Circadian rhythm of cutaneous pruritus. / 皮肤瘙痒的昼夜节律.

One of the most common and significant symptoms for skin disorders is pruritus. Additionally, it serves as a significant catalyst for the exacerbation or reoccurrence of skin diseases. Pruritus seriously affects patients' physical and mental health, and even the quality of life. It brings a heavy burden to the patients, the families, even the whole society. The pathogenesis and regulation mechanisms for pruritus are complicated and have not yet been elucidated. Previous clinical studies have shown that itch worsens at night in scabies, chronic pruritus, atopic dermatitis, and psoriasis, suggesting that skin pruritus may change with circadian rhythm. Cortisol, melatonin, core temperature, cytokines, and prostaglandins are the main regulatory factors of the circadian rhythm of pruritus. Recent studies have shown that some CLOCK genes, such as BMAL1, CLOCK, PER, and CRY, play an important role in the regulation of the circadian rhythm of pruritus by regulating the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) and nuclear factor kappa-B (NF-κB) signaling pathways. However, the mechanisms for circadian clock genes in regulation of circadian rhythm of pruritus have not been fully elucidated. Further studies on the mechanism of circadian clock genes in the regulation of circadian rhythm of pruritus will lay a foundation for elucidating the regulatory mechanisms for pruritus, and also provide new ideas for the control of pruritus and the alleviation of skin diseases.

CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.

tauFisher predicts circadian time from a single sample of bulk and single-cell pseudobulk transcriptomic data.

As the circadian clock regulates fundamental biological processes, disrupted clocks are often observed in patients and diseased tissues. Determining the circadian time of the patient or the tissue of focus is essential in circadian medicine and research. Here we present tauFisher, a computational pipeline that accurately predicts circadian time from a single transcriptomic sample by finding correlations between rhythmic genes within the sample. We demonstrate tauFisher's performance in adding timestamps to both bulk and single-cell transcriptomic samples collected from multiple tissue types and experimental settings. Application of tauFisher at a cell-type level in a single-cell RNAseq dataset collected from mouse dermal skin implies that greater circadian phase heterogeneity may explain the dampened rhythm of collective core clock gene expression in dermal immune cells compared to dermal fibroblasts. Given its robustness and generalizability across assay platforms, experimental setups, and tissue types, as well as its potential application in single-cell RNAseq data analysis, tauFisher is a promising tool that facilitates circadian medicine and research.

Variants in the circadian clock genes PER2 and PER3 associate with familial sleep phase disorders.

Delayed sleep phase disorder and advanced sleep phase disorder cause disruption of the circadian clock and present with extreme morning/evening chronotype with unclear role of the genetic etiology, especially for delayed sleep phase disorder. To assess if genotyping can aid in clinical diagnosis, we examined the presence of genetic variants in circadian clock genes previously linked to both sleep disorders in Slovenian patient cohort. Based on Morning-evening questionnaire, we found 15 patients with extreme chronotypes, 13 evening and 2 morning, and 28 controls. Sanger sequencing was used to determine the presence of carefully selected candidate SNPs in regions of the CSNK1D, PER2/3 and CRY1 genes. In a patient with an extreme morning chronotype and a family history of circadian sleep disorder we identified two heterozygous missense variants in PER3 gene, c.1243C>G (NM_001377275.1 (p.Pro415Ala)) and c.1250A>G (NM_001377275.1 (p.His417Arg)). The variants were significantly linked to Advanced sleep phase disorder and were also found in proband's father with extreme morningness. Additionally, a rare SNP was found in PER2 gene in a patient with clinical picture of Delayed sleep phase disorder. The novel variant in PER2 (NM_022817.3):c.1901-218 G>T was found in proband's parent with eveningness, indicating an autosomal dominant inheritance. We identified a family with autosomal dominant inheritance of two PER3 heterozygous variants that can be linked to Advanced sleep phase disorder. We revealed also a rare hereditary form of Delayed sleep phase disorder with a new PER2 variant with autosomal dominant inheritance, shedding the light into the genetic causality.

Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria.

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Circadian transcriptome of pancreatic adenocarcinoma unravels chronotherapeutic targets.

Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a poor outcome and an increasing incidence. A significant majority (>80%) of newly diagnosed cases are deemed unresectable, leaving chemotherapy as the sole viable option, though with only moderate success. This necessitates the identification of improved therapeutic options for PDA. We hypothesized that there are temporal variations in cancer-relevant processes within PDA tumors, offering insights into the optimal timing of drug administration - a concept termed chronotherapy. In this study, we explored the presence of the circadian transcriptome in PDA using patient-derived organoids and validated these findings by comparing PDA data from The Cancer Genome Atlas with noncancerous healthy pancreas data from GTEx. Several PDA-associated pathways (cell cycle, stress response, Rho GTPase signaling) and cancer driver hub genes (EGFR and JUN) exhibited a cancer-specific rhythmic pattern intricately linked to the circadian clock. Through the integration of multiple functional measurements for rhythmic cancer driver genes, we identified top chronotherapy targets and validated key findings in molecularly divergent pancreatic cancer cell lines. Testing the chemotherapeutic efficacy of clinically relevant drugs further revealed temporal variations that correlated with drug-target cycling. Collectively, our study unravels the PDA circadian transcriptome and highlights a potential approach for optimizing chrono-chemotherapeutic efficacy.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

Temperature-driven coordination of circadian transcriptional regulation.

The circadian clock is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce circadian oscillations in thousands of genes in a tissue-specific manner, orchestrating myriad biological processes. While previous studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome: genes are cycling in a temperature-specific manner, and the distributions of their phases also differ between the two conditions. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low temperatures and synchronization of rhythmic genes that are network neighbors. We report that the phase differences between cycling genes increase as a function of geodesic distance in the low temperature condition, suggesting increased coordination of cycling on the gene regulatory network. Our results suggest a potential mechanism whereby the circadian clock mediates the fly's response to seasonal changes in temperature.

Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid.

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

Investigating Circadian Gating of Temperature Responsive Genes.

Understanding gene expression dynamics in the context of the time of day and temperature response is an important part of understanding plant thermotolerance in a changing climate. Performing "gating" experiments under constant conditions and light-dark cycles allows users to identify and dissect the contribution of the time of day and circadian clock to the dynamic nature of stress-responsive genes. Here, we describe the design of specific laboratory experiments in plants (Arabidopsis thaliana and bread wheat, Triticum aestivum) to investigate temporal responses to heat (1 h at 37 °C) or cold (3 h at 4 °C), and we include known marker genes that have circadian-gated responses to temperature changes.

Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Daphnia uses its circadian clock for short-day recognition in environmental sex determination.

Some organisms have developed a mechanism called environmental sex determination (ESD), which allows environmental cues, rather than sex chromosomes or genes, to determine offspring sex.1,2,3,4 ESD is advantageous to optimize sex ratios according to environmental conditions, enhancing reproductive success.5,6 However, the process by which organisms perceive and translate diverse environmental signals into offspring sex remains unclear. Here, we analyzed the environmental perception mechanism in the crustacean, Daphnia pulex, a seasonal (photoperiodic) ESD arthropod, capable of producing females under long days and males under short days.7,8,9,10 Through breeding experiments, we found that their circadian clock likely contributes to perception of day length. To explore this further, we created a genetically modified daphnid by knocking out the clock gene, period, using genome editing. Knockout disrupted the daphnid's ability to sustain diel vertical migration (DVM) under constant darkness, driven by the circadian clock, and leading them to produce females regardless of day length. Additionally, when exposed to an analog of juvenile hormone (JH), an endocrine factor synthesized in mothers during male production, or subjected to unfavorable conditions of high density and low food availability, these knockout daphnids produced males regardless of day length, like wild-type daphnids. Based on these findings, we propose that recognizing short days via the circadian clock is the initial step in sex determination. This recognition subsequently triggers male production by signaling the endocrine system, specifically via the JH signal. Establishment of a connection between these two processes may be the crucial element in evolution of ESD in Daphnia.

Transcriptional rewiring of an evolutionarily conserved circadian clock.

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.

Modulation of the Circadian Rhythm and Oxidative Stress as Molecular Targets to Improve Vascular Dementia: A Pharmacological Perspective.

