Disruption of central and peripheral circadian clocks and circadian controlled estrogen receptor rhythms in night shift nurses in working environments.

Chronic disruption of circadian rhythms by night shift work is associated with an increased breast cancer risk. However, little is known about the impact of night shift on peripheral circadian genes (CGs) and circadian-controlled genes (CCGs) associated with breast cancer. Hence, we assessed central clock markers (melatonin and cortisol) in plasma, and peripheral CGs (PER1, PER2, PER3, and BMAL1) and CCGs (ESR1 and ESR2) in peripheral blood mononuclear cells (PBMCs). In day shift nurses (n = 12), 24-h rhythms of cortisol and melatonin were aligned with day shift-oriented light/dark schedules. The mRNA expression of PER2, PER3, BMAL1, and ESR2 showed 24-h rhythms with peak values in the morning. In contrast, night shift nurses (n = 10) lost 24-h rhythmicity of cortisol with a suppressed morning surge but retained normal rhythmic patterns of melatonin, leading to misalignment between cortisol and melatonin. Moreover, night shift nurses showed disruption of rhythmic expressions of PER2, PER3, BMAL1, and ESR2 genes, resulting in an impaired inverse correlation between PER2 and BMAL1 compared to day shift nurses. The observed trends of disrupted circadian markers were recapitulated in additional day (n = 20) and night (n = 19) shift nurses by measurement at early night and midnight time points. Taken together, this study demonstrated the misalignment of cortisol and melatonin, associated disruption of PER2 and ESR2 circadian expressions, and internal misalignment in peripheral circadian network in night shift nurses. Morning plasma cortisol and PER2, BMAL1, and ESR2 expressions in PBMCs may therefore be useful biomarkers of circadian disruption in shift workers.

Moderating effects of PER3 gene DNA methylation on the association between problematic mobile phone use and chronotype among Chinese young adults: Focus on gender differences.

Objective: To investigate the rates of problematic mobile phone use (PMPU) and chronotypes in young adults, and examine the associations of PMPU with chronotypes, as well as its gender differences. Furthermore, we explored the moderating role of PER3 gene DNA methylation on the associations. Methods: From April to May 2019, a total of 1,179 young adults were selected from 2 universities in Anhui and Jiangxi provinces. The Self-rating Questionnaire for Adolescent Problematic Mobile Phone Use (SQAPMPU) and reduced Morningness-Eveningness Questionnaire (rMEQ) were adopted to investigate PMPU and chronotypes in young adults, respectively. Moreover, 744 blood samples were collected to measure PER3 gene DNA methylation. Multivariate logistic regression models were established to analyze the associations between PMPU and chronotypes. Moderating analysis was used to determine whether PER3 gene DNA methylation moderated the relationships between PMPU and chronotypes. Results: The prevalence of PMPU, morning chronotypes (M-types), neutral chronotypes (N-types), and evening chronotypes (E-types) of young adults were 24.6%, 18.4%, 71.1%, and 10.5%, respectively. Multivariate logistic regression results indicated that PMPU was positively correlated with E-types (OR = 3.53, 95%CI: 2.08-6.00), and the association was observed only in females after stratified by gender (OR = 5.36, 95%CI: 2.70-10.67). Furthermore, PER3 gene DNA methylation has a negative moderating role between PMPU and chronotypes and has a sex-based difference. Conclusions: This study can provide valuable information for the prevention and control of circadian rhythm disturbance among young adults from the perspective of epidemiology and biological etiology.

Pharmacological Modulation of the Cytosolic Oscillator Affects Glioblastoma Cell Biology.

The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12-15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1 ε/δ (CK1ε/δ) with specific inhibitors (CHIR99021 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99021-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition compared to control cells. The combined treatment of CHIR99021 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects GBM cell biology.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

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.

Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice.

