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Background: Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs). Results: We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4−/− mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice. Conclusions: Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment. [ABSTRACT FROM AUTHOR]

Background: Sleep deprivation (SD) is a common public health problem that contributes to various physiological disorders and increases the risk of ocular diseases. However, whether sleep loss can damage corneal endothelial function remains unclear. This study aimed to determine the effect and possible mechanism of SD on the corneal endothelium. Methods: Male C57BL/6J mice were subjected to establish SD models. After 10 days, quantitative RT-PCR (qRT-PCR) and western blot or immunostaining for the expression levels of zonula occludens-1 (ZO-1), ATPase Na+/K + transporting subunit alpha 1 (Atp1a1), and core clock genes in the corneal endothelium were evaluated. Reactive oxygen species staining and mitochondrial abundance characterized the mitochondrial function. The regulatory role of Bmal1 was confirmed by specifically knocking down or overexpressing basic helix-loop-helix ARNT like 1 protein (Bmal1) in vivo. In vitro, a mitochondrial stress test was conducted on cultured human corneal endothelial cells upon Bmal1 knockdown. Results: SD damaged the barrier and pump functions of mouse corneal endothelium, accompanied by mitochondrial dysfunction. Interestingly, SD dramatically downregulated the core clock gene Bmal1 expression level. Bmal1 knockdown disrupted corneal endothelial function, while overexpression of Bmal1 ameliorated the dysfunction induced by SD. Mitochondrial bioenergetic deficiency mediated by Bmal1 was an underlying mechanism for SD induced corneal endothelial dysfunction. Conclusion: The downregulation of Bmal1 expression caused by SD led to corneal endothelial dysfunction via impairing mitochondrial bioenergetics. Our findings offered insight into how SD impairs the physiological function of the corneal endothelium and expanded the understanding of sleep loss leading to ocular diseases. [ABSTRACT FROM AUTHOR]

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments. [ABSTRACT FROM AUTHOR]

Obesity and overweight are common and complex conditions influenced by multiple genetic and environmental factors. Several genetic variants located in the genes involved in clock systems and fat taste perception can affect metabolic health. In particular, the polymorphisms in CLOCK and BMAL1 genes were reported to be significantly related to cardiovascular disease, metabolic syndrome, sleep reduction, and evening preference. Moreover, genetic variants in the CD36 gene have been shown to be involved in lipid metabolism, regulation of fat intake, and body weight regulation. The aim of this study is to evaluate, for the first time, the association between variants in some candidate genes (namely, BMAL1 rs7950226 (G>A), CLOCK rs1801260 (A>G), CLOCK rs4864548 (G>A), CLOCK rs3736544 (G>A), CD36 rs1984112 (A>G), CD36 rs1761667 (G>A)) and overweight/obesity (OB) in pregnant women. A total of 163 normal-weight (NW) and 128 OB participants were included. A significant correlation was observed between A-allele in CLOCK rs4864548 and an increased risk of obesity (OR: 1.97; 95% CI 1.22–3.10, p = 0.005). In addition, we found that subjects carrying the haplotype of rs1801260-A, rs4864548-A, and rs3736544-G are likely to be overweight or obese (OR 1.47, 95% CI 1.03–2.09, p = 0.030), compared with those with other haplotypes. Moreover, a significant relation was observed between third-trimester lipid parameters and genetic variants—namely, CD36 rs1984112, CD36 rs1761667, BMAL1 rs7950226, and CLOCK rs1801260. A multivariate logistic regression model revealed that CLOCK rs4864548 A-allele carriage was a strong risk factor for obesity (OR 2.05, 95% CI 1.07–3.93, p = 0.029); on the other hand, greater adherence to Mediterranean diet (OR 0.80, 95% CI 0.65–0.98, p = 0.038) and higher HDL levels (OR 0.96, 95% CI 0.94–0.99, p = 0.021) were related to a reduced risk of obesity. Interestingly, an association between maternal CLOCK rs4864548 and neonatal birthweight was detected (p = 0.025). These data suggest a potential role of the polymorphisms in clock systems and in fat taste perception in both susceptibility to overweight/obesity and influencing the related metabolic traits in pregnant women. [ABSTRACT FROM AUTHOR]

In children, teenagers, and young adults, environmental factors and genetic modifications have contributed to the development of obesity. There is a close relationship between obesity and circadian rhythm. To understand the role of CLOCK and BMAL1 in obesity, we analyzed the methylation status of CLOCK and BMAL1 in obese and control subjects. In this paper, we analyzed the methylation status of the CLOCK and BMAL1 genes by using MS-HRM in a total of 55 obese and 54 control subjects. In our study, we demonstrated that the level of fasting glucose and the level of HDL-cholesterol were associated with CLOCK methylation in obesity. We also showed a significant association between BMAL1 gene methylation and waist and hip circumference in obese subjects. This is the first study that shows the methylation of BMAL1 is associated with the obese phenotype. However, we could not show a direct association between CLOCK methylation and the obese phenotype. In this paper, a novel epigenetic interaction between circadian clock genes and obesity was demonstrated. [ABSTRACT FROM AUTHOR]

Sarcopenia is an age‐related disease characterized by a reduction in muscle mass, strength, and function and, therefore, a deterioration in skeletal muscle health and frailty. Although the cause of sarcopenia is still unknown and, thus, there is no treatment, increasing evidence suggests that chronodisruption, particularly alterations in Bmal1 clock gene, can lead to those deficits culminating in sarcopenia. To gain insight into the cause and mechanism of sarcopenia and the protective effect of a therapeutic intervention with exercise and/or melatonin, the gastrocnemius muscles of male and female skeletal muscle‐specific and inducible Bmal1 knockout mice (iMS‐Bmal1−/−) were examined by phenotypic tests and light and electron microscopy. Our results revealed a disruption of the normal activity/rest rhythm, a drop in skeletal muscle function and mass, and increased frailty in male and female iMS‐Bmal1−/− animals compared to controls. A reduction in muscle fiber size and increased collagenous tissue were also detected, accompanied by reduced mitochondrial oxidative capacity and a compensatory shift towards a more oxidative fiber type. Electron microscopy further supports mitochondrial impairment in mutant mice. Melatonin and exercise ameliorated the damage caused by loss of Bmal1 in mutant mice, except for mitochondrial damage, which was worsened by the latter. Thus, iMS‐Bmal1−/− mice let us to identify Bmal1 deficiency as the responsible for the appearance of sarcopenia in the gastrocnemius muscle. Moreover, the results support the exercise and melatonin as therapeutic tools to counteract sarcopenia, by a mechanism that does not require the presence of Bmal1. [ABSTRACT FROM AUTHOR]

Sarcopenia is an age-related condition that involves a progressive decline in muscle mass and function, leading to increased risk of falls, frailty, and mortality. Although the exact mechanisms are not fully understood, aging-related processes like inflammation, oxidative stress, reduced mitochondrial capacity, and cell apoptosis contribute to this decline. Disruption of the circadian system with age may initiate these pathways in skeletal muscle, preceding the onset of sarcopenia. At present, there is no pharmacological treatment for sarcopenia, only resistance exercise and proper nutrition may delay its onset. Melatonin, derived from tryptophan, emerges as an exceptional candidate for treating sarcopenia due to its chronobiotic, antioxidant, and anti-inflammatory properties. Its impact on mitochondria and organelle, where it is synthesized and crucial in aging skeletal muscle, further highlights its potential. In this review, we discuss the influence of clock genes in muscular aging, with special reference to peripheral clock genes in the skeletal muscle, as well as their relationship with melatonin, which is proposed as a potential therapy against sarcopenia. [ABSTRACT FROM AUTHOR]

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN-/-) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1-/- cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival. [ABSTRACT FROM AUTHOR]

