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Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm--associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health. [ABSTRACT FROM AUTHOR]

Molecular and physiological determinants of the timing of reproductive events, including the pre-ovulatory LH surge and seasonal fluctuations in fertility, are incompletely understood. We used the Cryptochrome 1-deficient duper mutant to examine the role of this core circadian clock gene in Syrian hamsters. We find that the phase of the LH surge and its stability upon shifts of the light: dark cycle are altered in duper mutants. The intensity of immunoreactive PER1 in GnRH cells of the preoptic area peaks earlier in the day in duper than wild type hamsters. We note that GnRH fibers coursing through the suprachiasmatic nucleus (SCN) contact vasopressin- and VIP-immunoreactive cells, suggesting a possible locus of circadian control of the LH surge. Unlike wild types, duper hamsters do not regress their gonads within 8 weeks of constant darkness, despite evidence of melatonin secretion during the subjective night. In light of the finding that the duper allele is a stop codon in Cryptochrome 1, our results suggest important neuroendocrine functions of this core circadian clock gene. [ABSTRACT FROM AUTHOR]

Environmental rhythms such as the daily light-dark cycle selected for endogenous clocks. These clocks predict regular environmental changes and provide the basis for well-timed adaptive homeostasis in physiology and behavior of organisms. Endogenous clocks are oscillators that are based on positive feedforward and negative feedback loops. They generate stable rhythms even under constant conditions. Since even weak interactions between oscillators allow for autonomous synchronization, coupling/synchronization of oscillators provides the basis of self-organized physiological timing. Amongst the most thoroughly researched clocks are the endogenous circadian clock neurons in mammals and insects. They comprise nuclear clockworks of transcriptional/translational feedback loops (TTFL) that generate ~24 h rhythms in clock gene expression entrained to the environmental day-night cycle. It is generally assumed that this TTFL clockwork drives all circadian oscillations within and between clock cells, being the basis of any circadian rhythm in physiology and behavior of organisms. Instead of the current gene-based hierarchical clock model we provide here a systems view of timing. We suggest that a coupled system of autonomous TTFL and posttranslational feedback loop (PTFL) oscillators/clocks that run at multiple timescales governs adaptive, dynamic homeostasis of physiology and behavior. We focus on mammalian and insect neurons as endogenous oscillators at multiple timescales. We suggest that neuronal plasma membrane-associated signalosomes constitute specific autonomous PTFL clocks that generate localized but interlinked oscillations of membrane potential and intracellular messengers with specific endogenous frequencies. In each clock neuron multiscale interactions of TTFL and PTFL oscillators/clocks form a temporally structured oscillatory network with a common complex frequency-band comprising superimposed multiscale oscillations. Coupling between oscillator/clock neurons provides the next level of complexity of an oscillatory network. This systemic dynamic network of molecular and cellular oscillators/clocks is suggested to form the basis of any physiological homeostasis that cycles through dynamic homeostatic setpoints with a characteristic frequency-band as hallmark. We propose that mechanisms of homeostatic plasticity maintain the stability of these dynamic setpoints, whereas Hebbian plasticity enables switching between setpoints via coupling factors, like biogenic amines and/or neuropeptides. They reprogram the network to a new common frequency, a new dynamic setpoint. Our novel hypothesis is up for experimental challenge. [ABSTRACT FROM AUTHOR]

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24 h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12 h light: 12 h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila. [ABSTRACT FROM AUTHOR]

Circadian clocks orchestrate a variety of physiological and behavioural functions within the 24-h day. These timekeeping systems have also been implicated in developmental and reproductive processes that span more (or less) than 24 h. Whether natural alleles of cardinal clock genes affect entire sets of life-history traits (i.e., reproductive arrest, developmental time, fecundity), thus providing a wider substrate for seasonal adaptation, remains unclear. Here we show that natural alleles of the timeless (tim) gene of Drosophila melanogaster, previously shown to modulate flies' propensity to enter reproductive dormancy, differentially affect correlated traits such as early-life fecundity and developmental time. Homozygous flies expressing the shorter TIM isoform (encoded by the s-tim allele) not only show a lower dormancy incidence compared to those homozygous for ls-tim (which produce both the short and an N-terminal additional 23-residues longer TIM isoform), but also higher fecundity in the first 12 days of adult life. Moreover, s-tim homozygous flies develop faster than ls-tim homozygous flies at both warm (25°C) and cold (15°C) temperatures, with the gap being larger at 15°C. In summary, this phenotypic analysis shows that natural variants of tim affect a set of life-history traits associated with reproductive dormancy in Drosophila. We speculate that this provides further adaptive advantage in temperate regions (with seasonal changes) and propose that the underlying mechanisms might not be exclusively dependent on photoperiod, as previously suggested. [ABSTRACT FROM AUTHOR]

The study of circadian rhythms in bacteria was transformed by studies of the cyanobacterium Synechococcus elongatus. However, in a number of respects S. elongatus is atypical, and while those unusual characteristics were helpful for rapid progress in the past, another commonly used cyanobacterial species, Synechocystis sp. PCC 6803, may be more representative and therefore more productive for future insights into bacterial clock mechanisms. In the past, circadian studies of Synechocystis have suffered from not having an excellent reporter of circadian gene expression, but we introduce here a new luminescence reporter that rivals the reporters that have been used so successfully in S. elongatus. Using this new system, we generate for the first time in Synechocystis circadian period mutants resulting from point mutations. The temperature compensation and dark-pulse resetting that mediates entrainment to the environment is characterized. Moreover, we analyse the complex organization of clock genes in Synechocystis and identify which genes are essential for circadian rhythmicity and adaptive fitness for entrainment and optimal phase alignment to environmental cycles (and which genes are not). These developments will provide impetus for new approaches towards understanding daily timekeeping mechanisms in bacteria. [ABSTRACT FROM AUTHOR]

There has been considerable progress in understanding follicular development, the ovulatory cycle and the oviposition cycles in the hen. In particular, there have been tremendous advances in understanding follicular development and recruitment of follicles to the hierarchy of large yellow follicles. There is a need to continue to follow the earlier literature while employing present techniques. Early work allowed an understanding of the ovulation and oviposition cycles. Models for ovulation were developed. However, while these have no passed the test of time, there is no present model that fully accounts to the cycles. Earlier work employed ahemoral light cycles to examine ovulation and oviposition cycles. Recent work has demonstrated that clock genes are expressed in the ovary. The control of incubation by prolactin has been largely elucidated in turkeys. There is evidence that other endocrine glands influence female reproduction in birds including the adrenal cortex, thyroid and pineal. However, there is much that remains to be fully understood. [ABSTRACT FROM AUTHOR]

Objective: Tooth morphogenesis and the formation of hard tissues have been reported to be closely related to circadian rhythms. This study investigates the spatiotemporal expression and relationship of p75NTR with core clock genes, mineralization-related or odontogenesis-related genes, and aims to derive the potential role of p75NTR in regulating circadian rhythm and incrementality growth line formation during tooth development. Materials and methods: The dynamic morphology of the rat dental germ was observed at seven stages (E14.5 d, E16.5 d, E18.5 d, P.N. 4 d, P.N. 7 d, P.N. 10 d, and P.N. 15 d). Next, the expressions of p75NTR and other target factors were traced. The ectomesenchymal stem cells (EMSCs) were isolated from the E18.5d rat dental germs and synchronized using 50% of fetal bovine serum. Then, they were cultured in light/light (L.L.), dark/dark (D.D.), and light/dark (L.D.) conditions for 48 h. The total RNA was collected every 4 h, and the circadian rhythm dynamics of target factors were observed. To reveal the mechanism further, p75NTR was down-regulated in p75NTRExIII−/− mice and up-regulated in immortalized mouse dental apical papilla progenitor cells. The change tendencies of other target factors were also detected. Results: The clock genes Bmal1, Clock, Per1, and Per2 were all expressed in tooth germs before the formation of dental hard tissues and demonstrated a regular oscillating expression pattern in EMSCs from dental germs. Their expression was affected by the L.D. stimulus, and most of them were promoted by D.D. conditions. p75NTR presented a similar expression pattern and a positive or negative relationship with most clock genes, mineralization-related and odontogenesis-related factors, such as brain and muscle ARNT-like protein-1 (Bmal1), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), MSH-like 1 (MSX1), dentin matrix acidic phosphoprotein 1 (Dmp1), and dentin sialophosphoprotein (Dspp). Moreover, the arrangement, morphology, and even boundary in pre-odontoblast/pre-ameloblast layers were disordered in the p75NTRExIII−/− mice. Conclusion: Circadian rhythm was found to affect tooth development. p75NTR might play a crucial role in regulating clock genes in the mineralization and formation of the dental hard tissues. p75NTR is actively involved in the odontoblast-ameloblast junction and cell polarity establishment during tooth morphogenesis. [ABSTRACT FROM AUTHOR]

