PER2 Research Articles

Silencing of NAMPT leads to up-regulation of insulin receptor substrate 1 gene expression in U87 glioma cells.

The aim of the present study was to investigate the effect of adipokine NAMPT (nicotinamide phosphoribosyltransferase) silencing on the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other proliferation related proteins in U87 glioma cells for evaluation of the possible significance of this adipokine in intergenic interactions. The silencing of NAMPT mRNA was introduced by NAMPT specific siRNA. The expression level of NAMPT, IGFBP3, IRS1, HK2, PER2, CLU, BNIP3, TPD52, GADD45A, and MKI67 genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Anti-visfatin antibody was used for detection of NAMPT protein by Western-blot analysis. It was shown that the silencing of NAMPT mRNA led to a strong down-regulation of NAMPT protein and significant modification of the expression of IRS1, IGFBP3, CLU, HK2, BNIP3, and MKI67 genes in glioma cells and a strong up-regulation of IGFBP3 and IRS1 and down-regulation of CLU, BNIP3, HK2, and MKI67 gene expressions. At the same time, no significant changes were detected in the expression of GADD45A, PER2, and TPD52 genes in glioma cells treated by siRNA specific to NAMPT. Furthermore, the silencing of NAMPT mRNA suppressed the glioma cell proliferation. Results of this investigation demonstrated that silencing of NAMPT mRNA with corresponding down-regulation of NAMPT protein and suppression of the glioma cell proliferation affected the expression of IRS1 gene as well as many other genes encoding the proliferation related proteins. It is possible that dysregulation of most of the studied genes in glioma cells after silencing of NAMPT is reflected by a complex of intergenic interactions and that NAMPT is an important factor for genome stability and regulatory mechanisms contributing to the control of glioma cell metabolism and proliferation.

Overexpression of the clock gene Per2 suppresses oral squamous cell carcinoma progression by activating autophagy via the PI3K/AKT/mTOR pathway.

The current studies reveal that the clock gene Per2 is expressed at lower levels in a variety of tumors and plays a significant tumor suppressor role. However, the biological functions and mechanism of Per2 in OSCC (OSCC: oral squamous cell carcinoma) remain unclear. In this study, OSCC cells with stable overexpression or silencing of Per2 were established to explore their biological functions and mechanism in vivo and in vitro. We discovered that the expression of Per2 decreases in OSCC cells. Overexpression of Per2 promoted autophagy and apoptosis in OSCC cells and inhibited proliferation. The opposite results were obtained in Per2-silenced OSCC cells. In Per2-overexpressing OSCC cells, the expression levels of PIK3CA, p-AKT, p-mTOR, p62 and Beclin1 were significantly reduced and the LC3B II/I ratio was significantly increased. In contrast, in Per2-silenced OSCC cells, the expression levels of PIK3CA, p-AKT, p-mTOR, p62 and Beclin1 were significantly enhanced and the LC3B II/I ratio was significantly reduced. When the AKT activator SC79 was added to Per2-overexpressing OSCC cells, the increased autophagy, apoptosis and decreased proliferation were significantly rescued. Furthermore, when autophinib, an autophagy inhibitor, was added to Per2-overexpressing OSCC cells, the decreased proliferation and increased apoptosis were significantly restored. An in vivo tumorigenesis assay also confirmed that overexpression of Per2 suppresses the growth of OSCC. In conclusion, our research results demonstrate that Per2 suppresses OSCC progression by motivating autophagy, as well as inhibiting cell proliferation and promoting apoptosis, which were mediated by autophagy, in a PI3K/AKT/mTOR pathway-dependent manner. Per2 could potentially be used as a valuable therapeutic marker for OSCC.

