Circadian Protein Period Research Articles

The contribution of clock genes BMAL1 and PER2 in osteoarthritis-associated pain.

Joint pain is the primary symptom of osteoarthritis (OA) and the main motivator for patients to seek medical care. OA-related pain significantly restricts joint function and diminishes quality of life. Despite the availability of various pain-relieving medications for OA, current treatment strategies often fall short in delivering adequate pain relief. Furthermore, long-term use of pain medications for OA management is frequently linked with notable side effects and toxicities, suggesting the need to explore new potential targets to treat pain in OA patients. In this context, clock genes, particularly brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1) and period circadian protein homolog 2 (PER2), known for their role in circadian rhythms, represent promising opportunities for pharmacological interventions due to their involvement in both the development and maintenance of OA pain. While BMAL1 and PER2 have been extensively studied in neuropathic and inflammatory pain, their specific contributions to OA pain remain less clear, demanding further investigation. This narrative review aims to synthesize the relationship between OA pain and the BMAL1 and PER2 signaling pathways, ultimately exploring the potential therapeutic role of clock genes in addressing this challenging condition.

Molecular circadian clock disruption in the leukocytes of individuals with type 2 diabetes and overweight, and its relationship with leukocyte–endothelial interactions

Aims/hypothesisAlterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.MethodsThis is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte–endothelial interaction assays were performed by perfusing participants’ leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.ResultsParticipants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte–endothelial interactions.Conclusions/interpretationOur study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte–endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.Graphical

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 (Δψ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta \\psi$$\\end{document}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 Δψ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta \\psi$$\\end{document}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.

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2.

The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.

Transfer of microRNA-22-3p by M2 macrophage-derived extracellular vesicles facilitates the development of ankylosing spondylitis through the PER2-mediated Wnt/β-catenin axis

Pathological osteogenesis and inflammation possess critical significance in ankylosing spondylitis (AS). The current study aimed to elucidate the mechanisms regarding extracellular vesicle (EV)-packaged microRNA-22-3p (miR-22-3p) from M2 macrophages in the osteogenic differentiation of mesenchymal stem cells (MSCs) in AS. EVs were initially isolated from M2 macrophages, which had been treated with either restored or depleted miR-22-3p. AS-BMSCs were subsequently treated with M2 macrophage-derived EVs to detect osteogenic differentiation in BMSCs using gain- or loss-of-function experiments. The binding affinity among miR-22-3p, period circadian protein 2 (PER2), and Wnt7b was identified. Finally, AS mouse models were established for testing the effects of M2-EV-miR-22-3p on the bone metastatic microenvironment in vivo. miR-22-3p from M2 macrophages could be transferred into BMSCs via EVs, which promoted the osteogenic differentiation of AS-BMSCs. miR-22-3p inhibited PER2, while PER2 blocked the Wnt/β-catenin signaling pathway via Wnt7b inhibition. M2-EV-shuttled miR-22-3p facilitated alkaline phosphatase activity and extracellular matrix mineralization via PER2-regulated Wnt/β-catenin axis, stimulating the BMSC osteogenic differentiation. Taken together, these findings demonstrate that miR-22-3p in M2 macrophage-released EVs downregulates PER2 to facilitate the osteogenesis of MSCs via Wnt/β-catenin axis.

