Expression Of Core Clock Genes Research Articles

Disruption of local circadian clocks in aristolochic acid-induced nephropathy in mice

BackgroundAristolochic acid I (AAI), an emerging biogenic contaminant widely present in Aristolochic plants, has been implicated in the progression of tubulointerstitial disease, known as aristolochic acid nephropathy (AAN). The circadian clock, a vital regulator of organ homeostasis, is susceptible to external chemical cues, including toxins. However, the reciprocal interactions between AAI and the circadian clock remain unexplored. MethodsWe initially assessed sex- and time-dependent nephropathy and behavioral responses in C57BL/6J mice exposed to AAI. Subsequently, we evaluated changes in the expression of circadian clock genes following treatment with AAI or its bioactive metabolite, aristolactam I, using real-time quantitative PCR and immunoblotting in renal tissues and cells. Additionally, real-time reporter assays were conducted on kidney explants from PER2::Luc knock-in reporter mice and Per2-dLuc/Bmal1-dLuc reporter cell lines. To further elucidate the regulatory role of circadian clocks in AAI-induced nephropathy, mice with global or kidney-specific knockout of Bmal1, as well as mice subjected to experimental jetlag, were utilized. ResultsOur findings revealed a sex-dependent nephrotoxicity of AAI, with males exhibiting greater vulnerability. AAI-induced nephropathy was accompanied by impaired spatial cognitive function, disruptions in free-running locomotor activity, altered renal expression of multiple core clock genes, and disturbances in the circadian rhythm of renal PER2::Luc activity. Notably, kidney-specific ablation of the core clock gene Bmal1 significantly exacerbated renal injury and inflammation, whereas disruptions to the central clock, either genetically (through conventional knockout of Bmal1) or environmentally (mimicking jetlag), had minimal effects on AAI nephrotoxicity. Furthermore, both AAI and its bioactive metabolite aristolactam I demonstrated the ability to disrupt circadian clocks in human osteosarcoma cells (U2OS) and mouse renal tubular epithelial cells (mRTEC). ConclusionCollectively, these findings highlight the detrimental impact of aristolochic acids on local renal circadian clocks, ultimately exacerbating kidney damage. This study provides novel insights into the molecular mechanisms underlying AAI nephrotoxicity, potentially opening avenues for therapeutic interventions aimed at modulating the renal circadian clock to treat AAN.

Clinical Implication of Time of Ischaemic Stroke Among Post-Stroke Survivors from Eastern India: A Circadian Perspective.

The circadian variation in stroke occurrence is a well-documented phenomenon. However, the circadian effect on stroke outcome, particularly on post-stroke cognition, has not yet been fully elucidated. We aim to evaluate the influence of diurnal variation of stroke onset upon post-stroke cognition and development of post-stroke depression. Based on 4-hourly time period of stroke occurrence, 249 recruited cohorts were categorized into 6 groups. Several clinical and cognitive parameters were compared among the groups. Then, the mRNA expression of core clock genes in Peripheral Blood Mononuclear Cells were quantified and correlated with post-stroke outcomes among 24 acute phase cases with day-time or night-time stroke occurrence. Furthermore, the genetic susceptibility towards a higher number of cases in the morning was examined by genotyping CLOCK (rs1801260T/C, rs4580704G/C) and CRY2 (rs2292912C/G) genes variants in cases and 292 controls. In our study, the peak for highest incidence although observed during the early morning from 4 to 8 am, the nocturnal-onset stroke cases showed more severity (12.2 ± 5.67) at the time of admission irrespective of arterial territory involved. The night onset cases were also found to be more susceptible to develop language impairment and post-stroke depression in due course of time. Upon transcript analysis, circadian genes (BMAL1 and CRY1) were found to be downregulated in night-time cases than day-time ones during the acute phase of onset. In addition, those mRNA levels also showed a correlation with raw scores for language and depression. However, the difference in incidence frequency along a day did not reveal any genetic correlation. Therefore, we suggest night-time stroke to be positively associated with higher immediate severity and poor cognitive outcome than day-time injury and propose downregulation of circadian genes during the acute phase could be the underlying molecular mechanism for this.

Comparative transcriptomic rhythms in the mouse and human prefrontal cortex.