Rasagiline antiremodeling action after myocardial infarction may be mediated by circadian rhythm and neurotrophic related gene expression changes

Rasagiline mesylate (RG) is a potent, selective, irreversible monoamine oxidase-B inhibitor, developed to prolong the action of dopamine in the brain. It may rescue degenerating dopamine neurons through inhibiting death signal transduction initiated by the mitochondria permeability transition pore. We have previously shown that it decreases post-experimental infarct remodeling (REM). We investigated whether this cardioprotective result could be due to centrally mediated actions. In a rat model undergoing experimental myocardial infarction (MI) by a permanent ligation of the left anterior descending coronary artery. RG was administered, intraperitoneally for 28 days (2 mg/kg) starting 24 hours after MI induction. Treatment with RG significantly ameliorated cardiac REM echocardiographically as we have previously shown. In order to identify potential new cardioprotective mechanisms of RG action after MI, a comparative RNA expression analysis of remote region cardiac tissue was performed in placebo (Normal saline- NS), RG and Sham samples. RNA-seq analysis results were also confirmed by real time PCR. Our analysis showed: – differential RNA expression of circadian rhythm in NS samples versus RG. Circadian related genes Per3, Ciart, Dbp, Hpd, Nr1d2, and Rev-erb were upregulated while Arntl gene was downregulated (all P < 0.01); – as regards neurotrophic genes, neuropeptide Y (Npy) and neurogranin (Nrgn) genes were downregulated in RG samples compared to NS while the Brain-Derived Neurotrophic Factor (Bdnf) was upregulated (all P < 0.01). Our findings indicate that MI injury influences in a protective manner the expression of circadian rhythm and neurotrophic factor genes in cardiac tissue; potential regulation of these genes is part of the novel cardioprotective mechanism of RG action described, further advocating the possible use of this drug post-MI.

Antibiotic Resistance Pattern and Frequency of PER-1, SHV-1 and AMPC Type B-Lactamase Genes in Pseudomonas aeruginosa Isolated from Clinical Samples

Background: The existence of Extended Spectrum B-lactamase (ESBL) genes plays an important role in spreading B-lactam antibiotic resistance in the producing strains of these enzymes. The resistance of gram-negative bacteria, such as Pseudomonas aeruginosa, to different antimicrobial agents, especially B-lactams, has increasingly been reported. Objective: This study was conducted to determine the prevalence of TEM-1and VEB-1 beta-lactamases gene in P. aeruginosa isolates through Polymerase Chain Reaction (PCR) method. Methods: 100 clinical isolates of P. aeruginosa were collected from different clinical samples. The antibiotic susceptibility was examined by the disc diffusion method. The presence of PER-1, SHV-1 and AMPC genes was detected by PCR method. Results: Out of the studied P. aeruginosa isolates, 7, 9 and 37 isolates were positive for PER-1, SHV-1 and AMPC B-lactamases resistance genes, respectively. Patients with urinary infection had the most resistant isolates. All isolates (100%) were sensitive to polymyxin B. Conclusion: Antibiotic resistance in isolates of Pseudomonas can be caused by B-lactamases resistance genes. Noticing the increasing rate of the ESBLs producing strains, using the appropriate treatment protocol based on the antibiogram pattern of the strains is highly recommended.

Is Idiopathic Hypersomnia a Circadian Rhythm Disorder?

The pathophysiology of idiopathic hypersomnia remains unclear, but some of its clinical features suggest the possibility of circadian dysfunction. This review will provide an overview of recent studies of circadian biology that have begun to elucidate the potential role of circadian rhythm dysfunction in idiopathic hypersomnia. Clinically, people with idiopathic hypersomnia tend to have both a late chronotype and prominent sleep inertia or sleep drunkenness. Melatonin and cortisol profiles in people with IH confirm this tendency toward phase delay. More recently, it has been suggested that the night phase as defined by melatonin profile or period length as defined by BMA1 in dermal fibroblasts may also be prolonged in people with IH. Additionally, amplitude of melatonin rhythm and circadian gene expression, particularly BMAL1, PER1, and PER2, may be impaired in this disease. Clinical features, melatonin profiles, and circadian gene expression all suggest abnormalities of the circadian system may be a contributor to the pathogenesis of IH.

Association Between Period 3 Gene Polymorphisms and Adverse Effects of Antidepressants for Major Depressive Disorder.