Modern lifestyle reduces environmental rhythmicity and may lead to circadian desynchrony. We are exposed to poor day-time lighting indoors and excessive night-time artificial light. We use air-conditioning to reduce ambient temperature cycle, and food is regularly available at all times. These disruptions of daily rhythms may lead to type 2 diabetes mellitus (T2DM), obesity, cardiometabolic diseases (CMD), depression and anxiety, all of which impose major public health and economic burden on societies. Therefore, we need appropriate animal models to gain a better understanding of their etiologic mechanisms, prevention, and management.We argue that the fat sand rat (Psammomys obesus), a diurnal animal model, is most suitable for studying the effects of modern-life conditions. Numerous attributes make it an excellent model to study human health disorders including T2DM, CMD, depression and anxiety. Here we review a comprehensive series of studies we and others conducted, utilizing the fat sand rat to study the underlying interactions between biological rhythms and health. Understanding these interactions will help deciphering the biological basis of these diseases, which often occur concurrently. We found that when kept in the laboratory (compared with natural and semi-wild outdoors conditions where they are diurnal), fat sand rats show low amplitude, nocturnal or arrhythmic activity patterns, dampened daily glucose rhythm, glucose intolerance, obesity and decreased survival rates. Short photoperiod acclimation exacerbates these pathologies and further dampens behavioral and molecular daily rhythms, resulting in CMD, T2DM, obesity, adipocyte dysfunction, cataracts, depression and anxiety. Increasing environmental rhythmicity by morning bright light exposure or by access to running wheels strengthens daily rhythms, and results in higher peak-to-trough difference in activity, better rhythmicity in clock genes expression, lower blood glucose and insulin levels, improved glucose tolerance, lower body and heart weight, and lower anxiety and depression. In summary, we have demonstrated that fat sand rats living under the correspondent of “human modern lifestyle” conditions exhibit dampened behavioral and biological rhythms and develop circadian desynchrony, which leads to what we have named “The Circadian Syndrome”. Environmental manipulations that increase rhythmicity result in improvement or prevention of these pathologies. Similar interventions in human subjects could have the same positive results and further research on this should be undertaken. [ABSTRACT FROM AUTHOR]

High-fat diet (HFD) feeding rewires circadian rhythms of peripheral organs including the liver and adipose tissue. While the liver has been extensively studied, it remains largely unknown whether and how HFD organizes circadian biology in adipose tissue. Here, we took a systems approach to profile the diurnal transcriptome of adipose tissue in diet-induced obese mice either fed a low-fat diet (LFD) that reduces weight or still fed HFD. We detected about 200 and 2,500 diurnal genes in HFD and LFD, respectively. Pathway analysis revealed that rhythmic pathways in HFD are represented by circadian rhythm, ribosome biogenesis, and nucleosome organization, whereas those in LFD are represented by myeloid cell function. Remarkably, the majority of the circadian clock genes, except Clock, exhibited robust diurnal rhythm in the adipose tissue of HFD-fed mice. Analysis of mRNAs and proteins in another cohort of HFD-fed mice confirmed that Clock lost rhythmicity at the transcript, but not protein level. Diet reversal to LFD specifically restored diurnal difference of the Clock transcripts in adipose tissue. We matched transcriptomics data with global profiling of neutral lipids and found that lipid metabolism catalyzed by triglycerol hydrolase Cesld is a key circadian feature that is activated by diet reversal. Together, our work defines the circadian signatures in the adipose tissue of diet-induced obese mice, and their flexibility upon dietary intervention, thereby shedding light on potential clock-modulated tissue-specific pathways during obesity. [ABSTRACT FROM AUTHOR]

Nocturnal light pollution has been rapidly increasing during the last decades and even though dim artificial light at night (ALAN) has been associated with metabolic diseases, its mechanism is still far from clear. Therefore, the aim of our study was to thoroughly analyze the effects of ALAN on energy metabolism, metabolites, metabolic hormones, and gene expression. Male Wistar rats were kept in either the standard light:dark (12:12) cycle or exposed to ALAN (~2 lx) during the whole 12-h dark phase for 2 weeks. Energy metabolism was measured in metabolic cages. In addition, we measured plasma and hepatic metabolites, clock and metabolic gene expression in the liver and epididymal adipose tissue, and plasma hormone levels. In ALAN rats, we observed an unexpected transitory daytime peak of locomotor activity and a suppression of the peak in locomotor activity at the beginning of the dark period. These changes were mirrored in the respiratory exchange ratio. Plasma metabolites became arrhythmic, and plasma and hepatic cholesterol levels were increased. Lost rhythmicity of metabolites was associated with disrupted behavioral rhythms and expression of metabolic genes. In the liver, the rhythms of metabolic sensors were either phase-advanced (Ppara, Pgc1a, Nampt) or arrhythmic (Sirt1, Lxra) after ALAN. The rhythmic pattern of Ppara and Sirt1 was abolished in the adipose tissue. In the liver, the amplitude of the daily rhythm in glycogen content was attenuated, the Glut2 rhythm was phase-advanced and Foxo1 lost its daily rhythmicity. Moreover, hepatic Foxo1 and Gck were up-regulated after ALAN. Interestingly, several parameters of lipid metabolism gained rhythmicity (adiponectin, Hmgcs2, Lpl, Srebf1c) in the liver, whereas Noct became arrhythmic in the adipose tissue. Peripheral clock genes maintained their robust oscillations with small shifts in their acrophases. Our data show that even a low level of ALAN can induce changes in the daily pattern of behavior and energy metabolism, and disturb daily rhythms of genes encoding key metabolic sensors and components of metabolic pathways in the liver and adipose tissue. Disturbed metabolic rhythms by ALAN could represent a serious risk factor for the development and progression of metabolic diseases. [ABSTRACT FROM AUTHOR]

Objectives: Obstructive sleep apnea (OSA) is an independent risk factor for metabolic syndrome (MetS). Recent studies have indicated that circadian clock genes were dysregulated in OSA. In addition, it is clear that the impairment of circadian clocks drives the progression of MetS. Therefore, we hypothesized that circadian rhythm disruption links OSA with MetS. Methods: A total of 118 participants, who underwent polysomnography (PSG) and were diagnosed as healthy snorers (control, n = 29) or OSA (n = 89) patients based on the apnea-hypopnea index (AHI), were enrolled in the present study. General information, anthropometric data, blood biochemical indicators, clock gene expressions, and levels of oxidative and inflammatory indicators were collected, determined, and compared in all the participants. Results: We found that Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Differentiated embryo chondrocyte 1 (Dec1) were upregulated, while Period 1 (Per1) was reduced in OSA patients. In addition, these changing trends were closely associated with the hypoxia indicator of AHI and have a significant impact on the presence of MetS components, such as hyperglycemia (Dec1 and Per1, p < 0.05 and 0.001, respectively), hypertension (Bmal1 and Dec1, p < 0.001 and 0.01, respectively), hyperlipidemia (Dec1, p < 0.01), and obesity (Dec1, p < 0.05). Notably, expressions of Dec1 correlated with IR and predicted the presence of MetS in OSA patients. Finally, we also observed that Dec1 expression was interrelated with levels of both oxidative indicators and inflammatory biomarkers (IL-6) in OSA. Conclusion: This study concluded that circadian clock disruptions, especially Dec1, link OSA with MetS in an oxidative and inflammatory-related manner. Circadian clock Dec1 can be used as a specific biomarker (p < 0.001) and therapeutic target in OSA combined with Mets patients. [ABSTRACT FROM AUTHOR]

