The physiological role of TRP channels in sleep and circadian rhythm.

TRP channels, are non-specific cationic channels that are involved in multiple physiological processes that include salivation, cellular secretions, memory extinction and consolidation, temperature, pain, store-operated calcium entry, thermosensation and functionality of the nervous system. Here we choose to look at the evidence that decisively shows how TRP channels modulate human neuron plasticity as it relates to the molecular neurobiology of sleep/circadian rhythm. There are numerous model organisms of sleep and circadian rhythm that are the results of the absence or genetic manipulation of the non-specific cationic TRP channels. Drosophila and mice that have had their TRP channels genetically ablated or manipulated show strong evidence of changes in sleep duration, sleep activity, circadian rhythm and response to temperature, noxious odours and pattern of activity during both sleep and wakefulness along with cardiovascular and respiratory function during sleep. Indeed the role of TRP channels in regulating sleep and circadian rhythm is very interesting considering the parallel roles of TRP channels in thermoregulation and thermal response with concomitant responses in growth and degradation of neurites, peripheral nerves and neuronal brain networks. TRP channels provide evidence of an ability to create, regulate and modify our sleep and circadian rhythm in a wide array of physiological and pathophysiological conditions. In the current review, we summarize previous results and novel recent advances in the understanding of calcium ion entry via TRP channels in different sleep and circadian rhythm conditions. We discuss the role of TRP channels in sleep and circadian disorders.

Circadian rhythm disruption upregulating Per1 in mandibular condylar chondrocytes mediating temporomandibular joint osteoarthritis via GSK3ß/ß-CATENIN pathway.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJOA) has a high incidence rate, but its pathogenesis remains unclear. Circadian rhythm is an important oscillation in the human body and influences various biological activities. However, it is still unclear whether circadian rhythm affects the onset and development of TMJOA. METHODS: We disrupted the normal rhythm of rats and examined the expression of core clock genes in the mandibular condylar cartilage of the jaw and histological changes in condyles. After isolating rat mandibular condylar chondrocytes, we upregulated or downregulated the clock gene Per1, examined the expression of cartilage matrix-degrading enzymes, tested the activation of the GSK3ß/ß-CATENIN pathway and verified it using agonists and inhibitors. Finally, after downregulating the expression of Per1 in the mandibular condylar cartilage of rats with jet lag, we examined the expression of cartilage matrix-degrading enzymes and histological changes in condyles. RESULTS: Jet lag led to TMJOA-like lesions in the rat mandibular condyles, and the expression of the clock gene Per1 and cartilage matrix-degrading enzymes increased in the condylar cartilage of rats. When Per1 was downregulated or upregulated in mandibular condylar chondrocytes, the GSK3ß/ß-CATENIN pathway was inhibited or activated, and the expression of cartilage matrix-degrading enzymes decreased or increased, which can be rescued by activator and inhibitor of the GSK3ß/ß-CATENIN pathway. Moreover, after down-regulation of Per1 in mandibular condylar cartilage in vivo, significant alleviation of cartilage degradation, cartilage loss, subchondral bone loss induced by jet lag, and inhibition of the GSK3ß/ß-CATENIN signaling pathway were observed. Circadian rhythm disruption can lead to TMJOA. The clock gene Per1 can promote the occurrence of TMJOA by activating the GSK3ß/ß-CATENIN pathway and promoting the expression of cartilage matrix-degrading enzymes. The clock gene Per1 is a target for the prevention and treatment of TMJOA.

Restored UBE2C expression in islets promotes ß-cell regeneration in mice by ubiquitinating PER1.

Insulin deficiency may be due to the reduced proliferation capacity of islet ß-cell, contributing to the onset of diabetes. It is therefore imperative to investigate the mechanism of the ß-cell regeneration in the islets. NKX6.1, one of the critical ß-cell transcription factors, is a pivotal element in ß-cell proliferation. The ubiquitin-binding enzyme 2C (UBE2C) was previously reported as one of the downstream molecules of NKX6.1 though the exact function and mechanism of UBE2C in ß-cell remain to be elucidated. Here, we determined a subpopulation of islet ß-cells highly expressing UBE2C, which proliferate actively. We also discovered that ß-cell compensatory proliferation was induced by UBE2C via the cell cycle renewal pathway in weaning and high-fat diet (HFD)-fed mice. Moreover, the reduction of ß-cell proliferation led to insulin deficiency in ßUbe2cKO mice and, therefore, developed type 2 diabetes. UBE2C was found to regulate PER1 degradation through the ubiquitin-proteasome pathway via its association with a ubiquitin ligase, CUL1. PER1 inhibition rescues UBE2C knockout-induced ß-cell growth inhibition both in vivo and in vitro. Notably, overexpression of UBE2C via lentiviral transduction in pancreatic islets was able to relaunch ß-cell proliferation in STZ-induced diabetic mice and therefore partially alleviated hyperglycaemia and glucose intolerance. This study indicates that UBE2C positively regulates ß-cell proliferation by promoting ubiquitination and degradation of the biological clock suppressor PER1. The beneficial effect of UBE2C on islet ß-cell regeneration suggests a promising application in treating diabetic patients with ß-cell deficiency.