Background: Today, modern lifestyles and disrupted sleep patterns cause circadian clock rhythm impairments that are associated with altered leptin levels, which subsequently affect a wide range of physiological processes and have significant health burdens on societies. Nevertheless, there has been no systematic review of circadian clock genes and proteins, leptin, and related signaling pathways. Methods: Accordingly, we systematically reviewed circadian clock proteins, leptin, and molecular mechanisms between them by searching Pubmed, Scopus, ProQuest, Web of Sciences, and Google Scholar until September 2022. After considering the inclusion and exclusion criteria, 20 animal studies were selected. The risk of bias was assessed in each study. Results: The results clarified the reciprocal interconnected relationship between circadian clock genes and leptin. Circadian clock genes regulate leptin expression and signaling via different mechanisms, such as CLOCK-BMAL1 heterodimers, which increase the expression of PPARs. PPARs induce the expression of C/EBPα, a key factor in upregulating leptin expression. CLOCK-BMAL1 also induces the expression of Per1 and Rev-erb genes. PER1 activates mTORC1 and mTORC1 enhances the expression of C/EBPα. In addition, REV-ERBs activate the leptin signaling pathway. Also, leptin controls the expression of circadian clock genes by triggering the AMPK and ERK/MAPK signaling pathways, which regulate the activity of PPARs. Moreover, the roles of these molecular mechanisms are elucidated in different physiological processes and organs. Conclusions: Crosstalk between circadian clock genes and leptin and their affecting elements should be considered in the selection of new therapeutic targets for related disorders, especially obesity and metabolic impairments. [ABSTRACT FROM AUTHOR]

Objectives: Growing evidence demonstrates a relationship between epilepsy and the circadian system. However, relatively little is known about circadian function in disease states, such as epilepsy. This study aimed to characterize brain and peripheral core circadian clock gene expression in rat models of genetic and acquired epilepsy. Methods: For the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) study, we used 40 GAERS and 40 non‐epileptic control (NEC) rats. For the kainic acid status epilepticus (KASE) study, we used 40 KASE and 40 sham rats. Rats were housed in a 7 am:7 pm light–dark cycle. Hypothalamus, hippocampus, liver, and small intestine samples were collected every 3 h throughout the light period. We then assessed core diurnal clock gene expression of per1, cry1, clock, and bmal1. Results: In the GAERS rats, all tissues exhibited significant changes in clock gene expression (P < 0.05) when compared to NEC. In the KASE rats, there were fewer effects of the epileptic condition in the hypothalamus, hippocampus, or small intestine (P > 0.05) compared with shams. Significance: These results indicate marked diurnal disruption to core circadian clock gene expression in rats with both generalized and focal chronic epilepsy. This could contribute to epileptic symptomology and implicate the circadian system as a viable target for future treatments. [ABSTRACT FROM AUTHOR]

BACKGROUND: Temporomandibular joint osteoarthritis is a common disease in stomatology and its pathogenesis is complex. Recently, it has been found that the decreased expression of clock gene Bmal1 can lead to osteoarthritis-like pathological changes of the temporomandibular joint in mice, but the mechanism of Bmal1 in temporomandibular joint osteoarthritis remains unclear. OBJECTIVE: To summarize the possible mechanism of clock gene Bmal1 in the development of temporomandibular joint osteoarthritis, so as to provide new insights into the pathogenesis of temporomandibular joint osteoarthritis. METHODS: Articles published from inception to 2022 were retrieved in PubMed, Web of Science, and CNKI by the keywords of “TMJOA, osteoarthritis, TMD, condylar cartilage, Bmal1, circadian, circadian clock, sleep” in English and Chinese. Fifty articles were finally selected for this review according to the criteria of inclusion and exclusion. RESULTS AND CONCLUSION: Circadian rhythm disturbance caused by sleep deprivation can lead to osteoarthritis-like pathological changes of the temporomandibular joint in mice, suggesting that circadian rhythm disturbance is a new risk factor for temporomandibular joint osteoarthritis. As a core clock factor of circadian rhythm, Bmal1 plays an important role in maintaining cartilage homeostasis of the temporomandibular joint. When Bmal1 expression is decreased, anabolism of the articular cartilage is decreased, catabolism is increased, condylar cartilage is degraded, and temporomandibular joint osteoarthritis is induced. Bmal1 overexpression can alleviate the pathological symptoms of osteoarthritis. Bmal1 can also affect chondrogenesis and endochondral ossification of the condyle cartilage through downstream signaling pathways, resulting in dysplasia of the condyle and mandible, which provides an anatomical basis for the occurrence and development of temporomandibular joint osteoarthritis. At the same time, Bmal1 can promote aging and obesity by regulating oxidative stress and fat metabolism, thereby increasing the susceptibility of temporomandibular joint osteoarthritis. Therefore, the clock gene Bmal1 may have direct and/or indirect effects on the occurrence and development of temporomandibular joint osteoarthritis. [ABSTRACT FROM AUTHOR]

Understanding of the relationship between the circadian molecular clock and allergic diseases continues to be studied and may be of practical importance for further selection of optimal treatment. The aim of this study was to evaluate the degree of mRNA expression of the circadian molecular clock genes bmal1 and per1 in buccal epithelial cells and to investigate the efficacy of protocol treatment in children with seasonal allergic rhinitis, taking into account the time dependence. Material and methods. The study included 20 patients aged 6 to 17 years with a confirmed diagnosis of seasonal allergic rhinitis (SAR). The control group included 7 healthy children of the same age with no history of allergic or chronic diseases. All patients with SAR were divided into 2 groups of 10 patients each, who received the protocol therapy in the morning (group 1) and in the evening (group 2) for 4 weeks. The expression of circadian molecular clock genes was studied by real-time polymerase chain reaction (real-time PCR) method by sampling the buccal epithelium of the children's oral cavity in the morning (8:00 am) and in the evening (8:00 pm), with a repeat examination of the samples 1 month after treatment. Statistical analysis was performed using the GraphPad Prism 5.00 computer program (GraphPad Software, Inc., San Diego, CA, USA). The research design was discussed and approved at the meeting of the medical and ethical commission of the Ukrainian Medical and Stomatological Academy, protocol No. 188 dated 11/25/2020, where permission to conduct medical and biological research was issued. The research design was discussed and approved at the meeting of the Medical and Ethical Committee of the Ukrainian Academy of Medicine and Dentistry, Protocol No. 188 dated 25.11.2000, where permission to conduct medical and biological research was granted. The study was conducted in accordance with the research plan of the Pediatrics Department no. 2 of Poltava State Medical University «Optimization of early diagnosis, treatment and prognosis of the most common childhood diseases», state registration number 0122U001876 (years of implementation 2021-2026) and research work - Research Institute of genetic and immunological bases of development of pathology and pharmacogenetics of PDMU «Development of methods of treatment and prevention of development of pulmonary fibrosis by activation of PPAR-gamma receptors», state registration number 0122U201686 (years of implementation 2023-2025). Results. Morning gene expression in healthy children showed statistically higher levels of per1 (0.780 ± 0.070) compared to bmal1 (0.293 ± 0.074; p<0.001), whereas in the evening the level of the latter (0.697 ± 0.130) was significantly higher than per1 (0.370 ± 0.044; p<0.05). Comparing the levels of per1 in this group, the expression of this gene was higher in the morning (0.780 ± 0.070) than in the evening (0.370 ± 0.044; p<0.001), while the expression of bmal1 was higher at 20:00 (0.697 ± 0.130 vs. 0.293 ± 0.074; p<0.05). In contrast, patients with SAR had a significantly higher level of bmal1 gene mRNA expression (1.036±0.161 vs. 0.293±0.074 in healthy children (p<0.05). Examination of gene expression in children with SAR in the evening revealed that bmal1 values showed similar levels of expression in all participants (0.775 ± 0.163 vs. 0.697 ± 0.130; p>0.05), while the mRNA level of the per1 gene was statistically higher in patients with rhinitis (1.293 ± 0.186; p<0.01). Conclusions. The results obtained indicate a dysregulation of the peripheral molecular circadian clock in the buccal epithelium of patients with SAR. The use of the protocol treatment in children with SAR in the evening demonstrated a better restoration of the expression of the positive and negative regulatory link of the molecular circadian clock. [ABSTRACT FROM AUTHOR]