The photoperiodic system is concealed in the highly complex black-box, comprising four functional subunits: 1) a photo/thermo-sensitive input unit, 2) a photoperiodic clock based on a circadian system, 3) a condenser unit counting the number of inductive signals, and 4) a neuroendocrine switch that triggers a phenotypic shift. This review aims to summarize the research history and current reach of our understanding on this subject to connect it with the molecular mechanism of the circadian clock rapidly being unveiled. The review also focuses on the mode of intersubunit information transduction. It will scan the recent advancement in research on each functional subunit, but special attention will be given to the circadian clock–endocrine conjunct and the role of melatonin signaling in the regulation of insect photoperiodism. Prothoracicotropic hormone (PTTH) probably plays the most crucial role in the regulation of pupal diapause, which is the simplest model system of diapause regulation by hormones investigated so far, particularly in the Chinese oak silkmoth (Antheraea pernyi). A search for the trigger to release the PTTH found some candidates, that is, indoleamines. Indolamine metabolism is controlled by arylalkylamine N -acetyltransferase (aaNAT). Indolamine dynamics and aaNAT enzymatic activity changed according to photoperiods. aaNAT activity and melatonin content in the brain showed not only a photoperiodic response but also a circadian fluctuation. aaNAT had multiple E-boxes, suggesting that it is a clock-controlled gene (ccg), which implies that cycle (cyc, or brain–muscle Arnt-like 1 = Bmal1)/Clock (Clk) heterodimer binds to E-box and stimulates the transcription of aaNAT , which causes the synthesis of melatonin. RNAi against transcription modulators, cyc, or Clk downregulated aaNAT transcription, while RNAi against repressor of cyc/Clk, per upregulated aaNAT transcription. Immunohistochemical localization showed that the circadian neurons carry epitopes of melatonin-producing elements such as aaNAT, the precursor serotonin, HIOMT, and melatonin as well as clock gene products such as cyc-ir, Per-ir, and dbt-ir, while PTTH-producing neurons juxtaposed against the clock neurons showed hMT2-ir in A. pernyi brain. Melatonin probably binds to the putative melatonin receptor (MT) that stimulates Ca2+ influx, which in turn activates PKC. This induces Rab 8 phosphorylation and exocytosis of PTTH, leading to termination of diapause. All the PTTH-expressing neurons have PKC-ir, and Rab8-ir. When diapause is induced and maintained under short days, serotonin binding to 5HTR1B suppresses PTTH release in a yet unknown way. RNAi against this receptor knocked out photoperiodism; short day response is blocked and diapause was terminated even under the short day condition. The result showed that a relatively simple system controls both induction and termination in pupal diapause of A. pernyi : the circadian system regulates the transcription of aaNAT as a binary switch, the enzyme produces a melatonin rhythm that gates PTTH release, and 5HTR1B and MT are probably also under photoperiodic regulation. Finally, we listed the remaining riddles which need to be resolved, to fully understand this highly complex system in future studies. [ABSTRACT FROM AUTHOR]

The expression of circadian clock genes, either centrally or in the periphery, has been shown to play an integral role in the control of behavior. Brain region-specific downregulation of clock genes revealed behavioral phenotypes associated with neuropsychiatric disorders and neurodegenerative disease. The specific function of the clock genes as well as the underlying mechanisms that contribute to the observed phenotypes, however, are not yet fully understood. We assessed anxiety- and depressive-like behavior and motor functions in male and female mice with a conditional ablation of Bmal1 or Per2 from medium spiny neurons (MSNs) of the striatum as well as mice lacking one copy of Gpr88. Whereas the conditional knockout of Bmal1 and Per2 had mild effects on affective behaviors, a pronounced effect on motor functions was found in Bmal1 knockout mice. Subsequent investigation revealed an attenuated response of Bmal1 knockout mice to dopamine receptor type 1 agonist treatment, independently of the expression of targets of the dopamine signaling pathway or mitochondrial respiration in MSNs. The study thus suggests a potential interaction of Bmal1 within the direct dopamine signaling pathway, which may provide the link to a shared, MSN-dependent mechanism regulating affective behavior and motor function in mice. [ABSTRACT FROM AUTHOR]

Circadian control allows organisms to anticipate and adapt to environmental changes through changes in physiology and behavior. The circadian system timing is entrained by cues, such as light, food, and temperature. An ambient temperature dramatically impacts the sleep–wake cycle and metabolic rhythmicity. As endotherms, mammals rely on tissues such as the liver to provide fuel for thermogenesis to maintain body temperature. The adaptive response of the circadian rhythm of liver metabolism to chronic cold exposure remains largely unexplored. Here, we investigated the circadian rhythm adaptation of hepatic metabolism in response to environmental cold stress using a mouse model of chronic cold exposure. We analyzed metabolites and transcripts of mouse livers at 24 h and found that long-term low-temperature exposure resulted in a synergistic and phase synchronization of transcriptional rhythms of many genes associated with metabolic pathways. Notably, transcription peaked in the early light phase when the body temperature was relatively low. Our results suggest that chronic cold does not alter the rhythmic expression of essential core clock genes in the liver, so the rewiring of clock control gene expression is another mechanism that optimizes the circadian rhythm of liver metabolism to meet the energy requirements of animal thermogenesis. [ABSTRACT FROM AUTHOR]

The expression of circadian clock genes, either centrally or in the periphery, has been shown to play an integral role in the control of behavior. Brain region-specific downregulation of clock genes revealed behavioral phenotypes associated with neuropsychiatric disorders and neurodegenerative disease. The specific function of the clock genes as well as the underlying mechanisms that contribute to the observed phenotypes, however, are not yet fully understood. We assessed anxiety- and depressive-like behavior and motor functions in male and female mice with a conditional ablation of Bmal1 or Per2 from medium spiny neurons (MSNs) of the striatum as well as mice lacking one copy of Gpr88. Whereas the conditional knockout of Bmal1 and Per2 had mild effects on affective behaviors, a pronounced effect on motor functions was found in Bmal1 knockout mice. Subsequent investigation revealed an attenuated response of Bmal1 knockout mice to dopamine receptor type 1 agonist treatment, independently of the expression of targets of the dopamine signaling pathway or mitochondrial respiration in MSNs. The study thus suggests a potential interaction of Bmal1 within the direct dopamine signaling pathway, which may provide the link to a shared, MSN-dependent mechanism regulating affective behavior and motor function in mice.

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila , PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus , an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect's circadian and photoperiodic systems. [ABSTRACT FROM AUTHOR]

The bivoltine strain of the domestic silkworm, Bombyx mori , exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per , tim , Clk , cyc , and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per , tim , Clk , cyc , and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms. [ABSTRACT FROM AUTHOR]

The circadian clock maintains our health by controlling physiological functions. Social jet lag is one factor that can disrupt the body clock. This is caused by the difference in sleeping hours between weekdays when we live according to social time and holidays when we live according to our body clock. The body clock can be altered by exercise, nutrition, and stress, and several studies have reported that these factors can be used to improve a disturbed body clock. Here we focused on exercise and examined whether continuous wheel-running could improve the disordered body clock in a mouse model that mimics social jet lag. The results showed that the wheel-running exercise group showed faster synchronization of the onset of activities on weekdays which had been delayed by social jet lag and the results were even more pronounced in the high-fat diet feeding condition. Also, when the expression rhythms of the clock genes were examined, they experienced a sudden time shift in the advance light condition or social jet lag condition, it was found that the wheel-running group had a higher ability to adapt to the advance direction. Thus, it is possible that the effective inclusion of exercise in human, especially those who eat high-fat foods, life can improve the disordered body clock in terms of social jet lag. [ABSTRACT FROM AUTHOR]

To better understand the complexity of clock genes in salmonids, a taxon with an additional whole genome duplication, an analysis was performed to identify and classify gene family members (clock, arntl, period, cryptochrome, nr1d, ror , and csnk1). The majority of clock genes, in zebrafish and Northern pike, appeared to be duplicated. In comparison to the 29 clock genes described in zebrafish, 48 clock genes were discovered in salmonid species. There was also evidence of species-specific reciprocal gene losses conserved to the Oncorhynchus sister clade. From the six period genes identified three were highly significantly rhythmic, and circadian in their expression patterns (per1a.1, per1a.2, per1b) and two was significantly rhythmically expressed (per2a, per2b). The transcriptomic study of juvenile Atlantic salmon (parr) brain tissues confirmed gene identification and revealed that there were 2,864 rhythmically expressed genes (p < 0.001), including 1,215 genes with a circadian expression pattern, of which 11 were clock genes. The majority of circadian expressed genes peaked 2 h before and after daylight. These findings provide a foundation for further research into the function of clock genes circadian rhythmicity and the role of an enriched number of clock genes relating to seasonal driven life history in salmonids. [ABSTRACT FROM AUTHOR]