Aims: Circadian rhythm genes including Period 3 (Per3) are associated with major depressive disorder (MDD) and have an effect on the patient's response to selective serotonin reuptake inhibitor (SSRI) antidepressants. The aim of this study was to identify possible associations between three single nucleotide polymorphisms (SNPs) of Per3 (rs10746473, rs228697, and rs228729), the MDD symptoms, and adverse effects of SSRIs. Materials and Methods: A total of 600 MDD patients who had been treated with SSRIs were enrolled. The 17-item Hamilton Rating Scale for Depression (HAMD17) was used to evaluate symptoms and treatment efficacy. In addition, the Treatment Emergent Symptom Scale/UKU Consumer Satisfaction Rating Scale (TESS/UKU) was used to assess adverse effects. The Per3 locus was genotyped by PCR and DNA sequencing. Results: The Per3 rs228697 CC genotype was associated with a higher sleep factor score when compared with the CG genotype (F = 4.027, p = 0.046). In addition, the rs228729 TC genotype was associated with a greater risk of suffering from excitement/agitation (p = 0.002, OR [odds ratio] = 4.049), akathisia (p = 0.014, OR = 4.905) and weight loss (p = 0.041, OR = 2.287) when compared with the CC genotype. Finally, the rs10746473 AA genotype patients were more likely to suffer from dizziness (p = 0.042, OR = 0.362) and the GA genotype patients from tachycardia (p = 0.015, OR = 0.340) when compared with those with GG genotype. Conclusion: The Per3 gene variants in patients can predict adverse effects of SSRIs and drug compliance.

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

Expression of miR-34a-5p is up-regulated in human colorectal cancer and correlates with survival and clock gene PER2 expression.

Colorectal cancer represents a leading cause of cancer death. MicroRNAs (miRNAs) are small non-coding RNA molecules that have been extensively studied in tumours, since changes in their levels can reveal patient prognosis. Cancer progression is also influenced by the circadian system whose functioning is based on the rhythmic expression of clock genes. Therefore, we performed macroarray screening of tumour and adjacent tissues in patients undergoing surgery for colorectal carcinoma. We identified 17 miRNAs showing expression that was more than 100 times higher in tumour tissue compared to adjacent tissue. From in silico analysis, miR-34a-5p was selected as showing a computer-predicted interaction with PER2. Real-time PCR revealed a negative correlation between expression of PER2 mRNA and miR-34a in patients with more advanced cancer stage. Expression of miR-34a was up-regulated in cancer tissue compared to adjacent tissue. High miR-34a expression was associated with better survival of patients. miR-34a showed lower expression levels in male patients with lymph node involvement, and a trend towards decreased expression in male patients with distant metastases. Male patients, but not female patients, with high expression of miR-34a and who were free of distant metastases and/or lymph node involvement showed better survival. Therefore, we proposed that expression of miR-34a was regulated in a sex-dependent manner and could be considered a marker of prognosis in earlier cancer stages in male patients.

Rhythmic Release of Corticosterone Induces Circadian Clock Gene Expression in the Cerebellum

Neurons of the cerebellar cortex contain a circadian oscillator, with circadian expression of clock genes being controlled by the master clock of the suprachiasmatic nucleus (SCN). However, the signaling pathway connecting the SCN to the cerebellum is unknown. Glucocorticoids exhibit a prominent SCN-dependent circadian rhythm, and high levels of the glucocorticoid receptor have been reported in the cerebellar cortex; we therefore hypothesized that glucocorticoids may control the rhythmic expression of clock genes in the cerebellar cortex. We here applied a novel methodology by combining the electrolytic lesion of the SCN with implantation of a micropump programmed to release corticosterone in a circadian manner mimicking the endogenous hormone profile. By use of this approach, we were able to restore the corticosterone rhythm in SCN-lesioned male rats. Clock gene expression in the cerebellum was abolished in rats with a lesioned SCN, but exogenous corticosterone restored the daily rhythm in clock gene expression in the cerebellar cortex, as revealed by quantitative real-time PCR and radiochemical in situ hybridization for the detection of the core clock genes Per1, Per2, and Arntl. On the contrary, exogenous hormone did not restore circadian rhythms in body temperature and running activity. RNAscope in situ hybridization further revealed that the glucocorticoid receptor colocalizes with clock gene products in cells of the cerebellar cortex, suggesting that corticosterone exerts its actions by binding directly to receptors in neurons of the cerebellum. However, rhythmic clock gene expression in the cerebellum was also detectable in adrenalectomized rats, indicating that additional control mechanisms exist. These data show that the cerebellar circadian oscillator is influenced by SCN-dependent rhythmic release of corticosterone.