Alterations in multiple subregions of the human prefrontal cortex (PFC) have been heavily implicated in psychiatric diseases. Moreover, emerging evidence suggests that circadian rhythms in gene expression are present across the brain, including in the PFC, and that these rhythms are altered in disease. However, investigation into the potential circadian mechanisms underlying these diseases in animal models must contend with the fact that the human PFC is highly evolved and specialized relative to that of rodents. Here, we use RNA sequencing to lay the groundwork for translational studies of molecular rhythms through a sex-specific, cross species comparison of transcriptomic rhythms between the mouse medial PFC (mPFC) and two subregions of the human PFC, the anterior cingulate cortex (ACC) and the dorsolateral PFC (DLPFC). We find that while circadian rhythm signaling is conserved across species and subregions, there is a phase shift in the expression of core clock genes between the mouse mPFC and human PFC subregions that differs by sex. Furthermore, we find that the identity of rhythmic transcripts is largely unique between the mouse mPFC and human PFC subregions, with the most overlap (20%, 236 transcripts) between the mouse mPFC and the human ACC in females. Nevertheless, we find that basic biological processes are enriched for rhythmic transcripts across species, with key differences between regions and sexes. Together, this work highlights both the evolutionary conservation of transcriptomic rhythms and the advancement of the human PFC, underscoring the importance of considering cross-species differences when using animal models.

Altered circadian expression of clock genes and clock-regulatory epigenetic modifiers in saliva of children with fetal alcohol spectrum disorders

Prenatal alcohol-exposed (AE) infants and children often demonstrate disrupted sleep patterns, including more frequent awakenings, reduced total sleep time, and more night-to-night sleep variability. Despite the strong connection between sleep patterns and circadian rhythmicity, relatively little is known about circadian rhythm disruptions in individuals with AE. Recently, several reports demonstrated that evaluating the expression patterns of human clock genes in biological fluids could reveal an individual’s circadian phenotype. Human saliva offers an emerging and easily available physiological sample that can be collected non-invasively for core-clock gene transcript analyses. We compared the expression patterns of core-clock genes and their regulatory genes in salivary samples of children aged 6–10 years-old with and without AE during the light cycle between ZT0-ZT11. We isolated the RNA from the samples and measured the expression patterns of core clock genes and clock regulating genes using the human specific primers with quantitative real-time PCR. Analysis of core clock genes expression levels in saliva samples from AE children indicates significantly altered levels in expression of core-clock BMAL1, CLOCK, PER1-3 and CRY1,2, as compared to those in age-matched control children. We did not find any sex difference in levels of clock genes in AE and control groups. Cosinor analysis was used to evaluate the rhythmic pattern of these clock genes, which identified circadian patterns in the levels of core clock genes in the control group but absent in the AE group. The gene expression profile of a salivary circadian biomarker ARRB1 was rhythmic in saliva of control children but was arhythmic in AE children. Altered expression patterns were also observed in clock regulatory genes: NPAS2, NFL3, NR1D1, DEC1, DEC2, and DBP, as well as chromatin modifiers: MLL1, P300, SIRT1, EZH2, HDAC3, and ZR1D1, known to maintain rhythmic expression of core-clock genes. Overall, these findings provide the first evidence that AE disturbs the circadian patten expression of core clock genes and clock-regulatory chromatin modifiers in saliva.

Effect of Alcohol on Clock Synchrony and Tissue Circadian Homeostasis in Mice.

Alcohol use disorder accounts for a growing worldwide health system concern. Alcohol causes damages to various organs, including intestine and liver, primarily involved in its absorption and metabolism. However, alcohol-related organ damage risk varies significantly among individuals, even when they report consuming comparable dosages of alcohol. Factor(s) that may modulate the risk of organ injuries from alcohol consumption could be responsible for inter-individual variations in susceptibility to alcohol-related organ damages. Accumulating evidence suggests disruptions in circadian rhythm can exacerbate alcohol-related organ damages. Here we investigated the interplay between alcohol, circadian rhythm, and key tissue cellular processes at baseline, after a regular and a shift in the light/dark cycle (LCD) in mice. Central/peripheral clock expression of core clock genes (CoClGs) was analyzed. We also studied circadian homeostasis of tissue cellular processes that are involved in damages from alcohol. These experiments reveal that alcohol affects the expression of CoClGs causing a central-peripheral dyssynchrony, amplified by shift in LCD. The observed circadian clock dyssynchrony was linked to circadian disorganization of key processes involved in the alcohol-related damages, particularly when alcohol was combined with LCD. These results offer insights into the mechanisms by which alcohol interacts with circadian rhythm disruption to promote organ injury.