The control of the biological rhythms begins with the activation of photo- and thermosensitive cells located in various organs of the fish such as brain, eye, and skin, but a central clock is still to be identified in teleosts. Thermal changes are stressors which increase cortisol and affect the rhythm of other hormones such as melatonin and growth hormone (GH), in both endo- and ectothermic organisms. Our aim was to investigate how temperature (23°C for 6 days) lower than the optimal (28°C) modulates expression of several gene pathways including growth hormone (gh1) and its receptors (ghra, ghrb), insulin-like growth factor1 (igf1a, igf1b) and its receptors (igf1ra, igf1rb), cortisol and its receptor (gr), the limiting enzyme of melatonin synthesis (arylalkylamine N-acetyltransferase, aanat) and melatonin receptors (mtnr1aa, mtnr1bb), as well as their relationship with clock genes in Danio rerio in early light and early dark phases of the day. Lower temperature reduced the expression of the hormone gene gh1 , and of the related receptors ghra, ghrb, igf1ra , and igf1rb. Cortisol levels were higher at the lower temperature, with a decrease of its receptor (gr) transcripts in the liver. Interestingly, we found higher levels of aanat transcripts in the brain at 23°C. Overall, lower temperature downregulated the transcription of hormone related genes and clock genes. The results suggest a strong correlation of temperature challenge with the clock molecular mechanism and the endocrine systems analyzed, especially the growth hormone and melatonin axes, in D. rerio tissues. [ABSTRACT FROM AUTHOR]

To better understand the complexity of clock genes in salmonids, a taxon with an additional whole genome duplication, an analysis was performed to identify and classify gene family members (clock, arntl, period, cryptochrome, nr1d, ror, and csnk1). The majority of clock genes, in zebrafish and Northern pike, appeared to be duplicated. In comparison to the 29 clock genes described in zebrafish, 48 clock genes were discovered in salmonid species. There was also evidence of species-specific reciprocal gene losses conserved to the Oncorhynchus sister clade. From the six period genes identified three were highly significantly rhythmic, and circadian in their expression patterns (per1a.1, per1a.2, per1b) and two was significantly rhythmically expressed (per2a, per2b). The transcriptomic study of juvenile Atlantic salmon (parr) brain tissues confirmed gene identification and revealed that there were 2,864 rhythmically expressed genes (p < 0.001), including 1,215 genes with a circadian expression pattern, of which 11 were clock genes. The majority of circadian expressed genes peaked 2 h before and after daylight. These findings provide a foundation for further research into the function of clock genes circadian rhythmicity and the role of an enriched number of clock genes relating to seasonal driven life history in salmonids.

The control of the biological rhythms begins with the activation of photo- and thermosensitive cells located in various organs of the fish such as brain, eye, and skin, but a central clock is still to be identified in teleosts. Thermal changes are stressors which increase cortisol and affect the rhythm of other hormones such as melatonin and growth hormone (GH), in both endo- and ectothermic organisms. Our aim was to investigate how temperature (23°C for 6 days) lower than the optimal (28°C) modulates expression of several gene pathways including growth hormone (gh1) and its receptors (ghra, ghrb), insulin-like growth factor1 (igf1a, igf1b) and its receptors (igf1ra, igf1rb), cortisol and its receptor (gr), the limiting enzyme of melatonin synthesis (arylalkylamine N-acetyltransferase, aanat) and melatonin receptors (mtnr1aa, mtnr1bb), as well as their relationship with clock genes in Danio rerio in early light and early dark phases of the day. Lower temperature reduced the expression of the hormone gene gh1, and of the related receptors ghra, ghrb, igf1ra, and igf1rb. Cortisol levels were higher at the lower temperature, with a decrease of its receptor (gr) transcripts in the liver. Interestingly, we found higher levels of aanat transcripts in the brain at 23°C. Overall, lower temperature downregulated the transcription of hormone related genes and clock genes. The results suggest a strong correlation of temperature challenge with the clock molecular mechanism and the endocrine systems analyzed, especially the growth hormone and melatonin axes, in D. rerio tissues.

The central circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, but an SCN-dependent molecular circadian oscillator is present in the cerebellar cortex. Recent findings suggest that circadian release of corticosterone is capable of driving the circadian oscillator of the rat cerebellum. To determine if additional neuroendocrine signals act to shape cerebellar clock gene expression, we here tested the role of the thyroid hormone triiodothyronine (T3) in regulation of the cerebellar circadian oscillator. In cultured cerebellar granule cells from mixed-gender neonatal rats, T3 treatment affected transcript levels of the clock genes Per2 , Arntl , Nr1d1 , and Dbp , suggesting that T3 acts directly on granule cells to control the circadian oscillator. We then used two different in vivo protocols to test the role of T3 in adult female rats: Firstly, a single injection of T3 did not influence clock gene expression in the cerebellum. Secondly, we established a surgical rat model combining SCN lesion with a programmable micropump infusing circadian physiological levels of T3; however, rhythmic infusion of T3 did not reestablish differential clock gene expression between day and night in SCN lesioned rats. To test if the effects of T3 observed in vitro were related to the developmental stage, acute injections of T3 were performed in mixed-gender neonatal rats in vivo ; this procedure significantly affected cerebellar expression of the clock genes Per1 , Per2 , Nr1d1 , and Dbp. Developmental comparisons showed rhythmic expression of all clock genes analyzed in the cerebellum of adult rats only, whereas T3 responsiveness was limited to neonatal animals. Thus, T3 shapes cerebellar clock gene profiles in early postnatal stages, but it does not represent a systemic circadian regulatory mechanism linking the SCN to the cerebellum throughout life. [ABSTRACT FROM AUTHOR]

Circadian clocks are cell-autonomous endogenous oscillators, generated and maintained by self-sustained 24-h rhythms of clock gene expression. In the fruit fly Drosophila melanogaster , these daily rhythms of gene expression regulate the activity of approximately 150 clock neurons in the fly brain, which are responsible for driving the daily rest/activity cycles of these insects. Despite their endogenous character, circadian clocks communicate with the environment in order to synchronize their self-sustained molecular oscillations and neuronal activity rhythms (internal time) with the daily changes of light and temperature dictated by the Earth's rotation around its axis (external time). Light and temperature changes are reliable time cues (Zeitgeber) used by many organisms to synchronize their circadian clock to the external time. In Drosophila , both light and temperature fluctuations robustly synchronize the circadian clock in the absence of the other Zeitgeber. The complex mechanisms for synchronization to the daily light–dark cycles are understood with impressive detail. In contrast, our knowledge about how the daily temperature fluctuations synchronize the fly clock is rather limited. Whereas light synchronization relies on peripheral and clock-cell autonomous photoreceptors, temperature input to the clock appears to rely mainly on sensory cells located in the peripheral nervous system of the fly. Recent studies suggest that sensory structures located in body and head appendages are able to detect temperature fluctuations and to signal this information to the brain clock. This review will summarize these studies and their implications about the mechanisms underlying temperature synchronization. [ABSTRACT FROM AUTHOR]

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world’s leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. We observed that timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte, an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock. Aedes aegypti is one of the world's leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affect Aedes locomotor activity. We observed that timeless completely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown of nocte , an important gene for Drosophila circadian synchronization by temperature cycles. Thus, timeless and nocte are important genes for synchronization by temperature cycles in Aedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock of Ae. aegypti differs significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior of Aedes with possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study. [ABSTRACT FROM AUTHOR]

Circadian rhythms form a self-sustaining, endogenous, time-keeping system that allows organisms to anticipate daily environmental changes. The core of the clock network consists of interlocking transcriptional-translational feedback loops that ensures that metabolic, behavioral, and physiological processes run on a 24 h timescale. The hierarchical nature of the clock manifests itself in multiple points of control on the daily cell division cycle, which relies on synthesis, degradation, and post-translational modification for progression. This relationship is particularly important for understanding the role of clock components in sensing stress conditions and triggering checkpoint signals that stop cell cycle progression. A case in point is the interplay among the circadian factor PERIOD2 (PER2), the tumor suppressor p53, and the oncogenic mouse double minute-2 homolog protein (MDM2), which is the p53’s negative regulator. Under unstressed conditions, PER2 and p53 form a stable complex in the cytosol and, along with MDM2, a trimeric complex in the nucleus. Association of PER2 to the C-terminus end of p53 prevents MDM2-mediated ubiquitylation and degradation of p53 as well as p53’s transcriptional activation. Remarkably, when not bound to p53, PER2 acts as substrate for the E3-ligase activity of MDM2; thus, PER2 is degraded in a phosphorylation-independent fashion. Unexpectedly, the phase relationship between PER2 and p53 are opposite; however, a systematic modeling approach, inferred from the oscillatory time course data of PER2 and p53, aided in identifying additional regulatory scenarios that explained, a priori, seemingly conflicting experimental data. Therefore, we advocate for a combined experimental/mathematical approach to elucidating multilevel regulatory cellular processes.