Circadian PER1 controls daily fat absorption with the regulation of PER1-PKA on phosphorylation of bile acid synthetase.

Several epidemiological studies suggest a correlation between eating time and obesity. Night eating syndrome characterized by a time-delayed eating pattern is positively associated with obesity in humans as well as in experimental animals. Here, we show that oil intake at night significantly makes more fat than that at day in wild-type mice, and circadian Period 1 (Per1) contributes to this day-night difference. Per1-knockout mice are protected from high-fat diet-induced obesity, which is accompanied by a reduction in the size of the bile acid pool, and the oral administration of bile acids restores fat absorption and accumulation. We identify that PER1 directly binds to the major hepatic enzymes involved in bile acid synthesis such as cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase. A biosynthesis rhythm of bile acids is accompanied by the activity and instability of bile acid synthases with PER1/PKA-mediated phosphorylation pathways. Both fasting and high fat stress enhance Per1 expression, increasing the fat absorption and accumulation. Our findings reveal that Per1 is an energy regulator and controls daily fat absorption and accumulation. Circadian Per1 controls daily fat absorption and accumulation, suggesting Per1 is a potential candidate of a key regulator in stress response and the relevant obesity risk.

Insertion of ISPa1635 in ISCR1 Creates a Hybrid Promoter for blaPER-1 Resulting in Resistance to Novel ß-lactam/ß-lactamase Inhibitor Combinations and Cefiderocol.

Eleven blaPER-1-positive Pseudomonas aeruginosa clinical isolates showed variable susceptibility to ceftazidime-avibactam (CZA). The genetic contexts of blaPER-1 were identical (ISCR1-blaPER-1-gst) except for the ST697 isolate HS204 (ISCR1-ISPa1635-blaPER-1-gst). The insertion of ISPa1635 in ISCR1 upstream of blaPER-1 created a hybrid promoter, which elevated the blaPER-1 transcription level and resulted in increased resistance to CZA, ceftolozane-tazobactam, cefepime-zidebactam, and cefiderocol. Diversity in the promoter activity of blaPER-1 partially explains the variable susceptibility to CZA in PER-producing isolates.

Stress hormone signalling inhibits Th1 polarization in a CD4 T-cell-intrinsic manner via mTORC1 and the circadian gene PER1.

Stress hormones are believed to skew the CD4 T-cell differentiation towards a Th2 response via a T-cell-extrinsic mechanism. Using isolated primary human naïve and memory CD4 T cells, here we show that both adrenergic- and glucocorticoid-mediated stress signalling pathways play a CD4 naïve T-cell-intrinsic role in regulating the Th1/Th2 differentiation balance. Both stress hormones reduced the Th1 programme and cytokine production by inhibiting mTORC1 signalling via two parallel mechanisms. Stress hormone signalling inhibited mTORC1 in naïve CD4 T cells (1) by affecting the PI3K/AKT pathway and (2) by regulating the expression of the circadian rhythm gene, period circadian regulator 1 (PER1). Both stress hormones induced the expression of PER1, which inhibited mTORC1 signalling, thus reducing Th1 differentiation. This previously unrecognized cell-autonomous mechanism connects stress hormone signalling with CD4 T-cell differentiation via mTORC1 and a specific circadian clock gene, namely PER1.

Structural Basis of PER-1-Mediated Cefiderocol Resistance and Synergistic Inhibition of PER-1 by Cefiderocol in Combination with Avibactam or Durlobactam in Acinetobacter baumannii.

Cefiderocol is a novel siderophore cephalosporin that displays activity against Gram-negative bacteria. To establish cefiderocol susceptibility levels of Acinetobacter baumannii strains from China, we performed susceptibility testing and genomic analyses on 131 clinical isolates. Cefiderocol shows high activity against the strains. The production of PER-1 is the key mechanism of cefiderocol resistance. In silico studies predicted that avibactam and durlobactam could inhibit cefiderocol hydrolysis by PER-1, which was confirmed by determining cefiderocol MICs in combination with inhibitors.