Circadian rhythm disruption upregulating Per1 in mandibular condylar chondrocytes mediating temporomandibular joint osteoarthritis via GSK3β/β-CATENIN pathway

BackgroundTemporomandibular 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.MethodsWe 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 GSK3β/β-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.ResultsJet 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 GSK3β/β-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 GSK3β/β-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 GSK3β/β-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 GSK3β/β-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.

PROVIT-CLOCK: A Potential Influence of Probiotics and Vitamin B7 Add-On Treatment and Metabolites on Clock Gene Expression in Major Depression

Introduction: An increasing body of evidence suggests a strong relationship between gut health and mental state. Lately, a connection between butyrate-producing bacteria and sleep quality has been discussed. The PROVIT study, as a randomized, double-blind, 4-week, multispecies probiotic intervention study, aims at elucidating the potential interconnection between the gut’s metabolome and the molecular clock in individuals with major depressive disorder (MDD). Methods: The aim of the PROVIT-CLOCK study was to analyze changes in core clock gene expression during treatment with probiotic intervention versus placebo in fasting blood and the connection with the serum- and stool-metabolome in patients with MDD (n = 53). In addition to clinical assessments in the PROVIT study, metabolomics analyses with 1H nuclear magnetic resonance spectroscopy (stool and serum) and gene expression (RT-qPCR) analysis of the core clock genes ARNTL, PER3, CLOCK, TIMELESS, NR1D1 in peripheral blood mononuclear cells of fasting blood were performed. Results: The gene expression levels of the clock gene CLOCK were significantly altered only in individuals receiving probiotic add-on treatment. TIMELESS and ARNTL gene expression changed significantly over the 4-week intervention period in both groups. Various positive and negative correlations between metabolites in serum/stool and core clock gene expression levels were observed. Conclusion: Changing the gut microbiome by probiotic treatment potentially influences CLOCK gene expression. The preliminary results of the PROVIT-CLOCK study indicate a possible interconnection between the gut microbiome and circadian rhythm potentially orchestrated by metabolites.

TauFisher predicts circadian time from a single sample of bulk and single-cell pseudobulk transcriptomic data

As the circadian clock regulates fundamental biological processes, disrupted clocks are often observed in patients and diseased tissues. Determining the circadian time of the patient or the tissue of focus is essential in circadian medicine and research. Here we present tauFisher, a computational pipeline that accurately predicts circadian time from a single transcriptomic sample by finding correlations between rhythmic genes within the sample. We demonstrate tauFisher’s performance in adding timestamps to both bulk and single-cell transcriptomic samples collected from multiple tissue types and experimental settings. Application of tauFisher at a cell-type level in a single-cell RNAseq dataset collected from mouse dermal skin implies that greater circadian phase heterogeneity may explain the dampened rhythm of collective core clock gene expression in dermal immune cells compared to dermal fibroblasts. Given its robustness and generalizability across assay platforms, experimental setups, and tissue types, as well as its potential application in single-cell RNAseq data analysis, tauFisher is a promising tool that facilitates circadian medicine and research.

Metabolic plasticity and obesity-associated changes in diurnal postexercise metabolism in mice

BackgroundCircadian disruption is widespread and increases the risk of obesity. Timing of therapeutic interventions may promote coherent and efficient gating of metabolic processes and restore energy homeostasis. AimTo characterize the diurnal postexercise metabolic state in mice and to identify the influence of diet-induced obesity on identified outcomes. MethodsC57BL6/NTac male mice (6 wks of age) were fed a standard chow or high-fat diet for 5 weeks. At week 5, mice were subjected to a 60-min (16 m/min, 5 % incline) running bout (or sham) during the early rest (day) or early active (night) phase. Tissue and serum samples were collected immediately post-exercise (n = 6/group). In vivo glucose oxidation was measured after oral administration of 13C-glucose via 13CO2 exhalation analysis in metabolic cages. Basal and isoproterenol-stimulated adipose tissue lipolysis was assessed ex vivo for 1 h following exercise. ResultsLean mice displayed exercise-timing-specific plasticity in metabolic outcomes, including phase-specificity in systemic glucose metabolism and adipose-tissue-autonomous lipolytic activity depending on time of day. Conversely, obesity impaired temporal postexercise differences in whole-body glucose oxidation, as well as the phase- and exercise-mediated induction of lipolysis in isolated adipose tissue. This obesity-induced alteration in diurnal metabolism, as well as the indistinct response to exercise, was observed concomitant with disruption of core clock gene expression in peripheral tissues. ConclusionsOverall, high-fat fed obese mice exhibit metabolic inflexibility, which is also evident in the diurnal exercise response. Our study provides physiological insight into exercise timing-dependent aspects in the dynamic regulation of metabolism and the influence of obesity on this biology.