Polymethoxyflavonoids, such as nobiletin (abundant in Citrus depressa), have been reported to have antioxidant, anti-inflammatory, anticancer, and anti-dementia effects, and are also a circadian clock modulator through retinoic acid receptor-related orphan receptor (ROR) α/γ. However, the optimal timing of nobiletin intake has not yet been determined. Here, we explored the time-dependent treatment effects of nobiletin and a possible novel mechanistic idea for nobiletin-induced circadian clock regulation in mice. In vivo imaging showed that the PER2::LUC rhythm in the peripheral organs was altered in accordance with the timing of nobiletin administration (100 mg/kg). Administration at ZT4 (middle of the light period) caused an advance in the peripheral clock, whereas administration at ZT16 (middle of the dark period) caused an increase in amplitude. In addition, the intraperitoneal injection of nobiletin significantly and potently stimulated corticosterone and adrenaline secretion and caused an increase in Per1 expression in the peripheral tissues. Nobiletin inhibited phosphodiesterase (PDE) 4A1A, 4B1, and 10A2. Nobiletin or rolipram (PDE4 inhibitor) injection, but not SR1078 (RORα/γ agonist), caused acute Per1 expression in the peripheral tissues. Thus, the present study demonstrated a novel function of nobiletin and the regulation of the peripheral circadian clock.

Bmal1 integrates circadian function and temperature sensing in the suprachiasmatic nucleus.

Circadian regulation and temperature dependency are important orchestrators of molecular pathways. How the integration between these two drivers is achieved, is not understood. We monitored circadian- and temperature-dependent effects on transcription dynamics of cold-response protein RNA Binding Motif 3 (Rbm3). Temperature changes in the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), induced Rbm3 transcription and regulated its circadian periodicity, whereas the core clock gene Per2 was unaffected. Rbm3 induction depended on a full Brain And Muscle ARNT-Like Protein 1 (Bmal1) complement: reduced Bmal1 erased Rbm3 responses and weakened SCN circuit resilience to temperature changes. By focusing on circadian and temperature dependency, we highlight weakened transmission between core clock and downstream pathways as a potential route for reduced circadian resilience.

Isoglutaminyl Cyclase Overexpression Enhances KYSE30 Cancer Cell Proliferation and Migration via the MAPK Signaling Pathway.

To understand how upregulated isoglutaminyl cyclase (isoQC) is involved in the initiation of diseases such as cancer, we developed a human KYSE30 carcinoma cell model in which isoQC was stably overexpressed. GO and KEGG analysis of the DEGs (228) and DEPs (254) respectively implicated isoQC on the proliferation invasion and metastasis of cells and suggested that isoQC might participate in the regulation of MAPK, RAS, circadian rhythm, and related pathways. At the functional level, isoQC-overexpressing KYSE30 cells showed enhanced proliferation, migration, and invasion capacity. Next, we decided to study the precise effect of isoQC overexpression on JNK, p-JNK, AKT, p-AKT, ERK, p-ERK, and PER2, as RNA levels of these proteins are significantly correlated with signal levels indicated in RNA-Seq analysis, and these candidates are the top correlated DEPs enriched in RT-qPCR analysis. We saw that only p-ERK expression was inhibited, while PER2 was increased. These phenotypes were inhibited upon exposure to PER2 inhibitor KL044, which allowed for the restoration of p-ERK levels. These data support upregulated isoQC being able to promote cancer cell proliferation and migration in vitro, likely by helping to regulate the MAPK and RAS signaling pathways, and the circadian protein PER2 might be a potential mediator.

GLYR1 transcriptionally regulates PER3 expression to promote the proliferation and migration of multiple myeloma.