Effect of urban environment on pineal machinery and clock genes expression of tree sparrow (Passer montanus)

Increasing urbanisation is altering the physiology of wild animals and the mechanisms involved are largely unknown. We hypothesised that altering the physiology of urban organisms is due to the effect of extra light at night on the circadian clock by modulating the expression of pineal machinery and clock genes. Two experiments were performed. In Experiment 1, immediately after being procured from their respective sites (urban and rural sites), birds were released individually in LLdim light conditions. Circadian rhythm period, activity duration, and total activity count were calculated and did not differ between urban and rural birds. In Experiment 2, birds (from urban and rural habitats) were sampled at six time points at regular 4-h intervals, beginning 1 h after sunrise. We measured daily variations in plasma melatonin levels. We also analysed the expression levels of Aanat, Mel1A and Mel1B as an indicator of melatonin biosynthesis and action machinery. Clock and clock-controlled genes (Bmal1, Clock, Per2, Per3, Cry1 and Npas2) were studied in the hypothalamus, the pineal gland, and retina to investigate the effects of urban habitats on the circadian clock. Our results show that there is a lower expression of Aanat in the pineal gland and relatively low plasma melatonin levels in urban birds. Further, clock genes are also differentially expressed in all three central tissues of urban birds. We propose that alterations in the melatonin biosynthesis machinery and the expression of clock genes could result in miscalculations in the internal timing of the organism, with environmental timings leading to altered physiology in urban wild animals.

Molecular biology of periodontal ligament fibroblasts and orthodontic tooth movement : Evidence and possible role of the circadian rhythm.

The circadian clock plays an important role in many physiological states and pathologies. The significance of its core genes in bone formation and tooth development has already been demonstrated. However, regulation of these genes and their influence on periodontal and bone remodeling in periodontal ligament (PDL) fibroblasts remains to be elucidated. Our hypothesis was that the circadian clock influences markers for periodontal and bone remodeling and therefore orthodontic tooth movement itself. Human PDL fibroblasts were cultured and synchronized in circadian rhythms with the help of adexamethasone shock. Cells were harvested at 4 h intervals. Reverse transcription and quantitative RT PCR (real time polymerase chain reaction) were performed to assess the mRNA levels of the clock genes ARNTL, CLOCK1, PER1, and PER2. Subsequently, mRNA expression of important marker genes for periodontal and bone remodeling, OPG, RANKL, OCN, OPN, RUNX2, COL1A1, IL1β, KI67, and POSTN, were examined at time points of ARNTL amplitude expression. Gene expression of core clock genes varied over 48 h in accordance with the circadian rhythm. Functional markers, except KI67, showed significant differences at time points of maximum fluctuation especially of ARNTL. PDL fibroblasts express circadian clock genes. Our results suggest that genes associated with bone and periodontal remodeling are influenced by the circadian rhythm. Further research will have to refine the understanding of this influence for orthodontic treatment.

Clock Genes Disruption in the Intensive Care Unit.

Intensive care unit (ICU) environment disrupts the circadian rhythms due to environmental and other nonphotic synchronizers. The main purpose of this article is to establish whether critically patients have desynchronization at the molecular level after 1 week of stay in the ICU. The rhythm of Clock, Bmal1, Cry1, and Per2 genes in neuro-ICU patients (n = 11) on the first day after admission in the unit (1 day) and 1 week later (1 week) was studied, 4 time points throughout the day, at 6, 12, 18, and 24 hours. Human whole blood samples were obtained from neuro-ICU patients. The total RNA was isolated and each sample was reverse transcribed to complementary DNA and quantitative polymerase chain reaction (PCRq) was performed. The possible rhythm was studied using Fourier Series. After 1 week, the clock gene rhythmicity completely disappeared. Messenger RNA (mRNA) expression for the 4 clock genes was shown rhythmicity at the first day after admission in the ICU. Circadian rhythmicity for none of them was observed but rather, ultradian rhythmicity was found. The expression of Clock, Bmal1, and Per2 mRNA after 1 week was similar in the 4-time point studies without significant fluctuation among the 4 time points analyzed. Rhythmic mRNA expression is present at the first day after admission in the ICU. However, ICU stay during 1 week affects the molecular machinery of the biological clock generating chronodisruption. Circadian disruption is associated with the risk of several pathologies, thus, it seems to be clear that ICU stay in constant conditions could adversely affect patient evolution and probably, circadian resynchronization restoring clock gene expression could lead to a better clinical evolution of the patient. Clock genes disruption is observed in neuro-ICU patients. Light therapy as well as melatonin treatment could reduce the impact of ICU stay period in biological clock, thereby improving patients' recovery.