The circadian rhythms generated by the master biological clock located in the brain's hypothalamus influence central physiological processes. At the molecular level, a core set of clock genes interact to form transcription-translation feedback loops that provide the molecular basis of the circadian rhythm. In animal models of disease, a desynchronization of clock genes in peripheral tissues with the central master clock has been detected. Interestingly, patients with vascular dementia have sleep disorders and irregular sleep patterns. These alterations in circadian rhythms impact hormonal levels, cardiovascular health (including blood pressure regulation and blood vessel function), and the pattern of expression and activity of antioxidant enzymes. Additionally, oxidative stress in vascular dementia can arise from ischemia-reperfusion injury, amyloid-beta production, the abnormal phosphorylation of tau protein, and alterations in neurotransmitters, among others. Several signaling pathways are involved in the pathogenesis of vascular dementia. While the precise mechanisms linking circadian rhythms and vascular dementia are still being studied, there is evidence to suggest that maintaining healthy sleep patterns and supporting proper circadian rhythm function may be important for reducing the risk of vascular dementia. Here, we reviewed the main mechanisms of action of molecular targets related to the circadian cycle and oxidative stress in vascular dementia.

The skin circadian clock gene F3 as a potential marker for psoriasis severity and its bidirectional relationship with IL-17 signaling in keratinocytes.

OBJECTIVE: Psoriasis is an immune-mediated skin disease where the IL-17 signaling pathway plays a crucial role in its development. Chronic circadian rhythm disorder in psoriasis pathogenesis is gaining more attention. The relationship between IL and 17 signaling pathway and skin clock genes remains poorly understood. METHODS: GSE121212 with psoriatic lesion and healthy controls was used as the exploration cohort for searching analysis. Datasets GSE54456, GSE13355, GSE14905, GSE117239, GSE51440, and GSE137218 were applied to validation analysis. Single-cell RNA sequencing (scRNA-seq) dataset GSE173706 was used to explore the F3 expression and related pathway activities in single-cell levels. Through intersecting with high-expression DEGs, F3 was selected as the signature skin circadian gene in psoriasis for further investigation. Functional analyses, including correlation analyses, prediction of transcription factors, protein-protein interaction, and single gene GSEA to explore the potential roles of F3. ssGSEA algorithm was performed to uncover the immune-related characteristics of psoriasis. We further explored F3 expression in the specific cell population in scRNA-seq dataset, besides this, AUCell analysis was performed to explore the pathway activities and the results were further compared between the specific cell cluster. Immunohistochemistry experiment, RT-qPCR was used to validate the location and expression of F3, small interfering RNA (siRNA) transfection experiment in HaCaT, and transcriptome sequencing analysis were applied to explore the potential function of F3. RESULTS: F3 was significantly down-regulated in psoriasis and interacted with IL-17 signaling pathway. Low expression of F3 could upregulate the receptor of JAK-STAT signaling, thereby promoting keratinocyte inflammation. CONCLUSION: Our research revealed a bidirectional link between the skin circadian gene F3 and the IL-17 signaling pathway in psoriasis, suggesting that F3 may interact with the IL-17 pathway by activating JAK-STAT within keratinocytes and inducing abnormal intracellular inflammation.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

Circadian Effects on Vascular Immunopathologies.

Circadian rhythms exert a profound impact on most aspects of mammalian physiology, including the immune and cardiovascular systems. Leukocytes engage in time-of-day-dependent interactions with the vasculature, facilitating the emigration to and the immune surveillance of tissues. This review provides an overview of circadian control of immune-vascular interactions in both the steady state and cardiovascular diseases such as atherosclerosis and infarction. Circadian rhythms impact both the immune and vascular facets of these interactions, primarily through the regulation of chemoattractant and adhesion molecules on immune and endothelial cells. Misaligned light conditions disrupt this rhythm, generally exacerbating atherosclerosis and infarction. In cardiovascular diseases, distinct circadian clock genes, while functioning as part of an integrated circadian system, can have proinflammatory or anti-inflammatory effects on these immune-vascular interactions. Here, we discuss the mechanisms and relevance of circadian rhythms in vascular immunopathologies.