Period circadian regulator 3 (PER3) functions as a tumor suppressor in various cancers. However, the role of PER3 in multiple myeloma (MM) has not been reported yet. Through this study, we aimed to investigate the potential role of PER3 in MM and the underlying mechanisms. RT-qPCR and western blotting were used to determine the mRNA and protein expression levels of PER3. Glyoxylate reductase 1 homolog (GLYR1) was predicted to be a transcription factor of PER3. The binding sites of GLYR1 on the promoter region of PER3 were analyzed using UCSC and confirmed using luciferase and chromatin immunoprecipitation assays. Viability, apoptosis, and metathesis were determined using CCK-8, colony formation, TUNEL, and transwell assays. We found that PER3 expression decreased in MM. Low PER3 levels may predict poor survival rates; PER3 overexpression suppresses the viability and migration of MM cells and promotes apoptosis. Moreover, GLYR1 transcriptionally activates PER3, and the knockdown of PER3 alleviates the effects of GLYR1 and induces its malignant behavior in MM cells. To conclude, GLYR1 upregulates PER3 and suppresses the aggressive behavior of MM cells, suggesting that GLYR1/PER3 signaling may be a potential therapeutic target for MM.

Prospective study of the association between chronotypes and depressive symptoms in Chinese university students: Moderating effects of PER1 gene DNA methylation.

Most studies have shown a link between chronotypes and mental health and have identified evening chronotypes (E-types) as a potential risk for depressive symptoms. However, the mechanisms behind this association remain unknown. Abnormal expression of the PER1 gene was not only associated with circadian rhythm disturbance, but also closely related to mental illness. Therefore, this study aimed to examine the association of chronotype with depressive symptoms, and further explore the moderating effects of the PER1 gene DNA methylation on chronotypes and depressive symptoms in Chinese university students. In a stratified cluster sampling design, chronotype and depressive symptoms were assessed in 1 042 university students from 2 universities in a two-year prospective survey from April 2019 to October 2020. The survey was conducted once every 6 months, corresponding to the time points in April 2019 (T0), October 2019 (T1), April 2020 (T2), and October 2020 (T3). At T0, the Morning and Evening Questionnaire 5 (MEQ-5) was adopted to assess chronotype. At T0-T3, the Patient Health Questionnaire 9 (PHQ-9) was adopted to investigate depressive symptoms. Meanwhile, at T0, participants were subjected to a health check-up trip in the hospital, and blood samples were taken from the students to measure the PER1 gene DNA methylation levels. Binary logistic regression was used to analyze the association of chronotypes with depressive symptoms. The depression/total depression group was coded as 1, while the remaining participants was defined as one group, and was coded as 0. The PROCESS plug-in of SPSS software was used to analyze the moderating effects of PER1 gene DNA methylation on the association of chronotype with depressive symptoms. After adjusting for covariates, the results indicated that T0 E-types were positively correlated with T0-T3 depression/total depression in female university students. Furthermore, the PER1 gene DNA methylation has negative moderating effects between T0 chronotype and T3 depressive symptoms and has a sex difference. This study can provide more favorable scientific value for the prevention and control of depression in university students.

Biological Clock Genes are Crucial and Promising Biomarkers for the Therapeutic Targets and Prognostic Assessment in Gastric Cancer.

BACKGROUND: Gastric cancer is one of the major public health problems worldwide. Circadian rhythm disturbances driven by circadian clock genes play a role in the development of cancer. However, whether circadian clock genes can serve as potential therapeutic targets and prognostic biomarkers for gastric cancer remains elusive. METHODS: In this study, we comprehensively analyzed the potential relationship between circadian clock genes and gastric cancer using online bioinformatics databases such as GEPIA, cBioPortal, STRING, GeneMANIA, Metascape, TIMER, TRRUST, and GEDS. RESULTS: Biological clock genes are expressed differently in human tumors. Compared with normal tissues, only PER1, CLOCK, and TIMELESS expression differences were statistically significant in gastric cancer (p < 0.05). PER1 (p = 0.0169) and CLOCK (p = 0.0414) were associated with gastric cancer pathological stage (p < 0.05). Gastric cancer patients with high expression of PER1 (p = 0.0028) and NR1D1 (p = 0.016) had longer overall survival, while those with high expression of PER1 (p = 0.042) and NR1D1 (p = 0.016) had longer disease-free survival. The main function of the biological clock gene is related to the circadian rhythms and melatonin metabolism and effects. CLOCK, NPAS2, and KAT2B were key transcription factors for circadian clock genes. In addition, we also found important correlations between circadian clock genes and various immune cells in the gastric cancer microenvironment. CONCLUSIONS: This study may establish a new gastric cancer prognostic indicator based on the biological clock gene and develop new drugs for the treatment of gastric cancer using biological clock gene targets.