The period circadian clock 2 gene responds to glucocorticoids and regulates osteogenic capacity.

IntroductionThe central regulatory system that generates biological rhythms is regulated by circadian clock genes expressed by cells in the suprachiasmatic nucleus. Signals from this system are converted to adrenocortical hormones through the sympathetic nervous system and transmitted to peripheral organs. Another system releases glucocorticoids (GCs) in response to stress through the HPA-axis. Here we investigated the second messenger GC, which is shared by these systems and influences the expression of circadian clock genes of cells of the musculoskeletal system and in viable bone tissue.MethodsWe used mouse-derived cell lines, which differentiate into osteoblasts (MC3T3-E1, C2C12, and 10T1/2) as well as primary cultures of mouse osteoblasts to determine the expression levels of circadian clock genes that respond to GC. Mice (mPer2m/m) with an inactivating mutation in the period circadian clock 2 gene (Per2) exhibit marked dysrhythmia. Here we compared the bone morphologies of mPer2m/m mice with those of wild-type (WT) mice.ResultsThe expression of major circadian clock genes was detected in each cell line, and their responsiveness to GC was confirmed. We focused on Per2, a negative regulator of the circadian clock and found that a Per2-loss-of-function mutation increased the proliferative capacity of osteoblasts. Treatment of mutant mice with slow-release GC and bisphosphonate affected the maturation of bone tissue, which reflects a tendency to retard calcification.ConclusionOur investigations of the mechanisms that regulate circadian rhythm function in tissues of the musculoskeletal system that respond to the stress hormone GC, reveal that Per2 is required for the maturation of bone tissue. Thus, the influences of the systems that control circadian rhythms and the responses to stress by regenerating tissue used for regenerative medicine must be considered and studied in greater detail.

D-Ser2-oxyntomodulin ameliorated Aβ31-35-induced circadian rhythm disorder in mice.

IntroductionThe 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.AimsThis 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.MethodsA 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.ResultsThe 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.ConclusionThis 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.

Illuminated night alters behaviour and negatively affects physiology and metabolism in diurnal zebra finches

Light at night (LAN) negatively impacts the behaviour and physiology; however, very little is known about molecular correlates of LAN-induced effects in diurnal animals. Here, we assessed LAN-induced effects on behaviour and physiology, and examined molecular changes in the liver of diurnal zebra finches (Taeniopygia guttata). Birds were exposed to dim LAN (dLAN: 12L = 150 lux: 12D = 5 lux), with controls on 12L (150 lux): 12D (0 lux). dLAN altered daily activity-rest and eating patterns, induced nocturnal eating and caused body fattening and weight gain, and reduced nocturnal melatonin levels. Concomitant increased nighttime glucose levels, decreased daytime thyroxine and triglycerides levels, and hepatic lipid accumulation suggested the impairment of metabolism under dLAN. Transcriptional assays evidenced dLAN-induced negative effects on metabolism in the liver, the site of metabolic homeostasis. Particularly, increased g6pc and foxo1 mRNA expressions suggested an enhanced gluconeogenesis, while increased egr1 and star expressions suggested enhanced cholesterol biosynthesis and lipid metabolism, respectively. Similarly, overexpressed sirt1 indicated protection from the metabolic damage due to elevated gluconeogenesis and cholesterol biosynthesis under dLAN. However, no effect on genes involved in lipogenesis (fasn) and insulin signalling pathway (socs3 and insig1) might indicate for the post transcriptional/post translational modification effects or the involvement of other genetic pathways in LAN-induced effects. We also found daily rhythm in the hepatic expression of selected clock and clock-controlled genes (per2, bmal1 and reverb-beta), with an elevated mesor and amplitude of per2 oscillation, suggesting a role of per2 in the liver metabolism. These results demonstrate dLAN-induced negative effects on the behaviour and physiology, and provide molecular insights into metabolic risks of the exposure to illuminated nights to diurnal animals including humans in an urban setting.