Activation of dopamine D2 receptors in the medial shell region of the nucleus accumbens increases Per1 expression to enhance alcohol consumption.

Circadian genes, including Per1, in the medial shell region of nucleus accumbens (mNAcSh), regulate binge alcohol consumption. However, the upstream mechanism regulating circadian genes-induced alcohol consumption is not known. Since activation of dopamine D2 receptors (D2R) increases Per1 gene expression, we hypothesised that local infusion of quinpirole, a D2R agonist, by increasing Per1 gene expression in the mNAcSh, will increase binge alcohol consumption in mice. We performed two experiments on male C57BL/6J mice, instrumented with bilateral guide cannulas above the mNAcSh, and exposed to a 4-day drinking-in-dark (DID) paradigm. The first experiment determined the effects of bilateral infusion of quinpirole (100 ng/300 nl/site) or DMSO (Vehicle group) in the mNAcSh on Per1 gene expression and alcohol consumption. The second experiment determined the effect of antisense-induced downregulation of Per1 in the mNAcSh on the quinpirole-induced increase in alcohol consumption. Control experiments were performed by exposing the animals to sucrose (10% w/v). After the experiment, animals were euthanised, brains removed and processed for localisation of injection sites and analysis of Per1 gene expression in the mNAcSh. As compared with the DMSO, local bilateral infusion of quinpirole significantly increased the expression of Per1 in the mNAcSh along with an increase in the amount of alcohol consumed in mice exposed to DID paradigm. In addition, local antisense-induced downregulation of Per1 significantly attenuated the effects of intro-accumbal infusion of quinpirole on alcohol consumption. Our results suggest that Per1 in the mNAcSh mediates D2R activation-induced increase in alcohol consumption.

Trends in the hospital-sector consumption of the WHO AWaRe Reserve group antibiotics in EU/EEA countries and the United Kingdom, 2010 to 2018.

BackgroundIn 2019, the World Health Organization published the 21st Model list of Essential Medicines and updated the Access, Watch Reserve (AWaRe) antibiotics classification to improve metrics and indicators for antibiotic stewardship activities. Reserve antibiotics are regarded as last-resort treatment options.AimWe investigated hospital-sector consumption quantities and trends of Reserve group antibiotics in European Union/European Economic Area countries and the United Kingdom (EU/EEA/UK).MethodsHospital-sector antimicrobial consumption data for 2010-2018 were obtained from the European Centre for Disease Prevention and Control. Antibacterials' consumption for systemic use (Anatomical Therapeutic Chemical classification (ATC) group J01) were included in the analysis and expressed as defined daily doses (DDD) per 1,000 inhabitants per day. We defined reserve antibiotics as per AWaRe classification and applied linear regression to analyse trends in consumption of reserve antibiotics throughout the study period.ResultsEU/EEA/UK average hospital-sector reserve-antibiotic consumption increased from 0.017 to 0.050 DDD per 1,000 inhabitants per day over the study period (p = 0.002). This significant increase concerned 15 countries. In 2018, four antibiotics (tigecycline, colistin, linezolid and daptomycin) constituted 91% of the consumption. Both absolute and relative (% of total hospital sector) consumption of reserve antibiotics varied considerably (up to 42-fold) between countries (from 0.004 to 0.155 DDD per 1,000 inhabitants per day and from 0.2% to 9.3%, respectively).ConclusionAn increasing trend in reserve antibiotic consumption was found in Europe. The substantial variation between countries may reflect the burden of infection with multidrug-resistant bacteria. Our results could guide national actions or optimisation of reserve antibiotic use.

Chronic Trazodone and Citalopram Treatments Increase Trophic Factor and Circadian Rhythm Gene Expression in Rat Brain Regions Relevant for Antidepressant Efficacy.