The fibrinolytic system is critical during the onset of fibrinolysis, a fundamental mechanism for fibrin degradation. Both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) trigger fibrinolysis, leading to proteolytic activation of plasminogen to plasmin and subsequently fibrin proteolysis. This system is regulated by several inhibitors; plasminogen activator inhibitor-1 (PAI-1), the most studied, binds to and inactivates both tPA and uPA. Through the action of plasmin, this system regulates several physiological processes: embryogenesis, activation of inflammatory cells, cell proliferation and death, synaptic plasticity, wound healing, and others. The deregulated intervention of fibrinolysis in the pathophysiology of various diseases has been widely studied; findings of altered functioning have been reported in different chronic non-communicable diseases (NCD), reinforcing its pleiotropic character and the importance of its physiology and regulation. The evidence indicates that fundamental elements of the fibrinolytic system, such as tPA and PAI-1, show a circadian rhythm in their plasmatic levels and their gene expression are regulated by circadian system elements, known as clock genes – Bmal, Clock, Cry-, and accessory clock genes such as Rev-Erb and Ror. The disturbance in the molecular machinery of the clock by exposure to light during the night alters the natural light/dark cycle and causes disruption of the circadian rhythm. Such exposure affects the synchronization and functioning of peripheral clocks responsible for the expression of the components of the fibrinolytic system. So, this circadian disturbance could be critical in the pathophysiology of chronic diseases where this system has been found to be deregulated.

Experimental and epidemiological studies have revealed a relationship between an adverse intrauterine environment and chronic non-communicable disease (NCD) like cardiovascular disease (CVD) in adulthood. An important risk factor for CVD is the deregulation of the fibrinolytic system particularly high levels of expression of plasminogen activator inhibitor 1 (Pai-1). Chronic exposure to altered photoperiod disrupts the circadian organization of physiology in the pregnant female, known as gestational chronodisruption, and cause long-term effects on the adult offspring’s circadian physiology. The Pai-1 expression is regulated by the molecular components of the circadian system, termed clock genes. The present study aimed to evaluate the long-term effects of chronic photoperiod shifts (CPS) during pregnancy on the expression of the clock genes and the fibrinolytic system in the liver of adult male offspring. Our results using an animal model demonstrated statistically significant differences at the transcriptional level in males gestated under CPS. At 90 days of postnatal age, the liver transcript levels of the clock gene Bmal1 were downregulated, whereas Rorα, Rorγ, Nfil3, and Pai-1 were upregulated. Our data indicate that CPS during pregnancy affects gene expression in the liver of male adult progeny, showing that alteration of the photoperiod in the mother’s environment leads to persistent effects in the offspring. In conclusion, these results reveal for the first time the long-term effects of gestational chronodisruption on the transcriptional activity of one well-established risk factor associated with CVD in the adult male offspring.

Heavy alcohol drinking dysregulates lipid metabolism, promoting hepatic steatosis – the first stage of alcohol-related liver disease (ALD). The molecular circadian clock plays a major role in synchronizing daily rhythms in behavior and metabolism and clock disruption can cause pathology, including liver disease. Previous studies indicate that alcohol consumption alters liver clock function, but the impact alcohol or clock disruption, or both have on the temporal control of hepatic lipid metabolism and injury remains unclear. Here, we undertook studies to determine whether genetic disruption of the liver clock exacerbates alterations in lipid metabolism and worsens steatosis in alcohol-fed mice. To address this question, male liver-specific Bmal1 knockout (LKO) and flox/flox (Fl/Fl) control mice were fed a control or alcohol-containing diet for 5 weeks. Alcohol significantly dampened diurnal rhythms of mRNA levels in clock genes Bmal1 and Dbp , phase advanced Nr1d1 /REV-ERBα, and induced arrhythmicity in Clock , Noct , and Nfil3 /E4BP4, with further disruption in livers of LKO mice. Alcohol-fed LKO mice exhibited higher plasma triglyceride (TG) and different time-of-day patterns of hepatic TG and macrosteatosis, with elevated levels of small droplet macrosteatosis compared to alcohol-fed Fl/Fl mice. Diurnal rhythms in mRNA levels of lipid metabolism transcription factors (Srebf1 , Nr1h2 , and Ppara) were significantly altered by alcohol and clock disruption. Alcohol and/or clock disruption significantly altered diurnal rhythms in mRNA levels of fatty acid (FA) synthesis and oxidation (Acaca/b , Mlycd , Cpt1a , Fasn , Elovl5/6 , and Fads1/2), TG turnover (Gpat1 , Agpat1/2 , Lpin1/2 , Dgat2 , and Pnpla2/3), and lipid droplet (Plin2/5 , Lipe , Mgll , and Abdh5) genes, along with protein abundances of p-ACC, MCD, and FASN. Lipidomics analyses showed that alcohol, clock disruption, or both significantly altered FA saturation and remodeled the FA composition of the hepatic TG pool, with higher percentages of several long and very long chain FA in livers of alcohol-fed LKO mice. In conclusion, these results show that the liver clock is important for maintaining temporal control of hepatic lipid metabolism and that disrupting the liver clock exacerbates alcohol-related hepatic steatosis. [ABSTRACT FROM AUTHOR]

Circadian rhythms form a self-sustaining, endogenous, time-keeping system that allows organisms to anticipate daily environmental changes. The core of the clock network consists of interlocking transcriptional-translational feedback loops that ensures that metabolic, behavioral, and physiological processes run on a 24 h timescale. The hierarchical nature of the clock manifests itself in multiple points of control on the daily cell division cycle, which relies on synthesis, degradation, and post-translational modification for progression. This relationship is particularly important for understanding the role of clock components in sensing stress conditions and triggering checkpoint signals that stop cell cycle progression. A case in point is the interplay among the circadian factor PERIOD2 (PER2), the tumor suppressor p53, and the oncogenic mouse double minute-2 homolog protein (MDM2), which is the p53's negative regulator. Under unstressed conditions, PER2 and p53 form a stable complex in the cytosol and, along with MDM2, a trimeric complex in the nucleus. Association of PER2 to the C-terminus end of p53 prevents MDM2-mediated ubiquitylation and degradation of p53 as well as p53's transcriptional activation. Remarkably, when not bound to p53, PER2 acts as substrate for the E3-ligase activity of MDM2; thus, PER2 is degraded in a phosphorylation-independent fashion. Unexpectedly, the phase relationship between PER2 and p53 are opposite; however, a systematic modeling approach, inferred from the oscillatory time course data of PER2 and p53, aided in identifying additional regulatory scenarios that explained, a priori , seemingly conflicting experimental data. Therefore, we advocate for a combined experimental/mathematical approach to elucidating multilevel regulatory cellular processes. [ABSTRACT FROM AUTHOR]

The fibrinolytic system is critical during the onset of fibrinolysis, a fundamental mechanism for fibrin degradation. Both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) trigger fibrinolysis, leading to proteolytic activation of plasminogen to plasmin and subsequently fibrin proteolysis. This system is regulated by several inhibitors; plasminogen activator inhibitor-1 (PAI-1), the most studied, binds to and inactivates both tPA and uPA. Through the action of plasmin, this system regulates several physiological processes: embryogenesis, activation of inflammatory cells, cell proliferation and death, synaptic plasticity, wound healing, and others. The deregulated intervention of fibrinolysis in the pathophysiology of various diseases has been widely studied; findings of altered functioning have been reported in different chronic non-communicable diseases (NCD), reinforcing its pleiotropic character and the importance of its physiology and regulation. The evidence indicates that fundamental elements of the fibrinolytic system, such as tPA and PAI-1, show a circadian rhythm in their plasmatic levels and their gene expression are regulated by circadian system elements, known as clock genes – Bmal, Clock , Cry -, and accessory clock genes such as Rev-Erb and Ror. The disturbance in the molecular machinery of the clock by exposure to light during the night alters the natural light/dark cycle and causes disruption of the circadian rhythm. Such exposure affects the synchronization and functioning of peripheral clocks responsible for the expression of the components of the fibrinolytic system. So, this circadian disturbance could be critical in the pathophysiology of chronic diseases where this system has been found to be deregulated. [ABSTRACT FROM AUTHOR]