Multidisciplinary and Comparative Investigations of Potential Psychobiotic Effects of Lactobacillus Strains Isolated From Newborns and Their Impact on Gut Microbiota and Ileal Transcriptome in a Healthy Murine Model.

Psychobiotics are probiotic microorganisms that may exert positive influence on the psychological status of the host. Studies have revealed immunological and microbiological correlations of gut microbiota and the gut-brain axis, and have investigated psychobiotics based on the findings of the gut-brain axis. Considering their mode of actions, the present study sets anti-inflammatory effect, neurotransmitter modulation, and gut microbiota modulation as three essential criteria to evaluate Lactobacillus casei ATG-F1 (F1), L. reuteri ATG-F3 (F3), and L. reuteri ATG-F4 (F4) isolated from newborns as psychobiotics candidates in a healthy mouse model and compares the results with a non-treated control group and an ampicillin-induced gut dysbiosis (Amp) group as a negative control. The F3 and F4 strains showed anti-inflammatory effects in vitro in RAW264.7 murine macrophages, and the level of anti-inflammatory cytokine interleukin (IL)-10 increased in ileums of mice orally administered with the F4 strain. Serum dopamine level significantly increased only in the F4-treated group as compared with the control group. Serum serotonin level was unaffected in Lactobacillus-treated groups, while a significant decrease in serum serotonin level was observed in the Amp group. Bacteroidetes population increased in fecal samples of the F4-treated group as compared with the control, and Bacteroidales S24-7 and Prevotellaceae population significantly increased at family level in fecal samples from the F4-treated group as compared with the control. In contrast, the Amp group showed an increase in the level of Proteobacteria and a decrease in the level of Bacteroidetes as compared with the control group. Transcriptome analysis revealed a distinctive clustering in ileums from the F4-treated group as compared to other experimental groups. In addition, the circadian rhythm pathway showed maximum enrichment in ileums of Lactobacillus-treated mice, and the F4-treated group showed the highest fold changes in circadian rhythm-related genes (Dbp, Per1, Per2, and Per3). Conclusively, L. reuteri ATG-F4 is suggested as a potential psychobiotics through demonstrations of anti-inflammatory effects, serum dopamine modulation, and gut microbiota modulation in a healthy murine model in the present study. Moreover, we carefully suggest gut circadian rhythm modulation as another important criterion of psychobiotics, which may have an important role in the gut-brain axis.

The protective effect of cycloastragenol on aging mouse circadian rhythmic disorder induced by d‐galactose

Aging process in mammals is associated with a decline in amplitude and a long period of circadian behaviors which are regulated by a central circadian regulator in the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. It is unclear whether enhancing clock function can retard aging. Using fibroblasts expressing per2::lucSV and senescent cells, we revealed cycloastragenol (CAG), a natural aglycone derivative from astragaloside IV, as a clock amplitude enhancing small molecule. CAG could activate telomerase to antiaging, but no reports focused on its effects on circadian rhythm disorders in aging mice. Here we analyze the potential effects of CAG on d-galactose-induced aging mice on the circadian behavior and expression of clock genes. For this purpose, CAG (20 mg/kg orally), was administered daily to d-galactose (150 mg/kg, subcutaneous) mice model of aging for 6 weeks. An actogram analysis of free-running activity of these mice showed that CAG significantly enhances the locomotor activity. We further found that CAG increase expressions of per2 and bmal1 genes in liver and kidney of aging mouse. Furthermore, CAG enhanced clock protein BMAL1 and PER2 levels in aging mouse liver and SCN. Our results indicated that the CAG could restore the behavior of circadian rhythm in aging mice induced by d-galactose. These data of present study suggested that CAG could be used as a novel therapeutic strategy for the treatment of age-related circadian rhythm disruption.

Abstract 1729: Investigating deregulated circadian clock machinery in cancer cells

Abstract The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response. Citation Format: Pauline J. van der Watt, Kate Davis, Virna Leaner. Investigating deregulated circadian clock machinery in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1729.

Circadian Rhythm Disruption and Sepsis in Severe Trauma Patients.