The interplay between the circadian clock and abiotic stress responses mediated by ABF3 and CCA1/LHY

Climate change is a global concern for all life on our planet, including humans and plants. Plants' growth and development are significantly affected by abiotic stresses, including adverse temperature, inadequate or excess water availability, nutrient deficiency, and salinity. The circadian clock is a master regulator of numerous developmental and metabolic processes in plants. In an effort to identify new clock-related genes and outputs through bioinformatic analysis, we have revealed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) play a crucial role in regulating a wide range of abiotic stress responses and target ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3), a key transcription factor in the plant hormone Abscisic acid (ABA)-signaling pathway. Specifically, we found that CCA1 and LHY regulate the expression of ABF3 under diel conditions, as well as seed germination under salinity. Conversely, ABF3 controls the expression of core clock genes and orchestrates the circadian period in a stress-responsive manner. ABF3 delivers the stress signal to the central oscillator by binding to the promoter of CCA1 and LHY. Overall, our study uncovers the reciprocal regulation between ABF3 and CCA1/LHY and molecular mechanisms underlying the interaction between the circadian clock and abiotic stress. This finding may aid in developing molecular and genetic solutions for plants to survive and thrive in the face of climate change.

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

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

Exercise alters molecular rhythms in the central nervous system that negatively correlate with depression-like behavior.

Major depressive disorder (MDD) ranks among the top 10 leading causes of death. However, exercise is known to improve depressive symptoms but the mechanism responsible is still unknown. To date, numerous studies have shown that molecular rhythms and exercise are associated with MDD. Thus, we hypothesized that exercise could affect the expression of central nervous system clock genes to improve depressive symptoms. Ninety adult male Sprague-Dawley rats (250 g) were divided into a control Normal Group, an unpredictable chronic mild stress (CMS) treated CMS Group and an Exercise Group, which was intervened by a moderate-intensity exercise training on a treadmill at 2 p.m. every day for 4 weeks after CMS treatment. The open field test, elevated plus maze and forced swim test were employed to test mood-related behaviors. The telemetry recording method recorded voluntary locomotor activity and core body temperature. Expression of core clock genes in the suprachiasmatic nucleus (SCN) was tested by qRT-PCR. Compared with the CMS Group, depressive symptoms were improved in the Exercise Group ( P < 0.05). Moreover, the periodic changes of molecular rhythms in the Exercise Group were close to those of rats in Normal Group. Next, exercise increased oscillations of expression of core clock genes in SCN after CMS treatment, and the amplitudes of core clock gene expression oscillations were negatively correlated with depressive-like behavior. Our findings suggested that exercise could change the expressions of central clock genes in MDD animals, and this effect was positively correlated with the improvement of depressive symptoms by exercise.

Time of day differences in the regulation of glutathione levels in the rat lens.

Evidence in non-ocular tissues indicate that the antioxidant glutathione (GSH) may be regulated in a circadian manner leading to the idea that GSH levels in the lens may also be controlled in a circadian manner to anticipate periods of oxidative stress. Male rat Wistar lenses (6 weeks) were collected every 4 hours over a 24-hour period at 6am, 10am, 2pm, 6pm, 10pm and 2am and quantitative-PCR, western blotting and immunohistochemistry performed to examine the expression of core clock genes and proteins (BMAL1, CLOCK, CRY1-2, PER 1-3) and their subcellular localisation over a 24-hour period. Western blotting of lenses was also performed to examine the expression of NRF2, a transcription factor involved in regulating genes involved in GSH homeostasis and GSH related enzymes (GCLC, GS and GR) over the 24-hour period. Finally, HLPC was used to measure GSH levels in the aqueous humour and lenses every 4 hours over a 24-hour period. The rat lens contains the core molecular components of a circadian clock with the expression of core clock proteins, NRF2 and GSH related enzymes fluctuating over a 24-hour period. BMAL1 expression was highest during the day, with BMAL1 localised to the nuclei at 10am. NRF2 expression remained constant over the 24-hour period, although appeared to move in and out of the nuclei every 4 hours. GSH related enzyme expression tended to peak at the start of night which correlated with high levels of GSH in the lens and lower levels of GSH in the aqueous humour. The lens contains the key components of a circadian clock, and time-of-day differences exist in the expression of GSH and GSH related enzymes involved in maintaining GSH homeostasis. GSH levels in the rat lens were highest at the start of night which represents the active phase of the rat when high GSH levels may be required to counteract oxidative stress induced by cellular metabolism. Future work to directly link the clock to regulation of GSH levels in the lens will be important in determining whether the clock can be used to help restore GSH levels in the lens.

Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

Sleep and circadian rhythmicity as entangled processes serving homeostasis.

Sleep is considered essential for the brain and body. A predominant concept is that sleep is regulated by circadian rhythmicity and sleep homeostasis, processes that were posited to be functionally and mechanistically separate. Here we review and re-evaluate this concept and its assumptions using findings from recent human and rodent studies. Alterations in genesthat are central to circadian rhythmicity affect not only sleep timing but also putative markers of sleep homeostasis such as electroencephalogram slow-wave activity (SWA). Perturbations of sleep change the rhythmicity in the expression of core clock genes in tissues outside the central clock. The dynamics of recovery from sleep loss vary across sleep variables: SWA and immediate early genes show an early response, but the recovery ofnon-rapid eye movement and rapid eye movement sleep follows slower time courses. Changes in the expression of many genes in response to sleep perturbations outlast the effects on SWA and time spent asleep. These findings are difficult to reconcile with the notion that circadian- and sleep-wake-driven processes are mutually independent and that the dynamics of sleep homeostasis are reflected in a single variable. Further understanding of how both sleep and circadian rhythmicity contribute to thehomeostasis of essential physiological variables may benefit from the assessment of multiple sleep and molecular variables over longer time scales.

Preservation of circadian rhythm in hepatocellular cancer

Circadian rhythms are controlled at the cellular level by a molecular clock consisting of several genes/proteins engaged in a transcription-translation-degradation feedback loop. These core clock proteins regulate thousands of tissue-specific genes. Regarding circadian control in neoplastic tissues, reports to date have demonstrated anomalous circadian function in tumor models and cultured tumor cells. We have extended these studies by analyzing circadian rhythmicity genome-wide in a mouse model of liver cancer, in which mice treated with diethylnitrosamine at 15days develop liver tumors by 6months. We injected tumor-bearing and control tumor-free mice with cisplatin every 2h over a 24-h cycle; 2h after each injection mice were sacrificed and gene expression was measured by XR-Seq (excision repair sequencing) assay. Rhythmic expression of several core clock genes was observed in both healthy liver and tumor, with clock genes in tumor exhibiting typically robust amplitudes and a modest phase advance. Interestingly, although normal hepatic cells and hepatoma cancer cells expressed a comparable number of genes with circadian rhythmicity (clock-controlled genes), there was only about 10% overlap between the rhythmic genes in normal and cancerous cells. "Rhythmic in tumor only" genes exhibited peak expression times mainly in daytime hours, in contrast to the more common pre-dawn and pre-dusk expression times seen in healthy livers. Differential expression of genes in tumors and healthy livers across time may present an opportunity for more efficient anticancer drug treatment as a function of treatment time.

Effects of light and circadian clock on the antiviral immune response in zebrafish.

The circadian clock mechanism, which is evolutionarily conserved across various organisms, plays a crucial role in synchronizing physiological responses to external conditions, primarily in response to light availability. By maintaining homeostasis of biological processes and behavior, the circadian clock serves as a key regulator. This biological mechanism also coordinates diurnal oscillations of the immune response during infections. However there is limited information available regarding the influence of circadian oscillation on immune regulation, especially in lower vertebrates like teleost fish. Therefore, the present study aimed to investigate the effects of light and the timing of infection induction on the antiviral immune response in zebrafish. To explore the relationship between the timing of infection and the response activated by viral pathogens, we used a zebrafish model infected with tilapia lake virus (TiLV). Our findings demonstrated that light availability significantly affects the antiviral immune response and the functioning of the molecular clock mechanism during TiLV infection. This is evident through alterations in the expression of major core clock genes and the regulation of TiLV replication and type I IFN pathway genes in the kidney of fish maintained under LD (light-dark) conditions compared to constant darkness (DD) conditions. Moreover, infection induced during the light phase of the LD cycle, in contrast to nocturnal infection, also exhibited similar effects on the expression of genes associated with the antiviral response. This study indicates a more effective mechanism of the zebrafish antiviral response during light exposure, which inherently involves modification of the expression of key components of the molecular circadian clock. It suggests that the zebrafish antiviral response to infection is regulated by both light and the circadian clock.

PPARG stimulation restored lung mRNA expression of core clock, inflammation- and metabolism-related genes disrupted by reversed feeding in male mice.