Pharmacological intervention of circadian rhythms is a potentially useful approach for ameliorating various health problems caused by disturbed circadian rhythms including sleep disorder and metabolic diseases. To find compounds that affect circadian rhythms, we screened mushroom extracts using mouse cells expressing the luciferase gene under the control of the mouse Bmal1 promoter. The culture filtrate extract from the basidiomycete Cyclocybe erebia enhanced the oscillation of bioluminescence caused by the expression of the luciferase gene and prolonged the period of bioluminescence. Bioassay-guided fractionation of the extract resulted in purification of compounds 1 and 2. Spectroscopic analyses along with single-crystal X-ray diffraction analysis, revealed that these compounds were diterpenoids with a unique skeleton and a fused ring system comprising 3-, 7-, and 5-membered rings. Compounds 1 and 2 were named cyclocircadins A and B, respectively. These findings suggested that natural diterpenoids could be a source of compounds with the activity affecting circadian rhythms. [Display omitted] • An extract of edible mushroom Cyclocybe erebia modulated circadian rhythms. • Two novel diterpenoids, cyclocircadins A and B, were identified. • The carbon skeleton of the compounds has not been reported from fungi kingdom. • The compounds enhanced and prolonged the periodic expression of the rhythm gene. [ABSTRACT FROM AUTHOR]

Aims The circadian clock is an internal biological timer that co-ordinates physiology and gene expression with the 24-h solar day. Circadian clock perturbations have been associated to vascular dysfunctions in mammals, and a function of the circadian clock in angiogenesis has been suggested. However, the functional role of the circadian clock in endothelial cells (ECs) and in the regulation of angiogenesis is widely unexplored. Methods and results Here, we used both in vivo and in vitro approaches to demonstrate that ECs possess an endogenous molecular clock and show robust circadian oscillations of core clock genes. By impairing the EC-specific function of the circadian clock transcriptional activator basic helix-loop-helix ARNT like 1 (BMAL1) in vivo , we detect angiogenesis defects in mouse neonatal vascular tissues, as well as in adult tumour angiogenic settings. We then investigate the function of circadian clock machinery in cultured EC and show evidence that BMAL and circadian locomotor output cycles protein kaput knock-down impair EC cell cycle progression. By using an RNA- and chromatin immunoprecipitation sequencing genome-wide approaches, we identified that BMAL1 binds the promoters of CCNA1 and CDK1 genes and controls their expression in ECs. Conclusion(s) Our findings show that EC display a robust circadian clock and that BMAL1 regulates EC physiology in both developmental and pathological contexts. Genetic alteration of BMAL1 can affect angiogenesis in vivo and in vitro settings. [ABSTRACT FROM AUTHOR]

While the suprachiasmatic nucleus (SCN) controls 24-h rhythms in breathing, including minute ventilation (VE), the mechanisms by which the SCN drives these daily changes are not well understood. Moreover, the extent to which the circadian clock regulates hypercapnic and hypoxic ventilatory chemoreflexes is unknown. We hypothesized that the SCN regulates daily breathing and chemoreflex rhythms by synchronizing the molecular circadian clock of cells. We used whole-body plethysmography to assess ventilatory function in transgenic BMAL1 knockout (KO) mice to determine the role of the molecular clock in regulating daily rhythms in ventilation and chemoreflex. Unlike their wild-type littermates, BMAL1 KO mice exhibited a blunted daily rhythm in VE and failed to demonstrate daily variation in the hypoxic ventilatory response (HVR) or hypercapnic ventilatory response (HCVR). To determine if the observed phenotype was mediated by the molecular clock of key respiratory cells, we then assessed ventilatory rhythms in BMAL1fl/fl; Phox2bCre/+ mice, which lack BMAL1 in all Phox2b-expressing chemoreceptor cells (hereafter called BKOP). BKOP mice lacked daily variation in HVR, similar to BMAL1 KO mice. However, unlike BMAL1 KO mice, BKOP mice exhibited circadian variations in VE and HCVR comparable to controls. These data indicate that the SCN regulates daily rhythms in VE, HVR, and HCVR, in part, through the synchronization of the molecular clock. Moreover, the molecular clock of Phox2b-expressing cells is specifically necessary for daily variation in the hypoxic chemoreflex. These findings suggest that disruption of circadian biology may undermine respiratory homeostasis, which, in turn, may have clinical implications for respiratory disease. Graphical Abstract [ABSTRACT FROM AUTHOR]

Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson's disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of BMAL1 and SIRT1 in the 6-OHDA-treated SH-SY5Y cells. Additionally, the SIRT1 inhibitor attenuated the effect of DT on BMAL1 and reduced the cell viability. The DT and SIRT1 activators activated SIRT1 and BMAL1, and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed. [ABSTRACT FROM AUTHOR]

Physiological activities of the body exhibit an obvious biological rhythm. At the core of the circadian rhythm, BMAL1 is the only clock gene whose deletion leads to abnormal physiological functions. However, whether intermittent heat stress influences cardiovascular function by altering the circadian rhythm of clock genes has not been reported. This study aimed to investigate whether intermittent heat stress induces autophagy and apoptosis, and the effects of BMAL1 on thoracic aortic autophagy and apoptosis. An intermittent heat stress model was established in vitro , and western blotting and immunofluorescence were used to detect the expression of autophagy, apoptosis, the AMPK/mTOR/ULK1 pathway, and BMAL1. After BMAL1 silencing, RT-qPCR was performed to detect the expression levels of autophagy and apoptosis-related genes. Our results suggest that heat stress induces autophagy and apoptosis in RTAECs. In addition, intermittent heat stress increased the phosphorylation of AMPK and ULK1, but reduced the phosphorylation of mTOR, AMPK inhibitor Compound C reversed the phosphorylation of AMPK, mTOR, and ULK1, and Beclin1 and LC3-II/LC3-I were downregulated. Furthermore, BMAL1 expression was elevated in vitro and shBMAL1 decreased autophagy and apoptosis. We revealed that intermittent heat stress induces autophagy and apoptosis, and that BMAL1 may be involved in the occurrence of autophagy and apoptosis. • Intermittent heat stress increased the expression of BMAL1, autophagy and apoptosis in RTAECs. • AMPK/mTOR/ULK1 pathway involved in the autophagy occurrence. • BMAL1 plays a role in autophagy and apoptosis in RTAECs under heat stress. [ABSTRACT FROM AUTHOR]