The Role of PER‐1, ET‐1 Axis in Regulating Endothelial Cell Function

ObjectiveResults from major epidemiological studies give evidence to the essential role that dietary salt plays in the development of hypertension. Contrastingly, the mechanisms that mediate individual susceptibility to salt‐sensitive hypertension are unknown. Blood pressure (BP) and sodium reabsorption by the kidneys are regulated by the circadian clock. Consistent with these observations, when mice with global knockout (KO) of the circadian protein period 1 (PER1) are exposed to a high salt diet, they demonstrate increased endothelin‐1 (ET‐1) levels, impaired natriuresis, and hypertension. However, the cumulative sodium balance of PER1 KO mice is not different from control mice. Thus, we hypothesize that PER1 might mediate endothelial cell dysfunction and susceptibility to salt sensitive hypertension.Materials and MethodsHuman Umbilical Vein Endothelial Cells (HUVEC) were transfected with siRNA targeting PER1 or non‐targeting siRNA controls. Endothelial cell function was assessed by measurement of nitric oxide bioavailability, proliferation, and migration. Nitric Oxide (NO) was quantified by DAF fluorescence, proliferation by EZQuant™, and migration by measuring velocity and distance travelled by single cells using time lapse imaging.ResultsNitric oxide bioavailability was increased with PER1 knockdown (Mean fluorescence 14433 ± 1344 vs. 7423 ± 472, p<0.005). Migration velocity and distance were decreased with PER1 siRNA compared to non‐target controls, though not statistically significant (Mean distance 118.9 uM ± 11.39 vs. 138 uM ± 10.88, p=0.2) and (Mean velocity 42.3 uM/sec ± 3.98 vs. 46 uM/sec ± 3.6, p=0.14).There were no differences in proliferation at 24, 48, or 72 hours (Fold change in cell numbers of PER1 siRNA treated cells compared to non‐target controls were 0.87 ± .05 at 24 hours, 0.87 ± .09 at 48 hours, and 0.90 ± .06 at 72 hours, p=NS).ConclusionsOur results suggest that knock down of PER1 increases NO bioavailability and decreases migration of endothelial cells. ET‐1 can increase NO bioavailability via ETB receptors. Experiments are ongoing to delineate the role of ET‐1/ETA and ET‐B in mediating the effect of PER1 on endothelial cell function.

Timing of Novel Drug 1A-116 to Circadian Rhythms Improves Therapeutic Effects against Glioblastoma.

The Ras homologous family of small guanosine triphosphate-binding enzymes (GTPases) is critical for cell migration and proliferation. The novel drug 1A-116 blocks the interaction site of the Ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase with some of its guanine exchange factors (GEFs), such as T-cell lymphoma invasion and metastasis 1 (TIAM1), inhibiting cell motility and proliferation. Knowledge of circadian regulation of targets can improve chemotherapy in glioblastoma. Thus, circadian regulation in the efficacy of 1A-116 was studied in LN229 human glioblastoma cells and tumor-bearing nude mice. Methods. Wild-type LN229 and BMAL1-deficient (i.e., lacking a functional circadian clock) LN229E1 cells were assessed for rhythms in TIAM1, BMAL1, and period circadian protein homolog 1 (PER1), as well as Tiam1, Bmal1, and Rac1 mRNA levels. The effects of 1A-116 on proliferation, apoptosis, and migration were then assessed upon applying the drug at different circadian times. Finally, 1A-116 was administered to tumor-bearing mice at two different circadian times. Results. In LN229 cells, circadian oscillations were found for BMAL1, PER1, and TIAM1 (mRNA and protein), and for the effects of 1A-116 on proliferation, apoptosis, and migration, which were abolished in LN229E1 cells. Increased survival time was observed in tumor-bearing mice when treated with 1A-116 at the end of the light period (zeitgeber time 12, ZT12) compared either to animals treated at the beginning (ZT3) or with vehicle. Conclusions. These results unveil the circadian modulation in the efficacy of 1A-116, likely through RAC1 pathway rhythmicity, suggesting that a chronopharmacological approach is a feasible strategy to improve glioblastoma treatment.

PER1 Is a Prognostic Biomarker and Correlated With Immune Infiltrates in Ovarian Cancer.