We have used the Cambridge Protein Trap resource (CPTI) to screen for flies whose locomotor rhythms are rhythmic in constant light (LL) as a means of identifying circadian photoreception genes. From the screen of ∼150 CPTI lines, we obtained seven hits, two of which targeted the glutamate pathway, Got1 (Glutamate oxaloacetate transaminase 1) and Gs2 (Glutamine synthetase 2). We focused on these by employing available mutants and observed that variants of these genes also showed high levels of LL rhythmicity compared with controls. It was also clear that the genetic background was important with a strong interaction observed with the common and naturally occurring timeless (tim) polymorphisms, ls-tim and s-tim. The less circadian photosensitive ls-tim allele generated high levels of LL rhythmicity in combination with Got1 or Gs2 , even though ls-tim and s-tim alleles do not, by themselves, generate the LL phenotype. The use of dsRNAi for both genes as well as for Gad (Glutamic acid decarboxylase) and the metabotropic glutamate receptor DmGluRA driven by clock gene promoters also revealed high levels of LL rhythmicity compared to controls. It is clear that the glutamate pathway is heavily implicated in circadian photoreception. TIM levels in Got1 and Gs2 mutants cycled and were more abundant than in controls under LL. Got1 but not Gs2 mutants showed diminished phase shifts to 10 min light pulses. Neurogenetic dissection of the LL rhythmic phenotype using the gal4/gal80 UAS bipartite system suggested that the more dorsal CRY-negative clock neurons, DNs and LNds were responsible for the LL phenotype. Immunocytochemistry using the CPTI YFP tagged insertions for the two genes revealed that the DN1s but not the DN2 and DN3s expressed Got1 and Gs2, but expression was also observed in the lateral neurons, the LNds and s-LNvs. Expression of both genes was also found in neuroglia. However, downregulation of glial Gs2 and Got1 using repo-gal4 did not generate high levels of LL rhythmicity, so it is unlikely that this phenotype is mediated by glial expression. Our results suggest a model whereby the DN1s and possibly CRY-negative LNds use glutamate signaling to supress the pacemaker s-LNvs in LL. [ABSTRACT FROM AUTHOR]

Experimental and epidemiological studies have revealed a relationship between an adverse intrauterine environment and chronic non-communicable disease (NCD) like cardiovascular disease (CVD) in adulthood. An important risk factor for CVD is the deregulation of the fibrinolytic system particularly high levels of expression of plasminogen activator inhibitor 1 (Pai-1). Chronic exposure to altered photoperiod disrupts the circadian organization of physiology in the pregnant female, known as gestational chronodisruption, and cause long-term effects on the adult offspring's circadian physiology. The Pai-1 expression is regulated by the molecular components of the circadian system, termed clock genes. The present study aimed to evaluate the long-term effects of chronic photoperiod shifts (CPS) during pregnancy on the expression of the clock genes and the fibrinolytic system in the liver of adult male offspring. Our results using an animal model demonstrated statistically significant differences at the transcriptional level in males gestated under CPS. At 90 days of postnatal age, the liver transcript levels of the clock gene Bmal1 were downregulated, whereas Ror α, Ror γ, Nfil3 , and Pai-1 were upregulated. Our data indicate that CPS during pregnancy affects gene expression in the liver of male adult progeny, showing that alteration of the photoperiod in the mother's environment leads to persistent effects in the offspring. In conclusion, these results reveal for the first time the long-term effects of gestational chronodisruption on the transcriptional activity of one well-established risk factor associated with CVD in the adult male offspring. [ABSTRACT FROM AUTHOR]

In vertebrates stress negatively affects body homeostasis and triggers a battery of metabolic responses, with liver playing a key role. This organ responds with altered metabolism, leading the animal to cope with the stress situation, which involves carbohydrate and lipid mobilization. However, metabolism among other physiological functions is under circadian control within the liver. Then, metabolic homeostasis at system level involves circadian timing systems within tissues and cells, and collaborate with each other. During chronic stress, cortisol maintains the liver metabolic response by modulating carbohydrate- and lipid-related metabolism. Stress also disrupts the circadian oscillator within the liver in mammals, whereas little information is available in other vertebrates, such as fish. To raise the complexity of this process, other candidates may mediate in such effect of stress. In fact, sirtuin1, a link between cellular sensing of energy status and circadian clocks, participates in the response to stress in mammals, but no information is available in fish. Considering the role played by liver in providing energy for the animal to deal with an adverse situation, and the existence of a circadian oscillator within this tissue, jeopardized liver circadian physiology during stress exposure might be expected. Whether the physiological response to stress is a well conserved process through the phylogeny and the mechanisms involved in such response is a question that remains to be elucidated. Then, we provide information at this respect in mammals and show comparable results in rainbow trout as fish animal model. Similar to that in mammals, stress triggers a series of responses in fish that leads the animal to cope with the adverse situation. Stress influences liver physiology in fish, affecting carbohydrate and lipid metabolism-related parameters, and the circadian oscillator as well. In a similar way than that of mammals different mediators participate in the response of liver circadian physiology to stress in fish. Among them, we confirm for the teleost rainbow trout a role of nuclear receptors (rev-erbβ), cortisol, and sirt1. However, further research is needed to evaluate the independent effect of each one, or the existence of any interaction among them.

A vast majority of the pathophysiological and metabolic processes in humans are temporally controlled by a master circadian clock located centrally in the hypothalamic suprachiasmatic nucleus of the brain, as well as by specialized peripheral oscillators located in other body tissues. This circadian clock system generates a rhythmical diurnal transcriptional-translational cycle in clock genes and protein expression and activities regulating numerous downstream target genes. Clock genes as key regulators of physiological function and dysfunction of the circadian clock have been linked to various diseases and multiple morbidities. Emerging omics technologies permits largescale multi-dimensional investigations of the molecular landscape of a given disease and the comprehensive characterization of its underlying cellular components (e.g., proteins, genes, lipids, metabolites), their mechanism of actions, functional networks and regulatory systems. Ultimately, they can be used to better understand disease and interpatient heterogeneity, individual profile, identify personalized targetable key molecules and pathways, discover novel biomarkers and genetic alterations, which collectively can allow for a better patient stratification into clinically relevant subgroups to improve disease prediction and prevention, early diagnostic, clinical outcomes, therapeutic benefits, patient's quality of life and survival. The use of “omics” technologies has allowed for recent breakthroughs in several scientific domains, including in the field of circadian clock biology. Although studies have explored the role of clock genes using circadiOmics (which integrates circadian omics, such as genomics, transcriptomics, proteomics and metabolomics) in human disease, no such studies have investigated the implications of circadian disruption in oral, head and neck pathologies using multi-omics approaches and linking the omics data to patient-specific circadian profiles. There is a burgeoning body of evidence that circadian clock controls the development and homeostasis of oral and maxillofacial structures, such as salivary glands, teeth and oral epithelium. Hence, in the current era of precision medicine and dentistry and patient-centered health care, it is becoming evident that a multi-omics approach is needed to improve our understanding of the role of circadian clock-controlled key players in the regulation of head and neck pathologies. This review discusses current knowledge on the role of the circadian clock and the contribution of omics-based approaches toward a novel precision health era for diagnosing and treating head and neck pathologies, with an emphasis on oral, head and neck cancer and Sjögren's syndrome.

Antarctic krill (Euphausia superba), a key species in the Southern Ocean, reduce their metabolism as an energy saving mechanism in response to the harsh environmental conditions during the Antarctic winter. Although the adaptive significance of this seasonal metabolic shift seems obvious, the driving factors are still unclear. In particular, it is debated whether the seasonal metabolic cycle is driven by changes in food availability, or if an endogenous timing system entrained by photoperiod might be involved. In this study, we used different long-term photoperiodic simulations to examine the influence of light regime and endogenous rhythmicity on the regulation of krill seasonal metabolic cycle. Krill showed a seasonal cycle of growth characterized by null-to-negative growth rates during autumn-winter and positive growth rates during spring-summer, which was manifested also in constant darkness, indicating strong endogenous regulation. Similar endogenous cycles were observed for the activity of the key-metabolic enzyme malate dehydrogenase (MDH) and for the expression levels of a selection of metabolic-related genes, with higher values in spring-summer and lower values in autumn-winter. On the other side, a seasonal cycle of oxygen consumption was observed only when krill were exposed to simulated seasonal changes in photoperiod, indicating that light-related cues might play a major role in the regulation of krill oxygen consumption. The influence of light-regime on oxygen consumption was minimal during winter, when light-phase duration was below 8 h, and it was maximal during summer, when light-phase duration was above 16 h. Significant upregulation of the krill clock genes clk, cry2, and tim1, as well as of the circadian-related opsins rh1a and rrh, was observed after light-phase duration had started to decrease in early autumn, suggesting the presence of a signaling cascade linking specific seasonal changes in the Antarctic light regime with clock gene activity and the regulation of krill metabolic dormancy over the winter.