Asthma often presents with a daily rhythm; however, the underlying mechanisms remain unclear. Circadian rhythm genes have been proposed to regulate inflammation and mucin expression. Here, ovalbumin (OVA)-induced mice and serum shock human bronchial epidermal cells (16HBE) were used in in vivo and in vitro models, respectively. We constructed a brain and muscle ARNT-like 1 (BMAL1) knockdown 16HBE cell line to analyze the effects of rhythmic fluctuations on mucin expression. Serum immunoglobulin E (IgE) and circadian rhythm genes in asthmatic mice showed rhythmic fluctuation amplitude. Mucin (MUC) 1 and MUC5AC expression was increased in the lung tissue of the asthmatic mice. MUC1 expression was negatively correlated with that of the circadian rhythm genes, particularly BMAL1 (r = −0.546, P = 0.006). There was also a negative correlation between BMAL1 and MUC1 expression (r = −0.507, P = 0.002) in the serum shock 16HBE cells. BMAL1 knockdown negated the rhythmic fluctuation amplitude of MUC1 expression and upregulated MUC1 expression in the 16HBE cells. These results indicate that the key circadian rhythm gene, BMAL1 , causes periodic changes in airway MUC1 expression in OVA-induced asthmatic mice. Targeting BMAL1 to regulate periodic changes in MUC1 expression may, therefore, improve asthma treatments. • Rhythm gene disorders in OVA asthmatic mice manifested by an increased amplitude of transcriptional oscillations. • MUC1 was accompanied by rhythmic oscillations in asthma due to BMAL1 regulation. • The serum IgE levels observed a rhythmic fluctuation in the OVA-challenged mice. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) is widely emerging in various grain crops, milk, and wine products, which can trigger different toxic effects on humans and animals by inhalation or ingestion. It also imposes a considerable financial loss on the agriculture and food industry each year. Previous studies have reported acute and chronic toxicity of DON in liver, and liver is not only the main detoxification organ for DON but also the circadian clock oscillator directly or indirectly regulates critical physiologically hepatic functions under different physiological and pathological conditions. However, researches on the association of circadian rhythm in DON-induced liver damage are limited. In the present study, mice were divided into four groups (CON, DON, Bmal1OE, and Bmal1OE + DON) and AAV8 was used to activate (Bmal1) expression in liver. Then mice were gavaged with 5 mg/kg bw/day DON or saline at different time points (ZT24 = 0, 4, 8, 12, 16, and 20 h) in 1 day and were sacrificed 30 min after oral gavage. The inflammatory cytokines, signal transducers, and activators of transcription Janus kinase/signal transducers and activator of transcription 3 (JAKs/STAT3) pathway and bile acids levels were detected by enzyme-linked immunosorbent assay (ELISA), western blotting, and target metabolomics, respectively. The DON group showed significantly elevated interleukin-1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) levels (P < 0.05 for both) and impaired liver function with rhythm disturbances compared to the CON and Bmal1OE groups. At the molecular level, expressions of some circadian clock proteins were significantly downregulated (P < 0.05 for both) and JAKs/STAT3 pathway was activated during DON exposure, accompanied by indicated circadian rhythm disturbance and inflammatory damage. Importantly, Bmal1 overexpression attenuated DON-induced liver damage, while related hepatic bile acids such as cholic acid (CA) showed a decreasing trend in the DON group compared with the CON group. Our study demonstrates a novel finding that Bmal1 plays a critical role in attenuating liver damage by inhibiting inflammatory levels and maintaining bile acids levels under the DON condition. Therefore, Bmal1 may also be a potential molecular target for reducing the hepatotoxic effects of DON in future studies. [ABSTRACT FROM AUTHOR]

Sex differences in alcohol use and abuse are pervasive and carry important implications for the prevention and treatment of alcohol use disorder (AUD), yet insight into underlying sexually dimorphic mechanisms is limited. Growing experimental and clinical evidence points to an important influence of circadian rhythms and circadian clock genes in the control of alcohol drinking behavior and AUD. Sex differences in the expression of circadian rhythms and in the molecular circadian clock that drive these rhythms have been reported in humans and animals. While studying the role of striatal circadian clock gene expression in the control of affective and goal-directed behaviors, we uncovered a novel sexually dimorphic function of the clock genes Bmal1 and Per2 in the control of voluntary alcohol consumption in mice, which may contribute to sex differences in alcohol drinking behavior. In this mini review, we briefly discuss relevant literature on AUD, circadian rhythms and clock genes, and on sex differences in these domains, and describe our own findings on clock genes as sexually dimorphic regulators of alcohol drinking behavior in mice. [ABSTRACT FROM AUTHOR]

Sarcopenia is an age-related condition that involves a progressive decline in muscle mass and function, leading to increased risk of falls, frailty, and mortality. Although the exact mechanisms are not fully understood, aging-related processes like inflammation, oxidative stress, reduced mitochondrial capacity, and cell apoptosis contribute to this decline. Disruption of the circadian system with age may initiate these pathways in skeletal muscle, preceding the onset of sarcopenia. At present, there is no pharmacological treatment for sarcopenia, only resistance exercise and proper nutrition may delay its onset. Melatonin, derived from tryptophan, emerges as an exceptional candidate for treating sarcopenia due to its chronobiotic, antioxidant, and anti-inflammatory properties. Its impact on mitochondria and organelle, where it is synthesized and crucial in aging skeletal muscle, further highlights its potential. In this review, we discuss the influence of clock genes in muscular aging, with special reference to peripheral clock genes in the skeletal muscle, as well as their relationship with melatonin, which is proposed as a potential therapy against sarcopenia.

The circadian rhythm, which is necessary for reproduction, is controlled by clock genes. In the mouse uterus, the oscillation of the circadian clock gene has been observed. The transcription of the core clock gene period (Per) and cryptochrome (Cry) is activated by the heterodimer of the transcription factor circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein-1 (Bmal1). By binding to E-box sequences in the promoters of Per1/2 and Cry1/2 genes, the CLOCK-BMAL1 heterodimer promotes the transcription of these genes. Per1/2 and Cry1/2 form a complex with the Clock/Bmal1 heterodimer and inactivate its transcriptional activities. Endometrial BMAL1 expression levels are lower in human recurrent-miscarriage sufferers. Additionally, it was shown that the presence of BMAL1-depleted decidual cells prevents trophoblast invasion, highlighting the importance of the endometrial clock throughout pregnancy. It is widely known that hormone synthesis is disturbed and sterility develops in Bmal1-deficient mice. Recently, we discovered that animals with uterus-specific Bmal1 loss also had poor placental development, and these mice also had intrauterine fetal death. Furthermore, it was shown that time-restricted feeding controlled the uterine clock's circadian rhythm. The uterine clock system may be a possibility for pregnancy complications, according to these results. We summarize the most recent research on the close connection between the circadian clock and reproduction in this review. [ABSTRACT FROM AUTHOR]

BACKGROUND: Rheumatoid arthritis is an inflammatory disease with a circadian variation of symptoms. Moxibustion has achieved remarkable efficacy in the treatment of rheumatoid arthritis, but it is unclear whether the specific mechanism is related to the regulation of the clock gene and the inhibition of pyroptosis. OBJECTIVE: To explore the effects of moxibustion at different time points on core clock genes Clock, Bmal1 and pyroptosis in rats with rheumatoid arthritis. METHODS: Forty adult Sprague-Dawley rats of both sexes were randomized into a control group, a model group, a moxibustion at 7:00-9:00 A.M. group and a moxibustion at 5:00-7:00 P.M. group, with 10 rats in each group. To establish the rheumatoid arthritis model, the rats were injected with Freund’s Complete Adjuvant at the right foot. Rats in the control group were given the normal saline at the right foot. At the 7th after modeling, the two moxibustion groups began to be treated with moxibustion at Zusanli and Shenshu acupoints at 7:00-9:00 A.M. or 5:00-7:00 P.M., once a day, for total 18 treatments with a 1-day break every 6 days. We measured the right foot volume of rats using drainage method before model establishment, after model establishment and after treatment. The hypothalamus and the synovium of the right knee joint were collected from the rats after treatment. The expressions of Clock, Bmal1, Nodlike receptor family pyrin domain-containing 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) in the hypothalamus or knee synovium were tested by real-time fluorescence quantitative PCR and western blot. RESULTS AND CONCLUSION: The mRNA expreession of Clock and BMAL1 in the hypothalamus had no obvious change among the four groups (P > 0.05). The relative mRNA expression of Clock and Bmal in the hypothalamus was higher than that in the synovium (P < 0.01). The expression of Clock and Bmal1 in the synovium was significantly down-regulated in the model group compared with the control group (P < 0.01). Moxibustion significantly increased the mRNA and protein expression of Clock and Bmal1 in the synoviums compared with the model group (P < 0.05). Compared with the moxibustion at 7:00-9:00 A.M. group, the mRNA and protein expressions of Clock and Bmal1 in the moxibustion at 5:00-7:00 P.M. group were slightly but insignificantly up-regulated (P > 0.05). The mRNA expressions of NLRP3, Caspase-1 and GSDMD in the synovium were significantly increased in the model group compared with the control group (P < 0.01). Compared with the model group, moxibustion at 5:00-7:00 P.M. could significantly reduce the relative mRNA expression of NLRP3, Caspase-1 and GSDMD in the synovium (P < 0.01). Compared with the moxibustion at 7:00-9:00 A.M. group, the relative mRNA expressions of NLRP3 and Caspase-1 were significantly lowered in the moxibustion at 5:00-7:00 P.M. group (P < 0.05, P < 0.01). All these findings indicate that moxibustion can treat rheumatoid arthritis rats by upregulating the expression of Clock and Bmal1 mRNA and protein in different tissues, down-regulating the mRNA expression of NLRP3, Caspase-1 and GSDMD in the classical signal pathway of pyroptosis, and reducing synovitis and pyroptosis. The anti-inflammatory mechanism of core clock genes Clock and BMAL1 in rheumatoid arthritis may be related to the inhibition of pyrolytic effectors. [ABSTRACT FROM AUTHOR]