Background: Period circadian protein homolog 1 (PER1) is an important component of the biorhythm molecular oscillation system and plays an important part in the development and progression of mammalian cancer. However, the correlations of PER1 with prognosis and tumor-infiltrating lymphocytes in ovarian cancer (OV) remain unclear.Methods: The Oncomine and TIMER databases were used to examine the expression of PER1 in OV. Kaplan–Meier Plotter and PrognoScan were used to evaluate the relationship between PER1 and prognosis. Kaplan–Meier Plotter was used to analyze the relationships between PER1 and clinicopathological features of OV patients. The relationship between PER1 expression and immune infiltration in OV was investigated using the TIMER database and CIBERSORT algorithm. The STRING database was used to analyze PER1-related protein functional groups, the GeneMANIA online tool was used to analyze gene groups with similar functions to those of PER1, and Network Analyst was used to identify transcription factors that regulate PER1. The correlation between PER1 and immunoinvasion of OV was analyzed using TIMER. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect PER1 expression.Results: PER1 was differentially expressed in different cancer tissues, and its expression in various OV subtypes was lower than that in normal ovarian tissue. OV patients with low PER1 expression had a reduced overall survival rate. Decreased PER1 expression in stage 1 and stage 1+2 OV patients was related to poor prognosis, while increased PER1 expression in stage 3+4 patients and TP53 mutation were related to poor overall survival and progression-free survival. We identified eight genes whose expression was strongly correlated with that of PER1, as well as four transcription factors that regulate PER1. In OV, PER1 expression levels were positively correlated with infiltration levels of cells including neutrophils, regulatory T cells, and M2 macrophages, and closely related to a variety of immune markers. Reduced expression of PER1 was significantly associated with poor overall survival.Conclusion: These findings suggest that PER1 could be used as a prognostic biomarker to determine prognosis and immune infiltration in OV patients.

The Circadian Protein PER1 Modulates the Cellular Response to Anticancer Treatments.

The circadian clock driven by the daily light–dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other’s expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.

Transcriptional and free radical responses to LVAD therapy

BackgroundMyocardial recovery with Left ventricular assistant device (LVAD) therapy is dichotomous with some patients obtaining remission from end-stage heart failure whereas most require transplantation or remain on pump support long term. Our goal was to determine transcriptional and free radical responses to LVAD treatment.MethodsTissues were collected from patients before and after LVAD placement in non-ischemic dilated cardiomyopathy patients (n = 14) along with controls (n = 3). RNA sequencing (RNASeq) analysis quantified transcriptional profiles by using a custom targeted panel of heart failure related genes on the PGM sequencer. The differential expression analysis between groups was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed genes to understand the biological pathways involved. Electron Paramagnetic Resonance (EPR) Spectroscopy was utilized to measure levels of free radicals in whole blood collected pre- and post-LVAD implantation (n = 16).ResultsThirty-five genes were differentially expressed in pre-LVAD failing hearts compared to controls. In response to LVAD therapy, only Pyruvate dehydrogenase kinase 4 (PDK4) and period circadian protein homolog 1 (PER1) were altered with 34 heart failure related genes still differentially expressed post-LVAD compared to controls. IPA showed that DNA methylation-related genes were upregulated in both pre- and post-LVAD and was persistent with a Z-score of 2.00 and 2.36 for DNA Methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B), respectively. Inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of − 2.00. Levels of free radicals in blood of pre- and post-LVAD patients did not change significantly.ConclusionLVAD therapy does not reverse many of the transcriptional changes associated with heart failure. Persistent changes in gene expression may be related to ongoing oxidative stress, continued DNA methylation, or changes in metabolism. PDK4 is a key regulator of glucose metabolism and its increased expression by LVAD therapy inhibited pyruvate metabolism.

Glucocorticoid receptor activation stimulates the sodium-chloride cotransporter and influences the diurnal rhythm of its phosphorylation.