The circadian rhythm system regulates lung function as well as local and systemic inflammations. The alteration of this rhythm might be induced by a change in the eating rhythm. Peroxisome proliferator-activated receptor gamma (PPARG) is a key molecule involved in circadian rhythm regulation, lung functions, and metabolic processes. We described the effect of the PPARG agonist pioglitazone (PZ) on the diurnal mRNA expression profile of core circadian clock genes (Arntl, Clock, Nr1d1, Cry1, Cry2, Per1, and Per2) and metabolism- and inflammation-related genes (Nfe2l2, Pparg, Rela, and Cxcl5) in the male murine lung disrupted by reversed feeding (RF). In mice, RF disrupted the diurnal expression pattern of core clock genes. It decreased Nfe2l2 and Pparg and increased Rela and Cxcl5 expression in lung tissue. There were elevated levels of IL-6, TNF-alpha, total cells, macrophages, and lymphocyte counts in bronchoalveolar lavage (BAL) with a significant increase in vascular congestion and cellular infiltrates in male mouse lung tissue. Administration of PZ regained the diurnal clock gene expression, increased Nfe2l2 and Pparg expression, and reduced Rela, Cxcl5 expression and IL-6, TNF-alpha, and cellularity in BAL. PZ administration at 7 p.m. was more efficient than at 7 a.m.

Circadian clock disruption in autoimmune thyroiditis.

A vicious cycle between circadian disruption and escalating immune responses has been described in diverse inflammatory disease. The current study aimed to explore the role of circadian clock disruption in autoimmune thyroiditis (AIT). Thirty AIT patients and 30 controls were enrolled and biopsied for thyroid tissues. Alterations of core clock genes expression in AIT thyroid tissues, and its association with serum and tissue inflammatory biomarkers were assessed. For animal studies, C57BL/6J mice administered with porcine thyroglobulin or PBS (as control) combined with adjuvants were sacrificed at four time points to investigate the circadian characteristic of experimental autoimmune thyroiditis (EAT). Light shift (LS) conditions were used to explore the influence of external circadian disturbance on EAT. The expression of clock genes BMAL1 and PER2 was significantly reduced in thyroid tissues from AIT patients and was negatively correlated to levels of thyroid peroxidase antibodies. In mouse models, diurnal fluctuations of proinflammatory cytokines were demonstrated, and further exposing mice to LS led to overproduction of TNF-α, IFN-γ, and anti-thyroglobulin antibodies. Circadian analysis revealed significant oscillations of Bmal1, Clock, Per2, Cry1, Ror, and Rev-erb, which was broadly disturbed in EAT, LS, and EAT + LS groups. This study demonstrates that expression pattern of clock genes was disrupted in AIT thyroid, and chronic circadian disruption may aggravate the inflammatory responses in AIT. Whether maintaining a regular circadian rhythm can alleviate autoimmune thyroid diseases warrants further research.

Circadian clock gene disruption in white blood cells of patients with celiac disease

Clock gene disruption has been reported in inflammatory and autoimmune diseases. Specifically, it has been shown that clock gene expression is down-regulated in intestinal tissue and peripheral blood mononuclear cells of patients with inflammatory bowel disease (IBD). We aimed to determine the systemic expression of the circadian clock genes in newly diagnosed untreated, young patients with celiac disease (CeD). We prospectively enrolled patients younger than 20 years old who underwent diagnostic endoscopic procedures either for CeD diagnosis or due to other gastrointestinal complaints, at the pediatric and adult gastroenterology units, the Tel Aviv Sourasky Medical Center from 8/2016–8/2022. Demographic data, anthropometric parameters, and endoscopic macroscopic and microscopic findings were obtained. Blood samples were obtained to determine tissue transglutaminase (tTG) and core clock gene (CLOCK, BMAL1, PER1, PER2, CRY1, CRY2) expression in white blood cells (WBC). Thirty individuals were analyzed (18 with newly diagnosed CeD and 12 controls). Expression of the clock genes CLOCK, BMAL1, CRY2, PER1 and PER2 was significantly reduced in CeD patients compared to controls, while CRY1 did not differ between the groups. In conclusion, newly diagnosed, untreated, young patients with CeD have reduced clock gene expression in WBC compared to controls. These results suggest that, in CeD, the inflammatory response is associated with systemic disruption of clock gene expression, as is manifested in other inflammatory and autoimmune diseases. ClinicalTrials.gov identifierNCT03662646.