Aims: Disturbances in circadian rhythms may promote cardiometabolic disorders in rotating night shift workers (r-NSWs). We hypothesized that timed light therapy might reverse disrupted circadian rhythms and glucose intolerance observed among r-NSWs). Methods: R-NSWs were randomly assigned to a protocol that included 12 weeks on followed by 12 weeks off light therapy (n = 13; 6 men; mean age, 39.5 ± 7.3 years) or a no-treatment control group (n = 9; 3 men; mean age 41.7 ± 6.3 years). Experimental and control participants underwent identical metabolic evaluations that included anthropometric, metabolic (including oral glucose tolerance tests), lipid, and inflammation-associated parameters together with an assessment of sleep quality and expression of circadian transcription factors REV-ERBα and BMAL1 in peripheral blood mononuclear cells (PBMCs) at baseline, 12 weeks, and 24 weeks of the protocol. Results: Twelve weeks of warm white-light exposure (10,000 lx at 35 cm for 30 min per day) had no impact on sleep, metabolic, or inflammation-associated parameters among r-NSWs in the experimental group. However, our findings revealed significant decreases in REV-ERBα gene expression (p = 0.048) and increases in the REV-ERBα/BMAL1 ratio (p = 0.040) compared to baseline in PBMCs isolated from this cohort. Diminished expression of REV-ERBα persisted, although the REV-ERBα/BMAL1 ratio returned to baseline levels after the subsequent 12-day wash-out period. Conclusions: Our results revealed that intermittent light therapy had no impact on inflammatory parameters or glucose tolerance in a defined cohort of r-NSWs. However, significant changes in the expression of circadian clock genes were detected in PBMCs of these subjects undergoing light therapy. [ABSTRACT FROM AUTHOR]

Humans and other organisms are continuously exposed to thousands of chemicals through the atmosphere, drinking water, food, or direct contact. A large proportion of such chemicals are present in very low concentrations and may have synergistic effects, even at their no-observed-adverse-effect level (NOAEL). Complex mixtures of contaminants are very difficult to assess by traditional toxicological methods. There is increasing attention on how different pollutants induce adverse physiological functions in the human body through effects on the circadian rhythm. However, it is very difficult to screen for compounds with circadian-rhythm-disrupting effects from a large number of chemicals or their complex mixtures. We established a stable firefly luciferase reporter gene knock-in U2-OS cell line by CRISPR/Cas9 to screen circadian-rhythm-disrupting pollutants. The luciferase gene was inserted downstream of the core clock gene BMAL1 and controlled by an endogenous promoter. Compared to detection systems using exogenous promoters, these cells enable the detection of compounds that interfere with the circadian rhythm system mediated by BMAL1 gene expression. The U2-OS knock-in cells showed BMAL1 and luciferase activity had parallel changes when treated with BMAL1 inhibitor and activator. Furthermore, the luciferase reporter gene has high sensitivity and is faster and more cost-effective than classic toxicology methods. The knock-in cell line can be used for high-throughput and efficient screening of circadian-rhythm-disrupting chemicals such as drugs and pollutants. [ABSTRACT FROM AUTHOR]

Radiofrequency electromagnetic fields (RF-EMF) exert pleiotropic effects on biological processes including circadian rhythms. miR-34a is a small non-coding RNA whose expression is modulated by RF-EMF and has the capacity to regulate clock gene expression. However, interference between RF-EMF and miR-34a-mediated regulation of the circadian oscillator has not yet been elucidated. Therefore, the present study was designed to reveal if 24 h exposure to 2.4 GHz RF-EMF influences miR-34a-induced changes in clock gene expression, migration and proliferation in colorectal cancer cell line DLD1. The effect of up- or downregulation of miR-34a on DLD1 cells was evaluated using real-time PCR, the scratch assay test and the MTS test. Administration of miR-34a decreased the expression of per2, bmal1, sirtuin1 and survivin and inhibited proliferation and migration of DLD1 cells. When miR-34a-transfected DLD1 cells were exposed to 2.4 GHz RF-EMF, an increase in cry1 mRNA expression was observed. The inhibitory effect of miR-34a on per2 and survivin was weakened and abolished, respectively. The effect of miR-34a on proliferation and migration was eliminated by RF-EMF exposure. In conclusion, RF-EMF strongly influenced regulation mediated by the tumour suppressor miR-34a on the peripheral circadian oscillator in DLD1 cells. [ABSTRACT FROM AUTHOR]

Objective: To observe the effect of overexpression of clock gene Bmal1 on the proliferation of vascular smooth muscle cells and investigate potential role mechanism of biological rhythms on vascular developmen through experiments. Methods: The method of lentiviral transfection packaging GV341-Bmal1 vector was used to construct rat thoracic aortic smooth muscle cells（A7R5） stably transfected with Bmal1 cell line. Real-time fluorescent quantitative PCR（RT-PCR） and immunofluorescence staining of Bmal1 on the cells grown on coverslips were used to determine whether the stable transfected cell line overexpressed Bmal1 stably. Immunofluorescence staining of Ki67 on the cells grown on coverslips was used to observe the effect of overexpression of clock gene Bmal1 on the proliferation of vascular smooth muscle cells. Results: The results of Real-time fluorescent quantitative PCR showed that the expression of Bmal1 in cells stably transfected with Bmal1 was 11.2 times higher compared to control group（P<0.01）. The results of immunofluorescence staining of the cells grown on coverslips showed that the expression of BMAL1 was significantly increased in stable transfected cell lines（P<0.05）. Furthermore, compared to the control group, the proportion of Ki67 positive cells was also increased significantly in vascular smooth muscle cells stably transfected with Bmal1（P<0.05）. Conclusion: We have established a vascular smooth muscle cell line with Bmal1 overexpressed stably by Lentiviral transfection method. Through observing immunofluorescence staining of Ki67 on the cells grown on coverslips, we have found that overexpression of Bmal1 promoted the proliferation of vascular smooth muscle cell. [ABSTRACT FROM AUTHOR]

Circadian information is stored in mammalian tissues by an autonomous network of transcriptional feedback loops that have evolved to optimally regulate tissue-specific functions. Currently, stable circadian rhythms of the expression of clock genes (Bmal1/Per2/Cry1, etc.), hormones, and metabolic genes (Glut4/leptin, etc.) have been demonstrated. Desynchronoses are disorders of the body's biorhythms, where the direction and degree of shift of various indicators of the oscillatory process are disturbed. Desynchronosis can be caused by natural conditions or man-made causes. The disruption of circadian rhythms is a risk factor for the appearance of physiological and behavioral disorders and the development of diseases, including epilepsy, and metabolic and oncological diseases. Evidence suggests that seizure activity in the epilepsy phenotype is associated with circadian dysfunction. Interactions between epilepsy and circadian rhythms may be mediated through melatonin, sleep–wake cycles, and clock genes. The correction of circadian dysfunction can lead to a decrease in seizure activity and vice versa. Currently, attempts are being made to pharmacologically correct desynchronosis and related psycho-emotional disorders, as well as combined somatic pathology. On the other hand, the normalization of the light regimen, the regulation of sleep–wake times, and phototherapy as additions to standard treatment can speed up the recovery of patients with various diseases. [ABSTRACT FROM AUTHOR]