The sodium-chloride cotransporter (NCC) in the distal convoluted tubule contributes importantly to sodium balance and blood pressure (BP) regulation. NCC phosphorylation determines transport activity and has a diurnal rhythm influenced by glucocorticoids. Disturbing this rhythm induces “nondipping” BP, an abnormality that increases cardiovascular risk. The receptor through which glucocorticoids regulate NCC is not known. In this study, we found that acute administration of corticosterone to male C57BL6 mice doubled NCC phosphorylation without affecting total NCC abundance in both adrenalectomized and adrenal-intact mice. Corticosterone also increased the whole kidney expression of canonical clock genes: period circadian protein homolog 1 (Per1), Per2, cryptochrome 1, and aryl hydrocarbon receptor nuclear translocator-like protein 1. In adrenal-intact mice, chronic blockade of glucocorticoid receptor (GR) with RU486 did not change total NCC but prevented corticosterone-induced NCC phosphorylation and activation of clock genes. Blockade of mineralocorticoid receptor (MR) with spironolactone reduced the total pool of NCC but did not affect stimulation by corticosterone. The diurnal rhythm of NCC phosphorylation, measured at 6-h intervals, was blunted by chronic GR blockade, and a similar dampening of diurnal variation was seen in GR heterozygous null mice. These effects on NCC phosphorylation did not reflect altered rhythmicity of plasma corticosterone or serum and glucocorticoid-induced kinase 1 activity. Both mineralocorticoids and glucocorticoids emerge as regulators of NCC, acting via distinct receptor pathways. MR activation provides maintenance of the NCC protein pool; GR activation dynamically regulates NCC phosphorylation and establishes the diurnal rhythm of NCC activity. This study has implications for circadian BP homeostasis, particularly in individuals with abnormal glucocorticoid signaling as is found in chronic stress and corticosteroid therapy.

A PAS-Containing Histidine Kinase is Required for Conidiation, Appressorium Formation, and Disease Development in the Rice Blast Fungus, Magnaporthe oryzae

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

Different as night and day: Behavioural and life history responses to varied photoperiods in Daphnia magna.

Nearly all animal species have utilized photoperiod to cue seasonal behaviours and life history traits. We investigated photoperiod responses in keystone species, Daphnia magna, to identify molecular processes underlying ecologically important behaviours and traits using functional transcriptomic analyses. Daphnia magna were photoperiod‐entrained immediately posthatch to a standard control photoperiod of 16 light/ 8 dark hours (16L:8D) relative to shorter (4L:20D, 8L:16D, 12L:12L) and longer (20L:4D) day length photoperiods. Short‐day photoperiods induced significantly increased light‐avoidance behaviours relative to controls. Correspondingly, significant differential transcript expression for genes involved in glutamate signalling was observed, a critical signalling pathway in arthropod light‐avoidance behaviour. Additionally, period circadian protein and proteins coding F‐box/LRR‐repeat domains were differentially expressed which are recognized to establish circadian rhythms in arthropods. Indicators of metabolic rate increased in short‐day photoperiods which corresponded with broadscale changes in transcriptional expression across system‐level energy metabolism pathways. The most striking observations included significantly decreased neonate production at the shortest day length photoperiod (4L:20D) and significantly increased male production across short‐day and equinox photoperiods (4L:20D, 8L:16D and 12L:12D). Transcriptional expression consistent with putative mechanisms of male production was observed including photoperiod‐dependent expression of transformer‐2 sex‐determining protein and small nuclear ribonucleoprotein particles (snRNPs) which control splice variant expression for genes like transformer. Finally, increased transcriptional expression of glutamate has also been shown to induce male production in Daphnia pulex via photoperiod‐sensitive mechanisms. Overall, photoperiod entrainment affected molecular pathways that underpin critical behavioural and life history traits in D. magna providing fundamental insights into biological responses to this primary environmental cue.

The Circadian tau Mutation in Casein Kinase 1 Is Part of a Larger Domain That Can Be Mutated to Shorten Circadian Period.

Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIε) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. We mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.