Glycyrrhizic acid treatment ameliorates anxiety-like behaviour via GLT1 and Per1/2-dependent pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicinal herb, Glycyrrhiza. URALENSIS: Fisch. (licorice root, chinese name: Gancao) has a variety of medicinal values and is widely used clinically. Its main active ingredient, glycyrrhizic acid (GA), is believed to have a neuroprotective effect. However, the underlying biological mechanisms of GA on stress-induced anxiety disorders are still unclear. AIM OF THE STUDY: To investigate the anti-anxiety effect of GA and its underlying mechanism. METHODS: We selected the anxiety model induced by repeated chronic restraint stress (CRS) for 2 h on each of 7 consecutive days. GA (4, 20, 100 mg/kg) was injected intraperitoneally once daily for 1 week. The potential GA receptors were identified using whole-cell patches and computer-assisted docking of molecules. High-throughput RNA sequencing, adeno-associated virus-mediated gene regulation, Western blotting, and RT-qPCR were used to assess the underlying molecular pathways. RESULTS: GA alleviate depression-like and anxiety-like behaviors in CRS mice. GA decreased synaptic transmission by facilitating glutamate reuptaking in mPFC. Meanwhile, long-term GA treatment increased the expression of clock genes Per1 and Per2. Suppressing both Per1 and Per2 abolished the anxiolytic effects of GA treatment. CONCLUSION: Our study suggests that GA may be developed for the treatment of stress-induced anxiety disorders, and its mechanism is related to GLT1 and Per1/2-dependent pathways. This presents a novel approach to discovering potent therapeutic drugs.

Antidepressant mechanism of traditional Chinese medicine: Involving regulation of circadian clock genes.

Numerous studies have demonstrated an intimate relationship between circadian rhythm disorders and the development and prevention of depression. The biological clock genes, which constitute the molecular basis of endogenous circadian rhythms, hold promising prospects for depression treatment. Based on an extensive review of recent domestic and international research, this article presents a comprehensive analysis of how traditional Chinese medicine (TCM) intervenes in depression by regulating circadian rhythms. The findings indicate that TCM exerts its antidepressant effects by targeting specific biological clock genes such as Bmal1, clock, Arntl, Per1, Per2, Per3, Nr1d1, Cry2, and Dbp, as well as regulating circadian rhythms of hormone secretion. However, most current research is still confined to basic experimental studies, lacking clinical double-blind control trials to further validate these viewpoints. Furthermore, there is insufficient research on the signal transduction pathway between biological clock genes and pathological changes in depression. Additionally, further clarification is needed regarding the specific targets of TCM on the biological clock genes.

Mucosal Genes Encoding Clock, Inflammation and Their Mutual Regulators Are Disrupted in Pediatric Patients with Active Ulcerative Colitis.