Approximately 50–80% of children with autism spectrum disorders (ASDs) exhibit sleep problems, but the contribution of circadian clock dysfunction to the development of ASDs remains largely unknown. The essential clock gene Bmal1 (Arntl or Mop3) has been associated with human sociability, and its missense mutation is found in ASD. Our recent study found that Bmal1-null mice exhibit a variety of autism-like phenotypes. Here, we further investigated whether an incomplete loss of Bmal1 function could cause significant autism-like behavioral changes in mice. Our results demonstrated that heterozygous Bmal1 deletion (Bmal1+/−) reduced the Bmal1 protein levels by ~50–75%. Reduced Bmal1 expression led to decreased levels of clock proteins, including Per1, Per2, Cry 1, and Clock but increased mTOR activities in the brain. Accordingly, Bmal1+/− mice exhibited aberrant ultrasonic vocalizations during maternal separation, deficits in sociability and social novelty, excessive repetitive behaviors, impairments in motor coordination, as well as increased anxiety-like behavior. The novel object recognition memory remained intact. Together, these results demonstrate that haploinsufficiency of Bmal1 can cause autism-like behavioral changes in mice, akin to those identified in Bmal1-null mice. This study provides further experimental evidence supporting a potential role for disrupted clock gene expression in the development of ASD. [ABSTRACT FROM AUTHOR]

Abstract Clock genes not only regulate the circadian rhythm of physiological activities but also participate in the pathogenesis of many diseases. Previous studies have documented the abnormal expression of clock genes in epilepsy. However, the molecular mechanism of brain and muscle Arnt-like protein 1 (Bmal1), one of the core clock genes, in the epileptogenesis and seizures of temporal lobe epilepsy (TLE) remain unclear. We first investigated the levels of Bmal1 and other clock proteins in the hippocampus of subjects with epilepsy to define the function of Bmal1. The levels of Bmal1 were decreased during the latent and chronic phases in the experimental group compared with those in the control group. Knockout of Bmal1 in hippocampal dentate gyrus (DG) neurons of Bmal1flox/flox mice by Synapsin 1 (Syn1) promoter AAV (adeno-associated virus) lowered the threshold of seizures induced by pilocarpine administration. High-throughput sequencing analysis showed that PCDH19 (protocadherin 19), a gene associated with epilepsy, was regulated by Bmal1. PCDH19 expression was also decreased in the hippocampus of epileptic mice. Furthermore, the higher levels of Bmal1 and PCDH19 were detected in patients with no hippocampal sclerosis (no HS) than in patients with HS International League Against Epilepsy (ILAE) type I and III. Altogether, these data suggest that decreased expression of clock gene Bmal1 may participate in epileptogenesis and seizures via PCDH19 in TLE.

Purpose of the study: to review in vivo studies on the relationship and role of various molecular genetic components of the circadian rhythm system in the initiation and development of malignant neoplasms. in contrast to clinical and epidemiological studies, animal models, including transgenic animal models, can model various changes and disturbances in the activity of clock genes and track the results of these changes.Material and Methods. the review includes data from studies carried out over the past 10 years in animal models, studying the mechanisms and effects of disturbances in the system of circadian rhythms related to the formation and development of tumors. the data sources for the review were the Medline, embase and scopus databases.Results. analysis of the literature has shown that interference with the work of the «biological clock» by changing the light cycle, disrupting the expression of clock genes and other manipulations is a factor predisposing to the development of tumors. in tumors of various types, the expression of clock genes is often mismatched, and it is unclear at what stage of their formation this occurs. in addition, the development of tumors disrupts the circadian homeostasis of the body. there are three key areas of research aimed at studying the role of circadian rhythms in tumor development: disturbance of circadian rhythms as a carcinogenic factor, disturbances in the clock gene system in a tumor, disturbances in the clock gene system of the whole organism, provoked by tumor development.Conclusion. the results of studies on animal models demonstrate that the relationship between the disturbance of circadian rhythms and the tumor process is complex since the causal relationship has not yet been studied. in this regard, the prospect of targeted pharmacological correction of circadian rhythms in clinical practice in cancer patients does not seem to be the nearest one.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging. [ABSTRACT FROM AUTHOR]

The regulation of renal function by circadian gene BMAL1 has been recently recognized; however, the role and mechanism of BMAL1 in renal ischaemia‐reperfusion injury (IRI) are still unknown. The purpose of this study was to clarify the pathophysiological role of BMAL1 in renal IRI. We measured the levels of BMAL1 and mitochondrial biogenesis‐related proteins, including SIRT1, PGC‐1α, NRF1 and TFAM, in rats with renal IRI. In rats, the level of BMAL1 decreased significantly, resulting in inhibition of SIRT1 expression and mitochondrial biogenesis. In addition, under hypoxia and reoxygenation (H/R) stimulation, BMAL1 knockdown decreased the level of SIRT1 and exacerbated the degree of mitochondrial damage and apoptosis. Overexpression of BMAL1 alleviated H/R‐induced injury. Furthermore, application of the SIRT1 inhibitor EX527 not only reduced the activities of SIRT1 and PGC‐1α but also further aggravated mitochondrial dysfunction and partially reversed the protective effect of BMAL1 overexpression. Moreover, whether in vivo or in vitro, the application of SIRT1 agonist resveratrol rescued the mitochondrial dysfunction caused by H/R or IRI by activating mitochondrial biogenesis. These results indicate that BMAL1 is a key circadian gene that mediates mitochondrial homeostasis in renal IRI through the SIRT1/PGC‐1α axis, which provides a new direction for targeted therapy for renal IRI. [ABSTRACT FROM AUTHOR]

The circadian clock is vital in the management of our daily physiological as well as metabolic processes. Disturbances of the clock can cause degenerative and age-related diseases. Increasing evidence has indicated that the intervertebral discs contain an internal biological clock related to degeneration. However, to date, no bioactive compounds have been found that can ameliorate intervertebral disc degeneration (IDD) by restoring the circadian clock. (-)-Epigallocatechin-3-gallate (EGCG) is a nutritious food with powerful antioxidant properties, as well as entraining biological clock to improve health. The purpose of this study was to determine whether the protective effects of EGCG on nucleus pulposus (NPCs) under oxidative stress is related to the circadian clock. First, we found that EGCG attenuated H2O2-induced extracellular matrix degradation in NPCs and inhibited H2O2-induced NPC apoptosis. Our in vivo experiments also confirmed this finding. Furthermore, EGCG attenuated H2O2-triggered dampening of phase shifts and daily oscillations in circadian clock gene transcription as well as protein expression levels. Intriguingly, core clock gene (Bmal1) knockdown notably blocked the protective effects of EGCG. To our knowledge, this study provides the first convincing evidence that EGCG prevents IDD in a Bmal1-dependent manner. In general, EGCG supplementation can be used as a nutritional prevention strategy for the rehabilitation of degenerative diseases related to the circadian clock. [ABSTRACT FROM AUTHOR]