Abuse potential of 2-(4-iodo-2, 5-dimethoxyphenyl)[sbnd]N-(2-methoxybenzyl)ethanamine (25I[sbnd]NBOMe); in vivo and ex vivo approaches

25INBOMe ("25-I", "N-Bomb"), one of new psychoactive substances (NPSs), is being abused for recreational purpose. However, the liability for abuse or dependence has not been systematically studied yet. The objective of the present study was to evaluate rewarding and reinforcing effects of 25INBOMe using conditioned place preference (CPP) and self-administration (SA) paradigms. In addition, ultrasonic vocalizations (USVs) were measured to investigate relationships between USVs and emotional state regarding dependence on psychoactive substances. To understand molecular mechanism involved in its action, dopamine (DA) level changes were analyzed using synaptosomes extracted from the striatal region of the brain. Expression level changes of SGK1 (serum/glucocorticoid regulated kinase 1) and PER2 (period circadian protein homolog 2), two putative biomarkers for drug dependence, were also analyzed. Results showed that 25INBOMe increased both CPP (0.3 mg/kg) and SA (0.03 mg/kg/infusion) and produced higher frequencies in USVs analysis. It also increased DA levels in the striatal region and changed expression levels of SGK1 and PER2. Results of the present study suggest that 25INBOMe might produce rewarding and reinforcing effects, indicating its dependence liability. In addition, frequencies of USV might be associated with emotional state of mice induced by psychoactive substances regarding substance dependence. This is the first systemic preclinical report on the dependence liability of 25INBOMe and the first attempt to introduce a possible relationship between USVs and emotional state of mice regarding substance dependency. Further studies are needed to clarify the mechanism involved in 25INBOMe dependency and determine the usefulness of USV measurement as a method for evaluating dependence liability.

Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.

Overexpression of PER3 reverses paclitaxel resistance of prostate cancer cells by inhibiting the Notch pathway.

To investigate the levels of period circadian protein homolog 3 (PER3) in paclitaxel-resistant prostate cancer patients and the effect of PER3 on paclitaxel-resistant prostate cancer cell lines. A total of 38 patients diagnosed with prostate cancer in our hospital from June 2013 to June 2016 were divided into paclitaxel-resistant group (n=19) and non-resistant group (n=19) according to the follow-up treatment effects. Fluorescent quantitative polymerase chain reaction (PCR) was performed to evaluate the levels of PER3 in drug-resistant and non-resistant groups as well as the relative levels of PER3 before and after treatment. PER3 was overexpressed or knocked down in a paclitaxel-resistant prostate cancer cell line, followed by measuring its IC50 as well as changes in cell cycle and apoptosis. Using Western blot, we detected downregulation of Notch pathway and related receptor proteins when PER3 was overexpressed. The results of fluorescence quantitative PCR showed that the expression of PER3 in the paclitaxel-resistant prostate cancer group was lower than that in the non-resistant group, and the relative expression of PER3 was decreased after treatment. Fluorescent quantitative PCR and Western blot showed that the expression of PER3 in paclitaxel-resistant prostate cancer cells was higher than that of the untreated counterparts. After overexpression of PER3 by transfecting prostate cancer-resistant cell lines with plasmids, the IC50 was significantly reduced, the cell cycle was arrested, and the apoptosis was significantly increased. Subsequently, we detected decreased expression of Notch1 in PER3 over-expressed paclitaxel-resistant cell lines by Western blot; this attenuated resistance in paclitaxel-resistant cell lines. PER3 can induce sensitivity of paclitaxel-resistant cell lines to paclitaxel by inhibiting the expression of Notch1.

Expression of circadian clock genes in human colorectal adenoma and carcinoma.

