recruitment of repressive chromatin modifiers to DNA-bound Clock-Bmal1.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker of the brain. It co-ordinates the daily rhythms of sleep and wakefulness, as well as physiology and behaviour, that set the tempo to our lives. Disturbance of this daily pattern, most acutely with jet-lag but more insidiously with rotational shift-work, can have severely deleterious effects for mental function and long-term health. The present review considers recent developments in our understanding of the properties of the SCN that make it a robust circadian time-keeper. It first focuses on the intracellular transcriptional/ translational feedback loops (TTFL) that constitute the cellular clockwork of the SCN neurone. Daily timing by these loops pivots around the negative regulation of the Period (Per) and Cryptochrome (Cry) genes by their protein products. The period of the circadian cycle is set by the relative stability of Per and Cry proteins, and this can be controlled by both genetic and pharmacological interventions. It then considers the function of these feedback loops in the context of cytosolic signalling by cAMP and intracellular calcium ([Ca(2+) ]i ), which are both outputs from, and inputs to, the TTFL, as well as the critical role of vasoactive intestinal peptide (VIP) signalling in synchronising cellular clocks across the SCN. Synchronisation by VIP in the SCN is paracrine, operating over an unconventionally long time frame (i.e. 24 h) and wide spatial domain, mediated via the cytosolic pathways upstream of the TTFL. Finally, we show how intersectional pharmacogenetics can be used to control G-protein-coupled signalling in individual SCN neurones, and how manipulation of Gq/[Ca(2+) ]i -signalling in VIP neurones can re-programme the circuit-level encoding of circadian time. Circadian pacemaking in the SCN therefore provides an unrivalled context in which to understand how a complex, adaptive behaviour can be organised by the dynamic activity of a relatively few gene products, operating in a clearly defined neuronal circuit, with both cell-autonomous and emergent, circuit-level properties., (© 2014 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of The British Society for Neuroendocrinology.)