The circadian clock system plays an important role in regulating testosterone synthesis in mammals. Male Bmal1 −/− mice are infertile with low serum testosterone levels and decreased expression of testicular steroidogenic genes, suggesting that circadian clock genes regulate testosterone biosynthesis by activating steroidogenic gene transcription. However, whether the circadian clock regulates testosterone production via other genes remains unknown. Using Bmal1 −/− mice and their wild-type (WT) siblings, we aimed to identify additional genes by which the circadian clock regulates testosterone synthesis. WT and Bmal1 −/− mouse testes sections had similar normal morphologies, although there was a decrease in testicular spermatozoa in the Bmal1 −/− mice. Low serum testosterone levels were detected in the Bmal1 −/− mice. RNA sequencing identified 37 and 48 genes that were differentially expressed between WT and Bmal1 −/− mouse testes at circadian time (CT2 and CT14), respectively. The cholesterol metabolism pathway was significantly enriched in the KEGG pathway analysis, and there was lower expression of three apolipoprotein genes (Apoa1 , Apoa2 , and Apoc3) at CT2 in the testes of Bmal1 −/− mice than in those of WT mice. These decreases in Apoa1 , Apoa2 , and Apoc3 expression were verified by quantitative polymerase chain reaction analysis, which also revealed downregulation of the expression of the circadian clock (Per2 , Dbp , and Nr1d1) and steroidogenic (StAR , Cyp11a1 , and Hsd17b3) genes. The expression of circadian clock genes was relatively stable in WT mice over a 20-h period, whereas there was clear circadian rhythmic expression of Apoa1 , Apoa2 , Apoc3 , StAR , Cyp11a1 , Hsd3b2 , and Hsd17b3. Bmal1 −/− mice showed severely reduced expression of testicular circadian clock genes at three time points (CT4, CT12, and CT20), and a reduction in mRNA expression levels of Apo (Apoa1 , Apoa2 , and Apoc3) and steroidogenic (StAR , Cyp11a1 , Hsd3b2 , and Hsd17b3) genes. Oil Red O staining showed decreased lipid aggregation in the Leydig cells of Bmal1 −/− mouse testes. Considering the vital role of Apo genes in high-density lipoprotein formation and cholesterol transport, the present data suggest that the circadian clock system regulates testosterone production by orchestrating the rhythmic expression of Apo genes. These data extend our understanding of the role of the circadian clock in regulating testosterone production in mammals. • Robust rhythmic expression of apolipoprotein genes was detected in WT mice testes. • The rhythmic expression of apolipoprotein genes was lost in Bmal1 −/− mice testes. • Reduced expression of apolipoprotein genes was observed in Bmal1 −/− mice testes. • The cholesterol metabolism pathway is severely impaired in Bmal1 −/− mice testes. • Lipid accumulation in Bmal1 −/− mice Leydig cells was clearly attenuated. [ABSTRACT FROM AUTHOR]

Testosterone is produced by Leydig cells (LCs) and undergoes diurnal changes in serum levels in rats, mice, and humans, but little is known in goats. The present study revealed that goat serum testosterone levels displayed diurnal rhythmic changes (peak time at ZT11.2). Immunohistochemical staining showed that BMAL1, a circadian clock protein, is highly expressed in goat LCs. ELISA revealed that both hCG (0–5 IU/ml) and 22R‐OH‐cholesterol (0–30 μM) addition stimulated testosterone synthesis in primary goat LCs in a dose‐dependent manner. Treating goat LCs with hCG (5 IU/ml) significantly increased intracellular cAMP levels. Additionally, real‐time quantitative polymerase chain reaction (PCR) analysis revealed that the circadian clock (BMAL1, PER1, PER2, DBP, and NR1D1) and steroidogenesis‐related genes (SF1, NUR77, StAR, HSD3B2, CYP17A1, CYP11A1, and HSD17B3) showed rhythmic expression patterns in goat LCs following dexamethasone synchronization. Several Bmal1‐Luc circadian oscillations were clearly observed in dexamethasone‐treated goat LCs transfected with the pLV6‐Bmal1‐Luc plasmid. BMAL1 knockdown significantly downregulated mRNA levels of PER2, NR1D1, DBP, StAR, HSD3B2, SF1, NUR77, and GATA4, and dramatically decreased StAR and HSD3B2 protein levels and testosterone production. In contrast, BMAL1 overexpression significantly increased the mRNA and protein expression levels of StAR and HSD17B3 and enhanced testosterone production. Reporter assays revealed that goat BMAL1, or in combination with mouse CLOCK, activated goat HSD17B3 transcription in vitro. These data indicate that BMAL1 contributes to testosterone production by regulating transcription of steroidogenesis‐related genes in goat LCs, providing a basis for further exploring the underlying mechanism by which the circadian clock regulates ruminant reproductive capability. [ABSTRACT FROM AUTHOR]

Clock genes not only regulate the circadian rhythm of physiological activities but also participate in the pathogenesis of many diseases. Previous studies have documented the abnormal expression of clock genes in epilepsy. However, the molecular mechanism of brain and muscle Arnt-like protein 1 (Bmal1), one of the core clock genes, in the epileptogenesis and seizures of temporal lobe epilepsy (TLE) remain unclear. We first investigated the levels of Bmal1 and other clock proteins in the hippocampus of subjects with epilepsy to define the function of Bmal1. The levels of Bmal1 were decreased during the latent and chronic phases in the experimental group compared with those in the control group. Knockout of Bmal1 in hippocampal dentate gyrus (DG) neurons of Bmal1flox/flox mice by Synapsin 1 (Syn1) promoter AAV (adeno-associated virus) lowered the threshold of seizures induced by pilocarpine administration. High-throughput sequencing analysis showed that PCDH19 (protocadherin 19), a gene associated with epilepsy, was regulated by Bmal1. PCDH19 expression was also decreased in the hippocampus of epileptic mice. Furthermore, the higher levels of Bmal1 and PCDH19 were detected in patients with no hippocampal sclerosis (no HS) than in patients with HS International League Against Epilepsy (ILAE) type I and III. Altogether, these data suggest that decreased expression of clock gene Bmal1 may participate in epileptogenesis and seizures via PCDH19 in TLE. [ABSTRACT FROM AUTHOR]

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care. [Display omitted] • The quantity and function of tumor-infiltrating T cells are time-of-day dependent • Rhythmic tumor infiltration of T cells depends on the endothelial circadian clock • Adjusting the time of injection during the day improves CAR T cell therapy efficacy • Enhanced tumor control by timed anti-PD-1 therapy depends on circadian T cell activity Efficacy of CAR T cell therapy and immune checkpoint blockade in cancer can be improved by adjusting the time of treatment during the day due to rhythmic infiltration and function of T cells regulated by the circadian clock in T cells and endothelial cells in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis, when left untreated, causes serious health problems that progress to cirrhosis and, in some cases, liver cancer. Activation of hepatic stellate cells may be a key characteristic in the development of hepatic fibrosis. Melatonin, a pineal hormone, exerts anti-fibrotic effects; however, the exact mechanisms remain unclear. In this study, the beneficial effects of melatonin against hepatic fibrosis and the underlying mechanisms were investigated using the human hepatic stellate cell line, LX-2, and in vivo murine animal models. The results showed that melatonin suppressed the expression of transforming growth factor (TGF)-β1-induced fibrosis markers and production of reactive oxygen species in LX-2 cells. Either 4-phenyl-2-propionamidotetralin, a melatonin receptor 2 selective antagonist, or melatonin receptor 2 small interfering RNA abolished the suppressive effects of melatonin, suggesting the involvement of melatonin receptor 2 in melatonin-induced anti-fibrotic and anti-oxidative actions. Melatonin increased the expression of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), a positive circadian clock gene. BMAL1 knockdown reduced the anti-fibrotic and anti-oxidative effects of melatonin, demonstrating the protective effects of melatonin against TGF-β1-induced hepatic stellate cell activation by exhibiting melatonin receptor 2-BMAL1-anti-oxidative effects. In high-fat diet-induced and carbon tetrachloride-induced hepatic fibrosis models, oral melatonin administration decreased the expression of hepatic fibrosis markers and increased the expression of messenger RNA and levels of proteins of BMAL1 and melatonin receptor 2. Thus, melatonin exerted protective effects against hepatic fibrosis through melatonin receptor 2 activation, followed by an upregulation of the BMAL1-anti-oxidative enzyme pathways. • Melatonin suppresses TGF-β1-induced fibrosis in hepatic stellate cells. • The role of membrane melatonin receptors in the anti-fibrosis is unknown. • Melatonin receptor 2 is involved in melatonin-induced anti-fibrotic effects. • BMAL1 upregulation is a key downstream signaling of melatonin receptor 2 activation. • Anti-oxidant effect of melatonin is implicated in BMAL1-mediated anti-fibrosis. [ABSTRACT FROM AUTHOR]

Abstract Background Disturbances in clock genes affect almost all patients with Alzheimer’s disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD. Methods In this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests. Results CLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice. Conclusion Clock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.