Circadian rhythms are fundamental biological systems in most organisms. Epidemiological and animal studies have demonstrated that disruption of circadian rhythms is linked to tumor progression and mammalian tumorigenesis. However, the clinical significance of in situ clock gene expression in precancerous and cancerous colorectal lesions remains unknown. The present study aimed to investigate mRNA transcript levels of circadian clock genes within human colorectal cancer and adenoma tissue sections. Using in situ hybridization, the expression of key clock genes, including period circadian protein homolog (Per) 1 and 2, cryptochrome 1 (Cry1), circadian locomoter output cycles protein kaput (Clock), brain and muscle ARNT-like protein 1 (Bmal1) and casein kinase 1ε (CK1ε) were retrospectively examined in 51 cases of colorectal carcinoma and 10 cases of adenoma. The expression of clock genes was almost undetectable in the majority of adenomas, whereas positive expression of clock genes was observed in 27–47% of carcinomas. Notably, positive Per1, Per2 and Clock staining in colorectal carcinomas were each significantly associated with a larger tumor size (P=0.012, P=0.011 and P=0.009, respectively). Tumors with positive Per2 and Clock expression tended to exhibit deeper depth of invasion and were generally more advanced than tumors that did not express these genes (P=0.052 and P=0.064, respectively). However, no statistically significant association was observed between clock gene expression and clinicopathological variables, including histopathological differentiation, lymph node metastasis, depth of invasion or disease stage, although Per2-positive tumors tended to be associated with poorer overall survival (P=0.060). The results of the current study suggest that dysregulated expression of clock genes may be important in human colorectal tumorigenesis.

Passionflower Extract Induces High-amplitude Rhythms without Phase Shifts in the Expression of Several Circadian Clock Genes in Vitro and in Vivo

Circadian rhythms play key roles in the regulation of physiological and behavioral systems including wake-sleep cycles. We evaluated the effects of passionflower (aerial parts of Passiflora incarnata Linnaeus) extract (PFE) on circadian rhythms using NIH3T3 cells and mice. PFE (100 μg/mL) induced high-amplitude rhythms in the expression of period circadian protein (Per) 2, cryptochrome (Cry) 1, superoxide dismutase (SOD) 1, and glutathione peroxidase (GPx) in vitro from 12 h after a treatment with serum-rich medium. Isovitexin 2"-O-glucoside, isoschaftoside, and homoorientin, which were purified from PFE, also significantly enhanced Per2 mRNA expression at 20 h. An oral treatment with PFE (100 mg/kg/day) at zeitgeber time (ZT) 0 h for 15 days improved sleep latencies and sleeping times in the pentobarbital-induced sleep test in mice, similar to muscimol (0.2 mg/kg, i.p.). PFE induced high-amplitude rhythms without obvious phase shifts in serum corticosterone levels and the expression of Per1, Per2, and Cry1 in the liver as well as NIH3T3 cells. However, in the cerebrum, PFE enhanced the circadian expression of brain-muscle ARNT-like protein (Bmal) 1, circadian locomotor output cycles kaput (Clock), and Per1. Regarding this difference, we suggest the involvement of several neurotransmitters that influence the circadian rhythm. Indeed, PFE significantly increased dopamine levels at ZT 18 h, and then affected the mRNA expression of the synthetic and metabolic enzymes such as monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), and glutamic acid decarboxylase (GAD). The results obtained show that PFE positively modulates circadian rhythms by inducing high-amplitude rhythms in the expression of several circadian clock genes.

The central clock controls the daily rhythm of Aqp5 expression in salivary glands.

Salivary secretion displays day-night variations that are controlled by the circadian clock. The central clock in the suprachiasmatic nucleus (SCN) regulates daily physiological rhythms by prompting peripheral oscillators to adjust to changing environments. Aquaporin 5 (Aqp5) is known to play a key role in salivary secretion, but the association between Aqp5 and the circadian rhythm is poorly understood. The aim of our study was to evaluate whether Aqp5 expression in submandibular glands (SMGs) is driven by the central clock in the SCN or by autonomous oscillations. We observed circadian oscillations in the activity of period circadian protein homolog 2 and luciferase fusion protein (PER2::LUC) in cultured SMGs with periodicity depending on core clock genes. A daily rhythm was detected in the expression profiles of Aqp5 in SMGs in vivo. In cultured SMGs ex vivo, clock genes showed distinct circadian rhythms, whereas Aqp5 expression did not. These data indicate that daily Aqp5 expression in the mouse SMG is driven by the central clock in the SCN.