Trazodone is an efficacious atypical antidepressant acting both as an SSRI and a 5HT2A and 5HT2C antagonist. Antagonism to H1-histaminergic and alpha1-adrenergic receptors is responsible for a sleep-promoting action. We studied long-term gene expression modulations induced by chronic trazodone to investigate the molecular underpinning of trazodone efficacy. Rats received acute or chronic treatment with trazodone or citalopram. mRNA expression of growth factor and circadian rhythm genes was evaluated by qPCR in the prefrontal cortex (PFCx), hippocampus, Nucleus Accumbens (NAc), amygdala, and hypothalamus. CREB levels and phosphorylation state were evaluated using Western blotting. BDNF levels were significantly increased in PFCx and hippocampus by trazodone and in the NAc and hypothalamus by citalopram. Likewise, TrkB receptor levels augmented in the PFCx after trazodone and in the amygdala after citalopram. FGF-2 and FGFR2 levels were higher after trazodone in the PFCx. The CREB phosphorylation state was increased by chronic trazodone in the PFCx, hippocampus, and hypothalamus. Bmal1 and Per1 were increased by both antidepressants after acute and chronic treatments, while Per2 levels were specifically augmented by chronic trazodone in the PFCx and NAc, and by citalopram in the PFCx, amygdala, and NAc. These findings show that trazodone affects the expression of neurotrophic factors involved in antidepressant responses and alters circadian rhythm genes implicated in the pathophysiology of depression, thus shedding light on trazodone's molecular mechanism of action.

Per1/Per2 Disruption Reduces Testosterone Synthesis and Impairs Fertility in Elderly Male Mice.

Circadian rhythm disorders caused by genetic or environmental factors lead to decreased male fertility but the mechanisms are poorly understood. The current study reports that the mechanism of Per1/Per2 Double knockout (DKO) reduced the reproductive capacity of elderly male mice. The sperm motility and spermatogenic capacity of male DKO mice were weak. Hormone-targeted metabolomics showed reduced plasma levels of free testosterone in DKO male mice compared with WT male mice. Transcriptomic analysis of testicular tissue showed the down-regulation of testosterone synthesis-related enzymes (Cyp11a1, Cyp17a1, Hsd17b3, Hsd3b1, and Star) in the steroid hormone synthesis pathway. Spermatogenesis genes, Tubd1 and Pafah1b were down-regulated, influencing tubulin dynamics and leading to impaired motility. Seleno-compound metabolic loci, Scly and Sephs2, were up-regulated and Slc7a11 and Selenop were down-regulated. Western-blotting showed that steroid acute regulatory protein (StAR) and p-CREB, PKA and AC1 were reduced in testicular tissue of DKO mice compared to WT. Therefore, Per1/Per2 disruption reduced testosterone synthesis and sperm motility by affecting the PKA-StAR pathway, leading to decreased fertility.

Per1/Per2 knockout Affects Spleen Immune Function in Elderly Mice via Inducing Spleen Lymphocyte Ferroptosis.

Disturbances in circadian rhythms are known to affect immune functions. However, the long-term impact of abnormal circadian rhythms on the immune-related functions of the spleen are poorly understood. Hence, we aimed to investigate the immune-related functions of spleen in Per1/Per2 double-knockout (DKO) and wild-type (WT) mice aged 4, 9, and 14 months. Compared to the WT mice, the DKO mice had smaller spleen white pulp (WP) and lymphocyte germinal area, as well as fewer immune cells with age-these differences were especially clear. The spleen lymphocyte mortality, malondialdehyde (MDA) levels, reactive oxygen species (ROS) levels, and ferritin-binding receptor (TFR1) levels were significantly higher in the 14-month-old DKO mice than in WT mice of the same age. Transcriptome analysis showed that most of the differentially expressed mRNAs were enriched in DNA damage repair-related pathways. In DKO mice, spleen cells showed up-regulation of pro-ferroptosis genes, such as Cd36,Atm, and Acsl4, and down-regulation of anti-ferroptosis genes, such as GPX4. We found that long-term abnormalities in the circadian rhythm can induce DNA damage and ferroptosis in mouse spleen.

The circadian clock gene Per1 modulates context fear memory formation within the retrosplenial cortex in a sex-specific manner.