In this study, we found that loss of the circadian clock gene Bmal1 causes disruptions throughout the growth hormone (GH) axis, from hepatic gene expression to production of urinary pheromones and pheromone-dependent behavior. First, we show that Bmal1 knockout (KO) males elicit reduced aggressive responses from wild-type (WT) males and secrete lower levels of major urinary proteins (MUPs); however, we also found that a liver-specific KO of Bmal1 (liver- Bmal1- KO) produces a similar reduction in MUP secretion without a defect in aggressive behavior, indicating that the decrease in elicited aggression arises from another factor. We then shifted our investigation to determine the cause of MUP dysregulation in Bmal1 KO animals. Because the pulse pattern of GH drives sexually dimorphic expression of hepatic genes including MUPs, we examined GH pulsatility. We found that Bmal1 KO males have a female-like pattern of GH release, whereas liver- Bmal1- KO mice are not significantly different from either WT or Bmal1 KO. Since differential patterns of GH release regulate the transcription of many sexually dimorphic genes in the liver, we then examined hepatic gene transcription in Bmal1 KO and liver- Bmal1 -KO mice. We found that while some female-predominant genes increase in the Bmal1 KO, there was no decrease in male-predominant genes, and little change in the liver- Bmal1- KO. We also found disrupted serum insulin growth factor 1 (IGF-1) and liver Igf1 messenger RNA in the Bmal1 KO mice, which may underlie the disrupted GH release. Overall, our findings differentiate between GH-pulse–driven and circadian-driven effects on hepatic genes, and the functional consequences of altered GH pulsatility. [ABSTRACT FROM AUTHOR]

• The circadian clock generally exists in mammals, and clock genes are expressed in a circadian rhythm. • Recently many studies have revealed that the circadian clock regulates the immune response. • There is a bi-directional relationship between inflammatory response and the circadian clock. • This work may provide a new direction for the discovery of circadian rhythm targets that may be used in the treatment of inflammatory diseases. The circadian clock is a complex cellular mechanism that controls a series of physiological processes, including inflammation. It can directly interact physically with the components of the key inflammatory pathway. Similarly, inflammation can also lead to circadian rhythm disorders, which may further amplify the inflammatory response and aggravate tissue damage. This review offers a structured overview that focusses on the core proteins of the circadian clock and their interactions with inflammatory players, and provides a potential mechanism for the pathological rhythms observed under inflammatory conditions. [ABSTRACT FROM AUTHOR]

We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3β, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed. [ABSTRACT FROM AUTHOR]

Objectives We aimed to characterize diurnal rhythms in CES1 expression and activity in mouse intestine, and to investigate a potential role of the core clock gene Bmal1 in generating diurnal rhythms. Methods The regulatory effects of intestinal Bmal1 on diurnal CES1 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Metabolic activity of CES1 in vitro and in vivo were determined by microsomal assays and pharmacokinetic studies, respectively. Transcriptional gene regulation was investigated using luciferase reporter assay. Key findings Total CES1 protein varied significantly according to time of the day in wild-type (Bmal1fl/fl) mice, peaking at ZT6. Of detectable Ces1 genes, Ces1d mRNA displayed a robust diurnal rhythm with a peak level at ZT6, whereas mRNAs of Ces1e, 1f and 1g showed no rhythms in wild-type mice. Loss of intestinal Bmal1 reduced the levels of total CES1 protein and Ces1d mRNA, and blunted their diurnal rhythms in mice. In vitro microsomal assays indicated that intestinal metabolism of mycophenolate mofetil (MMF, a known CES1 substrate) was more extensive at ZT6 than at ZT18. ZT6 dosing of MMF to wild-type mice generated a higher systemic exposure of mycophenolic acid (the active metabolite of MMF) as compared with ZT18 dosing. Intestinal ablation of Bmal1 down-regulated CES1 metabolism at ZT6, and abolished its time-dependency both in vitro and in vivo. Furthermore, Ces1d/CES1 rhythmicity and positive regulation of Ces1d/CES1 by BMAL1 were confirmed in CT26 and Caco-2 cells. Mechanistically, BMAL1 trans-activated Ces1d/CES1 probably via binding to the E-box elements in the gene promoters. Conclusions Bmal1 controls diurnal rhythms in expression and activity of intestinal CES1. Our findings have implications for understanding the crosstalk between circadian clock and xenobiotic metabolism in the intestine. [ABSTRACT FROM AUTHOR]

Streptococcus oralis (S. oralis) has been recognized as a fatal pathogen to cause multiorgan failure by contributing to the formation of microthrombus. Coagulation and fibrinolysis systems have been found under the control of circadian clock genes. This study aimed to explore the correlation between BMAL1 and coagulation factor biosynthesis in S. oralis infection. Mice were administered S. oralis to induce sepsis, and HepG2 cells were also infected by S. oralis. The expression of BMAL1 of hepatocytes was downregulated in the S. oralis infection group, leading to the downregulation of coagulation factor VII (FVII) and the upregulation of the coagulation factor XII (FXII) in vitro and in vivo. Furthermore, we confirmed that the deficiency of BAML1 contributed to the elevation of FVII and the decline in FXII by constructing BMAL1-deficiency (Bmal1 −/−) mice. The current result showed that BMAL1 regulates FVII directly. Thus, a novel insight into the coagulation abnormality in S. oralis infection was gained that may optimize the treatment of sepsis by rescuing the expression of BMAL1 in the liver. [ABSTRACT FROM AUTHOR]

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]

The circadian clock is a global regulatory mechanism that controls the expression of 50 to 80% of transcripts in mammals. Some of the genes controlled by the circadian clock are oncogenes or tumor suppressors. Among these Myc has been the focus of several studies which have investigated the effect of clock genes and proteins on Myc transcription and MYC protein stability. Other studies have focused on effects of Myc mutation or overproduction on the circadian clock in comparison to their effects on cell cycle progression and tumorigenesis. Here we have used mice with mutations in the essential clock genes Bmal1, Cry1, and Cry2 to gain further insight into the effect of the circadian clock on this important oncogene/oncoprotein and tumorigenesis. We find that mutation of both Cry1 and Cry2,which abolishes the negative armof the clock transcription- translation feedback loop (TTFL), causes down-regulation of c-MYC, and mutation of Bmal1, which abolishes the positive arm of TTFL, causes up-regulation of the c-MYC protein level in mouse spleen. These findings must be taken into account in models of the clock disruption-cancer connection. [ABSTRACT FROM AUTHOR]

Dentine‐ and enamel‐forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so‐called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine‐ and enamel‐ secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1−/−). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild‐type, Bmal+/–, and Bmal1−/−). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue‐specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates. [ABSTRACT FROM AUTHOR]

The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently timeof- day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytimerestricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day-dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment. [ABSTRACT FROM AUTHOR]

Introduction: The occurrence of circadian rhythm disorder in patients with Alzheimer's disease (AD) is closely related to the abnormal deposition of amyloid‐β (Aβ), and d‐Ser2‐oxyntomodulin (Oxy) is a protease‐resistant oxyntomodulin analogue that has been shown to exert neuroprotective effects. Aims: This study aimed to explore whether Oxy, a new GLP‐1R/GCGR dual receptor agonist, can improve the Aβ‐induced disrupted circadian rhythm and the role of GLP‐1R. Methods: A mouse wheel‐running experiment was performed to explore the circadian rhythm, and western blotting and real‐time PCR were performed to assess the expression of the circadian clock genes Bmal1 and Per2. Furthermore, a lentivirus encoding an shGLP‐1R‐GFP‐PURO was used to interfere with GLP‐1R gene expression and so explore the role of GLP‐1R. Results: The present study has confirmed that Oxy could restore Aβ31‐35‐induced circadian rhythm disorders and improve the abnormal expression of Bmal1 and Per2. After interfering the GLP‐1R gene, we found that Oxy could not improve the Aβ31‐35‐induced circadian rhythm disorder and abnormal expression of clock genes. Conclusion: This study demonstrated that Oxy could improve Aβ31‐35‐induced circadian rhythm disorders, and GLP‐1R plays a critical role. This study thus describes a novel target that may be potentially used in the treatment of AD. [ABSTRACT FROM AUTHOR]