The marine bristle worm Platynereis dumerilii is a useful functional model system for the study of the circadian clock and its interplay with others, e.g., circalunar clocks. The focus has so far been on the worm's head. However, behavioral and physiological cycles in other animals typically arise from the coordination of circadian clocks located in the brain and in peripheral tissues. Here, we focus on peripheral circadian rhythms and clocks, revisit and expand classical circadian work on the worm's chromatophores, investigate locomotion as read-out and include molecular analyses. We establish that different pieces of the trunk exhibit synchronized, robust oscillations of core circadian clock genes. These circadian core clock transcripts are under strong control of the light-dark cycle, quickly losing synchronized oscillation under constant darkness, irrespective of the absence or presence of heads. Different wavelengths are differently effective in controlling the peripheral molecular synchronization. We have previously shown that locomotor activity is under circadian clock control. Here, we show that upon decapitation worms exhibit strongly reduced activity levels. While still following the light-dark cycle, locomotor rhythmicity under constant darkness is less clear. We also observe the rhythmicity of pigments in the worm's individual chromatophores, confirming their circadian pattern. These size changes continue under constant darkness, but cannot be re-entrained by light upon decapitation. Our works thus provides the first basic characterization of the peripheral circadian clock of P. dumerilii. In the absence of the head, light is essential as a major synchronization cue for peripheral molecular and locomotor circadian rhythms, while circadian changes in chromatophore size can continue for several days in the absence of light/dark changes and the head. Thus, in Platynereis the dependence on the head depends on the type of peripheral rhythm studied. These data show that peripheral circadian rhythms and clocks should also be considered in "non-conventional" molecular model systems, i.e., outside Drosophila melanogaster , Danio rerio , and Mus musculus , and build a basic foundation for future investigations of interactions of clocks with different period lengths in marine organisms. [ABSTRACT FROM AUTHOR]

In vertebrates stress negatively affects body homeostasis and triggers a battery of metabolic responses, with liver playing a key role. This organ responds with altered metabolism, leading the animal to cope with the stress situation, which involves carbohydrate and lipid mobilization. However, metabolism among other physiological functions is under circadian control within the liver. Then, metabolic homeostasis at system level involves circadian timing systems within tissues and cells, and collaborate with each other. During chronic stress, cortisol maintains the liver metabolic response by modulating carbohydrate- and lipid-related metabolism. Stress also disrupts the circadian oscillator within the liver in mammals, whereas little information is available in other vertebrates, such as fish. To raise the complexity of this process, other candidates may mediate in such effect of stress. In fact, sirtuin1, a link between cellular sensing of energy status and circadian clocks, participates in the response to stress in mammals, but no information is available in fish. Considering the role played by liver in providing energy for the animal to deal with an adverse situation, and the existence of a circadian oscillator within this tissue, jeopardized liver circadian physiology during stress exposure might be expected. Whether the physiological response to stress is a well conserved process through the phylogeny and the mechanisms involved in such response is a question that remains to be elucidated. Then, we provide information at this respect in mammals and show comparable results in rainbow trout as fish animal model. Similar to that in mammals, stress triggers a series of responses in fish that leads the animal to cope with the adverse situation. Stress influences liver physiology in fish, affecting carbohydrate and lipid metabolism-related parameters, and the circadian oscillator as well. In a similar way than that of mammals different mediators participate in the response of liver circadian physiology to stress in fish. Among them, we confirm for the teleost rainbow trout a role of nuclear receptors (rev-erbβ), cortisol, and sirt1. However, further research is needed to evaluate the independent effect of each one, or the existence of any interaction among them. [ABSTRACT FROM AUTHOR]

A vast majority of the pathophysiological and metabolic processes in humans are temporally controlled by a master circadian clock located centrally in the hypothalamic suprachiasmatic nucleus of the brain, as well as by specialized peripheral oscillators located in other body tissues. This circadian clock system generates a rhythmical diurnal transcriptional-translational cycle in clock genes and protein expression and activities regulating numerous downstream target genes. Clock genes as key regulators of physiological function and dysfunction of the circadian clock have been linked to various diseases and multiple morbidities. Emerging omics technologies permits largescale multi-dimensional investigations of the molecular landscape of a given disease and the comprehensive characterization of its underlying cellular components (e.g., proteins, genes, lipids, metabolites), their mechanism of actions, functional networks and regulatory systems. Ultimately, they can be used to better understand disease and interpatient heterogeneity, individual profile, identify personalized targetable key molecules and pathways, discover novel biomarkers and genetic alterations, which collectively can allow for a better patient stratification into clinically relevant subgroups to improve disease prediction and prevention, early diagnostic, clinical outcomes, therapeutic benefits, patient's quality of life and survival. The use of "omics" technologies has allowed for recent breakthroughs in several scientific domains, including in the field of circadian clock biology. Although studies have explored the role of clock genes using circadiOmics (which integrates circadian omics, such as genomics, transcriptomics, proteomics and metabolomics) in human disease, no such studies have investigated the implications of circadian disruption in oral, head and neck pathologies using multi-omics approaches and linking the omics data to patient-specific circadian profiles. There is a burgeoning body of evidence that circadian clock controls the development and homeostasis of oral and maxillofacial structures, such as salivary glands, teeth and oral epithelium. Hence, in the current era of precision medicine and dentistry and patient-centered health care, it is becoming evident that a multi-omics approach is needed to improve our understanding of the role of circadian clock-controlled key players in the regulation of head and neck pathologies. This review discusses current knowledge on the role of the circadian clock and the contribution of omics-based approaches toward a novel precision health era for diagnosing and treating head and neck pathologies, with an emphasis on oral, head and neck cancer and Sjögren's syndrome. [ABSTRACT FROM AUTHOR]

Antarctic krill (Euphausia superba), a key species in the Southern Ocean, reduce their metabolism as an energy saving mechanism in response to the harsh environmental conditions during the Antarctic winter. Although the adaptive significance of this seasonal metabolic shift seems obvious, the driving factors are still unclear. In particular, it is debated whether the seasonal metabolic cycle is driven by changes in food availability, or if an endogenous timing system entrained by photoperiod might be involved. In this study, we used different long-term photoperiodic simulations to examine the influence of light regime and endogenous rhythmicity on the regulation of krill seasonal metabolic cycle. Krill showed a seasonal cycle of growth characterized by null-to-negative growth rates during autumn-winter and positive growth rates during spring-summer, which was manifested also in constant darkness, indicating strong endogenous regulation. Similar endogenous cycles were observed for the activity of the key-metabolic enzyme malate dehydrogenase (MDH) and for the expression levels of a selection of metabolic-related genes, with higher values in spring-summer and lower values in autumn-winter. On the other side, a seasonal cycle of oxygen consumption was observed only when krill were exposed to simulated seasonal changes in photoperiod, indicating that light-related cues might play a major role in the regulation of krill oxygen consumption. The influence of light-regime on oxygen consumption was minimal during winter, when light-phase duration was below 8 h, and it was maximal during summer, when light-phase duration was above 16 h. Significant upregulation of the krill clock genes clk , cry 2, and tim 1, as well as of the circadian-related opsins rh 1a and rrh , was observed after light-phase duration had started to decrease in early autumn, suggesting the presence of a signaling cascade linking specific seasonal changes in the Antarctic light regime with clock gene activity and the regulation of krill metabolic dormancy over the winter. [ABSTRACT FROM AUTHOR]