Circadian rhythms are important regulators of immune functions. Admission to an intensive care unit may impact molecular clock activity and host response. Our objective was to assess and compare the immune circadian rhythms in trauma patients who develop and in those who do not develop sepsis. Blood samples were collected from severe trauma patients within 4 days after admission, with collections taking place every 4 h over a 24-h period. Cortisol and cytokines were measured with immunoassays. Whole-blood expression of 3 clock genes (Bmal1, Per2, and Per3) was studied by reverse transcription quantitative polymerase chain reaction. Neutrophils, monocytes, and lymphocytes were analyzed by flow cytometry. Patients with and without sepsis were compared with the cosinor mixed model to estimate mesors, amplitudes, and acrophases. Thirty-eight patients were enrolled in the study, and 13 developed at least 1 septic episode. The septic patients had higher levels of cortisol than the nonseptic patients (mesor at 489 nmol/L vs. 405 nmol/L, P < 0.05) and delayed acrophases (22 h vs. 15 h, P < 0.05). They also had lower lymphocyte counts (mesor at 785 vs. 1,012 cells/μL, P < 0.05), higher neutrophil counts (mesor at 7,648 vs. 7,001 cells/μL, P < 0.05), and monocyte counts (mesor at 579 vs. 473 cells/μL, P < 0.05) than the nonseptic patients. Although no amplitude difference was identified, the acrophases were significantly different between the 2 groups for lymphocytes, interleukin 10 and tumor necrosis factor. We demonstrated that all trauma patients had impaired circadian rhythms of cortisol, cytokines, leukocytes, and clock genes. Early circadian disruption was associated with the occurrence of sepsis and might be a marker of sepsis severity.

Abstract 1888: Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture

Abstract It is important to know whether breast cancer cells within tumors have functional circadian clocks. If so, they may benefit from the ability provided by the clock to predict when important daily events occur such as nutrient availability in blood following meals or elevated immune cell surveillance at night. If instead the circadian clock is nonfunctional, then clinical treatments could be directed towards activating the clock because evidence indicates circadian rhythm loss leads to more aggressive tumor growth. Here, we examined the role of circadian clocks in metastasis-related events, focusing on epithelial-mesenchymal transition (EMT), including cancer stem cell (CSC) generation, and mesenchymal-epithelial transition (MET). Because of their differing circadian properties two tumor-forming cell lines were compared, MCF-7 human breast cancer and C6 rat glioma cells. C6 monolayer and tumorsphere cultures express distinct circadian rhythms in gene expression. In contrast, monolayer MCF-7 cell cultures are reported to be arrhythmic and so appear to lack a functional circadian clock. Nevertheless, MCF-7 cells may contain inducible circadian oscillators that fail to generate circadian rhythms under standard culture conditions but may function in tumor microenvironments. Tumorspheres simulate the three-dimensional tumor structure and were used here to test for favorable conditions enabling circadian rhythms in cancer cells. Circadian rhythms in EMT, MET, mitosis, migration, and cell death were evaluated through time-lapse imaging over several days. Bioluminescence imaging and quantitative RT-PCR were used to test for circadian rhythms in expression of the core circadian clock gene Per2 in MCF-7 cells. Following treatment with serum-free culture medium designed for stem cells, EMT and CSC-related protein markers of MCF-7 and C6 cells were confirmed by immunofluorescence: MSI1 and CD133 in MCF-7; OCT4, TWIST1, ZEB1, vimentin in C6. Morphometry showed distinct changes in cell shape following EMT as shown by nearly all MCF-7 cells having an average roundness of 0.815 ±0.09 SD and a C6 roundness of 0.855 ±0.029. This medium increased the number of MCF-7 tumorspheres expressing circadian rhythms by 31.5%, and average amplitude of rhythms increased 11.6-fold. MET was then induced by returning cells to serum-containing medium, which produced cells with a flattened cell morphology indicating epithelial or pseudoepithelial differentiation. A circadian clock in C6 cells appeared to gate MET events after the return to serum (95% confidence limit of period: 20.8-26.9 hrs, p&amp;lt;0.05). The circadian timing properties of MCF-7 tumorspheres suggest that the circadian clock may also be present in tumors and that cell-cell interactions or paracrine factors could sustain otherwise poorly performing circadian clocks in breast cancer. Citation Format: Arpan De, Vishal Premdev Sharma, Dilshan Harshajith Beligala, Angelia Marie Lee, Michael Eric Geusz. Circadian rhythm modulation of breast cancer gene expression depends on stem cell properties induced in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1888.