Patients with active ulcerative colitis (UC) display a misalignment of the circadian clock, which plays a vital role in various immune functions. Our aim was to characterize the expression of clock and inflammation genes, and their mutual regulatory genes in treatment-naïve pediatric patients with UC. Using the Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) platform and R algorithms, we analyzed rectal biopsy transcriptomic data from two cohorts (206 patients with UC vs. 20 healthy controls from the GSE-109142 study, and 43 patients with UC vs. 55 healthy controls from the GSE-117993 study). We compared gene expression levels and correlation of clock genes (BMAL1, CLOCK, PER1, PER2, CRY1, CRY2), inflammatory genes (IκB, IL10, NFκB1, NFκB2, IL6, TNFα) and their mutual regulatory genes (RORα, RORγ, REV-ERBα, PGC1α, PPARα, PPARγ, AMPK, SIRT1) in patients with active UC and healthy controls. The clock genes BMAL1, CLOCK, PER1 and CRY1 and the inflammatory genes IκB, IL10, NFκB1, NFκB2, IL6 and TNFα were significantly upregulated in patients with active UC. The genes encoding the mutual regulators RORα, RORγ, PGC1α, PPARα and PPARγ were significantly downregulated in patients with UC. A uniform pattern of gene expression was found in healthy controls compared to the highly variable expression pattern in patients with UC. Among the healthy controls, inflammatory genes were positively correlated with clock genes and they all showed reduced expression. The difference in gene expression levels was associated with disease severity and endoscopic score but not with histological score. In patients with active UC, clock gene disruption is associated with abnormal mucosal immune response. Disrupted expression of genes encoding clock, inflammation and their mutual regulators together may play a role in active UC.

Cellular iron depletion enhances behavioral rhythm by limiting brain Per1 expression in mice.

AIMS: Disturbances in the circadian rhythm are positively correlated with the processes of aging and related neurodegenerative diseases, which are also associated with brain iron accumulation. However, the role of brain iron in regulating the biological rhythm is poorly understood. In this study, we investigated the impact of brain iron levels on the spontaneous locomotor activity of mice with altered brain iron levels and further explored the potential mechanisms governing these effects in vitro. RESULTS: Our results revealed that conditional knockout of ferroportin 1 (Fpn1) in cerebral microvascular endothelial cells led to brain iron deficiency, subsequently resulting in enhanced locomotor activity and increased expression of clock genes, including the circadian locomotor output cycles kaput protein (Clock) and brain and muscle ARNT-like 1 (Bmal1). Concomitantly, the levels of period circadian regulator 1 (PER1), which functions as a transcriptional repressor in regulating biological rhythm, were decreased. Conversely, the elevated brain iron levels in APP/PS1 mice inhibited autonomous rhythmic activity. Additionally, our findings demonstrate a significant decrease in serum melatonin levels in Fpn1cdh5 -CKO mice compared with the Fpn1flox/flox group. In contrast, APP/PS1 mice with brain iron deposition exhibited higher serum melatonin levels than the WT group. Furthermore, in the human glioma cell line, U251, we observed reduced PER1 expression upon iron limitation by deferoxamine (DFO; iron chelator) or endogenous overexpression of FPN1. When U251 cells were made iron-replete by supplementation with ferric ammonium citrate (FAC) or increased iron import through transferrin receptor 1 (TfR1) overexpression, PER1 protein levels were increased. Additionally, we obtained similar results to U251 cells in mouse cerebellar astrocytes (MA-c), where we collected cells at different time points to investigate the rhythmic expression of core clock genes and the impact of DFO or FAC treatment on PER1 protein levels. CONCLUSION: These findings collectively suggest that altered iron levels influence the circadian rhythm by regulating PER1 expression and thereby modulating the molecular circadian clock. In conclusion, our study identifies the regulation of brain iron levels as a potential new target for treating age-related disruptions in the circadian rhythm.

Importance of Per2 in cardiac mitochondrial protection during stress.