The circadian system controls temporal homeostasis in all vertebrates. The light-dark (LD) cycle is the most important zeitgeber ("time giver") of circadian system, but feeding time also acts as a potent synchronizer in the functional organization of the teleost circadian system. In mammals is well known that food intake during the rest phase promotes circadian desynchrony which has been associated with metabolic diseases. However, the impact of a misalignment of LD and feeding cycles in the entrainment of fish circadian oscillators is largely unknown. The objective of this work was to investigate how a time-lag feeding alters temporal homeostasis and if this could be considered a stressor. To this aim, goldfish maintained under a 12 h light-12 h darkness were fed at mid-photophase (SF6) or mid-scotophase (SF18). Daily rhythms of locomotor activity, clock genes expression in hypothalamus, liver, and head kidney, and circulating cortisol were studied. Results showed that SF6 fish showed daily rhythms of bmal1a and clock1a in all studied tissues, being in antiphase with rhythms of per1 genes, as expected for proper functioning clocks. The 12 h shift in scheduled feeding induced a short phase advance (4–5-h) of the clock genes daily rhythms in the hypothalamus, while in the liver the shift for clock genes expression rhythms was the same that the feeding time shift (∼12 h). In head kidney, acrophases of per genes underwent a 12-h shift in SF18 animals, but only 6 h shift for clock1a. Plasma cortisol levels showed a significant daily rhythm in animals fed at SF6, but not in SF18 fish fed, which displayed higher cortisol values throughout the 24-h. Altogether, results indicate that hypothalamus, liver, and head kidney oscillate in phase in SF6 fish, but these clocks are desynchronized in SF18 fish, which could explain cortisol alterations. These data reinforce the hypothesis that the misalignment of external cues (daily photocycle and feeding time) alters fish temporal homeostasis and it might be considered a stressor for the animals. [ABSTRACT FROM AUTHOR]

Oscillations of brain proteins in circadian rhythms are important for determining several cellular and physiological processes in anticipation of daily and seasonal environmental rhythms. In addition to the suprachiasmatic nucleus, the primary central oscillator, the cerebellum shows oscillations in gene and protein expression. The variety of local circuit rhythms that the cerebellar cortex contains influences functions such as motivational processes, regulation of feeding, food anticipation, language, and working memory. The molecular basis of the cerebellar oscillator has been demonstrated by “clock gene” expression within cells of the cerebellar layers. Genetic and epidemiological evidence suggests that disruption of circadian rhythms in humans can lead to many pathological conditions. Despite this importance, data about clock gene and protein expression in the cerebellum of diurnal (day-active) species, specifically primates, is currently poorly explored, mainly in regard to cellular identity, as well as the relationship with other molecules also involved in cerebellar functions. These studies could contribute to clarification of the possible mechanisms behind cerebellar rhythmicity. Considering that calcium binding proteins (CaBPs) play crucial roles in preserving and modulating cerebellar functions and that clock gene expression can be controlled by afferent projections or paracrine circadian signals such as the hormone melatonin, the present study aimed to describe cellular identities, distribution patterns and day/night expression changes in PER1, PER2, CaBPs, and MT1 and MT2 melatonin receptors in the cerebellar cortex of a diurnal primate using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. PER1 and PER2 immunoreactive (IR) cells were observed in the Purkinje cells of the cerebellum, and MT1 and MT2 receptors were localized around Purkinje cells in the Pj layer in Bergmann cells. This identity was confirmed by the S100β-IR of these cells. The highest expression of PER seen in the daytime analysis coincided with the highest expression of melatonin receptors. CaBPs showed day/night morphological and density changes in the cerebellar cortex. The presence of the same temporal variations in the expression of PER in the Pj neurons and in MT1 and MT2 receptors in Bergmann cells indicates a possible relation between these cells during the rhythmic processing of the cerebellum, in addition to the CaBP temporal morphological and density changes.

Circadian rhythms are biological oscillations with a period of ~24 h. These rhythms are orchestrated by a circadian timekeeper in the suprachiasmatic nucleus of the hypothalamus, the circadian “master clock,” which exactly adjusts clock outputs to solar time via photic synchronization. At the molecular level, circadian rhythms are generated by the interaction of positive and negative feedback loops of transcriptional and translational processes of the so-called “clock genes.” A large number of clock genes encode numerous proteins that regulate their own transcription and that of other genes, collectively known as “clock-controlled genes.” In addition to the sleep/wake cycle, many cellular processes are regulated by circadian rhythms, including synaptic plasticity in which an exquisite interplay between neurons and glial cells takes place. In particular, there is compelling evidence suggesting that glial cells participate in and regulate synaptic plasticity in a circadian fashion, possibly representing the missing cellular and physiological link between circadian rhythms with learning and cognition processes. Here we review recent studies in support of this hypothesis, focusing on the interplay between glial cells, synaptic plasticity, and circadian rhythmogenesis.

Circadian plasticity of the visual system of Drosophila melanogaster depends on functioning of both the neuronal and glial oscillators. The clock function of the former is already quite well-recognized. The latter, however, is much less known and documented. In this study we focus on the glial oscillators that reside in the distal part of the second visual neuropil, medulla (dMnGl), in vicinity of the PIGMENT-DISPERSING FACTOR (PDF) releasing terminals of the circadian clock ventral Lateral Neurons (LNvs). We reveal the heterogeneity of the dMnGl, which express the clock protein PERIOD (PER) and the pan-glial marker REVERSED POLARITY (REPO) at higher (P1) or lower (P2) levels. We show that the cells with stronger expression of PER display also stronger expression of REPO, and that the number of REPO-P1 cells is bigger during the day than during the night. Using a combination of genetic markers and immunofluorescent labeling with anti PER and REPO Abs, we have established that the P1 and P2 cells can be associated with two different types of the dMnGl, the ensheathing (EnGl), and the astrocyte-like glia (ALGl). Surprisingly, the EnGl belong to the P1 cells, whereas the ALGl, previously reported to play the main role in the circadian rhythms, display the characteristics of the P2 cells (express very low level of PER and low level of REPO). Next to the EnGl and ALGl we have also observed another type of cells in the distal medulla that express PER and REPO, although at very low levels. Based on their morphology we have identified them as the T1 interneurons. Our study reveals the complexity of the distal medulla circadian network, which appears to consist of different types of glial and neuronal peripheral clocks, displaying molecular oscillations of higher (EnGl) and lower (ALGl and T1) amplitudes. [ABSTRACT FROM AUTHOR]

Circadian rhythms are biological oscillations with a period of ~24 h. These rhythms are orchestrated by a circadian timekeeper in the suprachiasmatic nucleus of the hypothalamus, the circadian "master clock," which exactly adjusts clock outputs to solar time via photic synchronization. At the molecular level, circadian rhythms are generated by the interaction of positive and negative feedback loops of transcriptional and translational processes of the so-called "clock genes." A large number of clock genes encode numerous proteins that regulate their own transcription and that of other genes, collectively known as "clock-controlled genes." In addition to the sleep/wake cycle, many cellular processes are regulated by circadian rhythms, including synaptic plasticity in which an exquisite interplay between neurons and glial cells takes place. In particular, there is compelling evidence suggesting that glial cells participate in and regulate synaptic plasticity in a circadian fashion, possibly representing the missing cellular and physiological link between circadian rhythms with learning and cognition processes. Here we review recent studies in support of this hypothesis, focusing on the interplay between glial cells, synaptic plasticity, and circadian rhythmogenesis. [ABSTRACT FROM AUTHOR]

Circadian rhythms are essential for health and regulate various physiological functions. These rhythms are regulated by a negative-feedback loop involving clock genes in the suprachiasmatic nucleus (SCN) and peripheral tissues. The rate of secretion of salivary substances, ions, and water follows a circadian rhythm, however, the relationship between the molecular mechanism of salivary secretion and peripheral circadian rhythm is not yet clear. Anoctamin 1 (ANO1, also known as TMEM16A) and Aquaporin 5 (AQP5) play an important role in the transport of ions and water in the submandibular glands (SGs). We examined the interaction between the rhythmic expression pattern of the clock genes, Ano1 and Aqp5, in rat whole SGs as well as isolated acinar and ductal cells. Circadian rhythmic expression for Bmal1, Per1, Per2, Clock, Cry1, Cry2, Rora, and Rev-erba mRNAs, also called the clock genes, was observed in rat SGs by semiquantitative RT-PCR analysis. We also observed rhythmic patterns in Ano1 and Aqp5 mRNA expression. The expression of ANO1 protein also showed circadian rhythm, as confirmed by western blot analysis. We could not observe any time delay between the peak expression of ANO1 protein and its mRNA. Expression levels of the clock gene mRNAs in the ductal cells was higher than that in acinar cells, however, rhythmic oscillations were observed in both. Our results suggest that SGs have peripheral clocks, and rhythmic expressions of Ano1 and Aqp5 along with the clock genes, may play an important role in the circadian regulation of salivary secretion. [ABSTRACT FROM AUTHOR]