Context memory formation is a complex process that requires transcription in many subregions of the brain including the dorsal hippocampus and retrosplenial cortex. One critical gene necessary for memory formation is the circadian gene Period1 (Per1), which has been shown to function in the dorsal hippocampus to modulate spatial memory in addition to its well-documented role in regulating the diurnal clock within the suprachiasmatic nucleus (SCN). We recently found that alterations in Per1 expression in the dorsal hippocampus can modulate spatial memory formation, with reduced hippocampal Per1 impairing memory and overexpression of Per1 ameliorating age-related impairments in spatial memory. Whether Per1 similarly functions within other memory-relevant brain regions is currently unknown. Here, to test whether Per1 is a general mechanism that modulates memory across the brain, we tested the role of Per1 in the retrosplenial cortex (RSC), a brain region necessary for context memory formation. First, we demonstrate that context fear conditioning drives a transient increase in Per1 mRNA expression within the anterior RSC that peaks 60 m after training. Next, using HSV-CRISPRi-mediated knockdown of Per1, we show that reducing Per1 within the anterior RSC before context fear acquisition impairs memory in both male and female mice. In contrast, overexpressing Per1 with either HSV-CRISPRa or HSV-Per1 before context fear acquisition drives a sex-specific memory impairment; males show impaired context fear memory whereas females are not affected by Per1 overexpression. Finally, as Per1 levels are known to rhythmically oscillate across the day/night cycle, we tested the possibility that Per1 overexpression might have different effects on memory depending on the time of day. In contrast to the impairment in memory we observed during the daytime, Per1 overexpression has no effect on context fear memory during the night in either male or female mice. Together, our results indicate that Per1 modulates memory in the anterior retrosplenial cortex in addition to its documented role in regulating memory within the dorsal hippocampus, although this role may differ between males and females.

Antisense-induced knockdown of cAMP response element-binding protein downregulates Per1 gene expression in the shell region of nucleus accumbens resulting in reduced alcohol consumption in mice.

INTRODUCTION: We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption. METHODS: To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24 h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining. RESULTS: Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed. CONCLUSIONS: Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption.

Antisense-Induced Downregulation of Clock Genes in the Shell Region of the Nucleus Accumbens Reduces Binge Drinking in Mice.

INTRODUCTIONS: Binge drinking is a deadly pattern of alcohol consumption. Evidence suggests that genetic variation in clock genes is strongly associated with alcohol misuse; however, the neuroanatomical basis for such a relationship is unknown. The shell region of the nucleus accumbens (NAcSh) is well known to play a role in binge drinking. Hence, we examined whether clock genes in the NAcSh regulate binge drinking. METHODS: To address this question, 2 experiments were performed on male C57BL/6J mice. In the first experiment, mice exposed to alcohol or sucrose under the 4-day drinking-in-the-dark (DID) paradigm were euthanized at 2 different time points on day 4 [7 hours after light (pre-binge drinking) or dark (post-binge drinking) onset]. The brains were processed for RT-PCR to examine the expression of circadian clock genes (Clock, Per1, and Per2) in the NAcSh and suprachiasmatic nucleus (SCN). In the second experiment, mice were exposed to alcohol, sucrose, or water as described above. On day 4, 1 hour prior to the onset of alcohol exposure, mice were bilaterally infused with either a mixture of circadian clock gene antisense oligodeoxynucleotides (AS-ODNs; antisense group) or nonsense/random ODNs (R-ODNs; control group) through surgically implanted cannulas above the NAcSh. Alcohol/sucrose/water consumption was measured for 4 hours. Blood alcohol concentration was measured to confirm binge drinking. Microinfusion sites were histologically verified using cresyl violet staining. RESULTS: As compared to sucrose, mice euthanized post-binge drinking (not pre-binge drinking) on day 4 displayed a greater expression of circadian genes in the NAcSh but not in the SCN. Knockdown of clock genes in the NAcSh caused a significantly lower volume of alcohol to be consumed on day 4 than in the control treatment. No differences were found in sucrose or water consumption. CONCLUSIONS: Our results suggest that clock genes in the NAcSh play a crucial role in binge drinking.

[Effects of Acrolein on the Proliferation of and Per1 Gene Expression in Pulmonary Epithelial Cells].

OBJECTIVE: To investigate the effect of acrolein on the proliferation of pulmonary epithelial cells and its possible mechanism. METHODS: Two strains of pulmonary epithelial cells, A549 cells and MLE15 cells, were used as in vitro models of pulmonary epithelial cell, and were treated with 80 µmol/L acrolein or phosphate buffer saline (PBS) as the control. The proliferation of pulmonary epithelial cells were determined with CCK-8 kit after cell culturing resumed for 12 h, 24 h, 36 h and 48 h post acrolein treatment, and the expression of period circadian regulator gene 1 ( Per1) was examined using Western blot test 24 h after acrolein treatment. In addition, after acrolein treatment, the cells were restored with transforming growth factor-ß (TGF-ß) added in the medium, and the cell proliferation and the expression of Per1 protein were also examined. RESULTS: The proliferation of A549 cells and MLE15 cells decreased significantly after being treated with 80 µmol/L acrolein for 30 min, and the expression of Per1 protein was also downregulated significantly ( P<0.05). The addition of TGF-ß after acrolein treatment did not significantly change the reduction in cell proliferation caused by acrolein, but the expression of Per1 protein in pulmonary epithelial cells was significantly higher than that in cells restored without TGF-ß ( P<0.05). CONCLUSION: Acrolein treatment resulted in the decreased proliferation of pulmonary epithelial cells and the Per1 expression in pulmonary epithelial cells. Although TGF-ß addition did not reverse the reduction of cell proliferation after acrolein treatment, the Per1 expression levels were recovered to a certain extent compared to that in cells restored in medium without TGF-ß after acrolein treatment.

RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). METHODS: The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. RESULTS: ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. CONCLUSION: ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

Mechanisms of circadian clock interactions with aryl hydrocarbon receptor signalling.

Per-Arnt-Sim (PAS) domain-containing proteins are critical to homeostatic regulatory networks that mediate responsiveness to environmental change. PAS domains are multifunctional structural motifs that allow protein-protein interactions amongst family members, typically forming heterodimeric transcription factors to affect the transcription of target genes. Prototypical PAS domain-dependent pathways include the circadian clock network and metabolic regulation of the xenobiotic response through the aryl hydrocarbon receptor (AhR). Both pathways are increasingly linked to health, and alteration in their function contributes to development of disease. The AhR demonstrates promiscuity in ligand binding and selectivity during heterodimer formation, which allows varied combinations of protein-protein interactions with other Per-Arnt-Sim (PAS) domain-containing proteins and crosstalk amongst signalling pathways, including the molecular clockworks. AhR and the circadian signalling pathways are highly integrated and reciprocally regulated. AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists. Conversely, AhR influences amplitude and phase of rhythms in circadian clock genes, hormones, and behaviour. Understanding the molecular interactions between AhR and the clock provides insight into physiological regulation of rhythmic processes and provides an innovative approach to development of therapeutics.

Distribution of Pantoea agglomerans isolates and molecular detection of blaPER-1 ESßL gene: A statistical study.

THE PURPOSE: to study the distribution of Pantoea agglomerans (P. agglomerans) statistically and the presence of blaPER-1 type ESßL in the clinical and environmental isolates. METHODS: During a period of 2014-2015, 895 blood specimens and 438 hospital environmental samples were collected from one children's hospital in Baghdad city. The results of statistical analysis showed there was no relationship between the infection with P. agglomerans and the sex, while there was a relationship between the infection with the P. agglomerans and the place of residence and also the age of patients. RESULT: A total of 23 P. agglomerans were isolated during the study, out of 23 isolates, 13 (56.52%) and 10 (43.48%) were isolated from blood specimens and from hospital environment. All 23 isolates had 100% sensitivity rate to Imipenem and the highest resistant rate was (95.65%) to Ampicillin. Out of 23 P. agglomerans, 14 (60.87%) isolates were positive ESßL producing by the screening test. CONCLUSION: The result of molecular screening of the gene blaPER-1 showed the presence of this gene only in phenotypically ESBL producing isolates, while all negative ESßL producing isolates don't harboring blaPER-1 gene. Out of 14 positive ESßL producing P. agglomerans isolates, 5 (35.71%) were harboring blaPER-1 gene and 9 (64.29%) of positive ESßL producing isolates were don't harboring blaPER-1 gene (significant difference at ≤0.05).

CRISPR/Cas-mediated Fubp1 silencing disrupts circadian oscillation of Per1 protein via downregulating Syncrip expression.

Most living organisms have physiological and behavioral circadian rhythms controlled by molecular clocks. In mammals, several core clock genes show self-perpetuating oscillation profiles of their messenger RNAs (mRNAs) and proteins through an auto-regulatory transcription-translation feedback loop (TTFL). As a critical component in the molecular clock system, Period 1 (Per1) contributes to the maintenance of circadian rhythm duration predominantly in peripheral clocks. Alterations in Per1 expression and oscillating patterns lead to the development of cancers as well as circadian rhythm abnormalities. In this study, we demonstrate that the phasic profile of Per1 protein was clearly disrupted in CRISPR/Cas-mediated Fubp1-deficient cells. Although Fubp1 does not show rhythmic expression, Fubp1 upregulates the mRNA and protein level of Syncrip, the main post-transcriptional regulator of Per1 protein oscillation. In addition to the diverse physiological functions of Fubp1, including cell-cycle regulation and cellular metabolic control, our results suggest new roles for Fubp1 in the molecular clock system.