During myocardial injury, inflammatory mediators and oxidative stress significantly increase to impair cardiac mitochondria. Emerging evidence has highlighted interplays between circadian protein-period 2 (Per2) and mitochondrial metabolism. However, besides circadian rhythm regulation, the direct role of Per2 in mitochondrial performance particularly following acute stress, remains unknown. In this study, we aim to determine the importance of Per2 protein's regulatory role in mitochondrial function following exposure to inflammatory cytokine TNFα and oxidative stressor H2O2 in human cardiomyocytes. Global warm ischemia (37 °C) significantly impaired complex I activity with concurrently reduced mitochondrial Per2 in adult mouse hearts. TNFα or H2O2 decreased Per2 protein levels and damaged mitochondrial respiratory function in adult mouse cardiomyocytes. Next, mitochondrial membrane potential ([Formula: see text] M) using JC-1 fluorescence probe and mitochondrial respiration capacity via Seahorse Cell Mito Stress Test were then detected in Per2 or control siRNA transfected AC16 Human Cardiomyocytes (HCM) that were subjected to 2 h-treatment of TNFα (100 ng/ml) or H2O2 (100 µM). After 4 h-treatment, cell death was also measured using Annexin V and propidium iodide apoptosis kit through flow cytometry. We found that knockdown of Per2 enhanced TNFα-induced cell death and TNFα- or H2O2-disrupted [Formula: see text]M, as well as TNFα- or H2O2-impaired mitochondrial respiration function. In conclusion, Per2 knockdown increases likelihood of cell death and mitochondrial dysfunction in human cardiomyocytes exposed to either TNFα or H2O2, supporting the protective role of Per2 in HCM during stress with a focus on mitochondrial function.

DNA polymerase beta connects tumorigenicity with the circadian clock in liver cancer through the epigenetic demethylation of Per1.

The circadian-controlled DNA repair exhibits a strong diurnal rhythm. Disruption in circadian clock and DNA repair is closely linked with hepatocellular carcinoma (HCC) progression, but the mechanism remains unknown. Here, we show that polymerase beta (POLB), a critical enzyme in the DNA base excision repair pathway, is rhythmically expressed at the translational level in mouse livers. Hepatic POLB dysfunction dampens clock homeostasis, whereas retards HCC progression, by mediating the methylation of the 4th CpG island on the 5'UTR of clock gene Per1. Clinically, POLB is overexpressed in human HCC samples and positively associated with poor prognosis. Furthermore, the hepatic rhythmicity of POLB protein expression is orchestrated by Calreticulin (CALR). Our findings provide important insights into the molecular mechanism underlying the synergy between clock and food signals on the POLB-driven BER system and reveal new clock-dependent carcinogenetic effects of POLB. Therefore, chronobiological modulation of POLB may help to promote precise interventions for HCC.

Circadian clock in choroid plexus is resistant to immune challenge but dampens in response to chronodisruption.

The choroid plexus (ChP) in the brain ventricles has a major influence on brain homeostasis. In this study, we aimed to determine whether the circadian clock located in ChP is affected by chronodisruption caused by misalignment with the external light/dark cycle and/or inflammation. Adult mPer2Luc mice were maintained in the LD12:12 cycle or exposed to one of two models of chronic chronodisruption - constant light for 22-25 weeks (cLL) or 6-hour phase advances of the LD12:12 cycle repeated weekly for 12 weeks (cLD-shifts). Locomotor activity was monitored before the 4th ventricle ChP and suprachiasmatic nuclei (SCN) explants were recorded in real time for PER2-driven population and single-cell bioluminescence rhythms. In addition, plasma immune marker concentrations and gene expression in ChP, prefrontal cortex, hippocampus and cerebellum were analyzed. cLL dampened the SCN clock but did not shorten the inactivity interval (sleep). cLD-shifts had no effect on the SCN clock, but transiently affected sleep duration and fragmentation. Both chronodisruption protocols dampened the ChP clock. Although immune markers were elevated in plasma and hippocampus, levels in ChP were unaffected, and unlike the liver clock, the ChP clock was resistant to lipopolysaccharide treatment. Importantly, both chronodisruption protocols reduced glucocorticoid signaling in ChP. The data demonstrate the high resistance of the ChP clock to inflammation, highlighting its role in protecting the brain from neuroinflammation, and on the other hand its high sensitivity to chronodisruption. Our results provide a novel link between human lifestyle-induced chronodisruption and the impairment of ChP-dependent brain homeostasis.