Rhythmic Gene Expression Research Articles

REM Sleep Rebound Following Acute Intermittent Hypoxia in Freely Behaving Rats

Core components of the circadian clock and molecules involved in neuroplasticity elicited by acute intermittent hypoxia (AIH) exhibit 24‐hour rhythms in gene expression in the phrenic/diaphragm motor system. Ongoing investigations of time‐of‐day effects on AIH‐induced motor plasticity must account for changes in time spent across vigilance states since sleep architecture profoundly influences other biological rhythms across the rest‐active cycle. Previous investigations from our group reported a marginal increase in wakefulness during the AIH exposure in unanesthetized rats (10, 5‐min hypoxic episodes; 5 min intervals). Following AIH, we also observed a prolonged increase in slow‐wave sleep lasting hours, but changes in REM were not assessed (Terada et al., J. Appl. Physiol., 2011). Given the significant effects disruptions of the sleep‐wake cycle can have on daily biological rhythms, further characterization of sleep architecture following AIH is needed. In this preliminary analysis, from an extensive study of diaphragm long‐term facilitation (LTF) in rats at opposite stages of the diurnal cycle (mid‐active vs. mid‐rest), we added second set of intracortical EEG leads (ECoG) not used by Terada et al. (2011) to enhance detection of hippocampal theta bursts and sleep spindling. In 8 unanesthetized, instrumented rats exposed to AIH, ECoG, and nuchal EMG signals acquired via radiotelemetry were analyzed in 10‐second epochs using Fast Fourier transformation spectral analysis. Power spectra were averaged in frequency bands: delta (1.5‐4.0Hz), theta (4.0‐10.0Hz), beta (10.0‐30.0 Hz), and sigma (10.0‐14.0Hz). We assessed vigilance state at baseline, during AIH, and 90 minutes post‐AIH; there were significant effects of AIH protocol period (p < 0.05) and sleep state (p<0.001). There were marginal, non‐significant trends towards increased active wakefulness during AIH, with decreased quiet wakefulness, slow‐wave sleep, and REM. Time spent in REM sleep rebounded to levels nearly two‐fold above baseline at 60 and 90 min post‐AIH; there were non‐significant trends towards reduced quiet wakefulness without apparent change in NREM sleep post‐AIH. In this preliminary analysis, we report an acute AIH protocol (3, 5 min hypoxic episodes, 13% O2; 5‐min normoxic intervals) has minimal impact on NREM sleep during or following AIH but increases REM post‐AIH. Additional analyses will be performed during and following AIH during the mid‐active and mid‐rest phases, using the 5‐min AIH protocol and a modified protocol with similar cumulative duration of hypoxia (15, 1 min hypoxic episodes; 1 min intervals) often used in human studies of repetitive AIH as a neurotherapeutic modality to improve breathing and non‐respiratory motor function in humans living with chronic spinal cord injury and ALS. REM sleep is essential for mechanisms of neuroplasticity in neural networks controlling diverse physiological systems. These results warrant future research to delineate the influence REM may play in initiating and maintaining memory in the respiratory system.

Circadian Regulation of Gene Expression and Metabolism in the Liver.

Circadian rhythms are approximately 24-hour cycles of variation in physiological processes, gene expression, and behavior. They result from the interplay of internal biological clocks with daily environmental rhythms, including light/dark and feeding/fasting. Note that 24-hour rhythms of liver metabolic processes have been known for almost 100 years. Modern studies reveal that, like metabolism, hepatic gene expression is highly rhythmic. Genetic or environmental changes can disrupt the circadian rhythms of the liver, leading to metabolic disorders and hepatocellular carcinoma. In this review, we summarize the current understanding of mechanisms regulating rhythmic gene expression in the liver, highlighting the roles of transcription factors that comprise the core clock molecular as well as noncanonical regulators. We emphasize the plasticity of circadian rhythms in the liver as it responds to multiple inputs from the external and internal environments as well as the potential of circadian medicine to impact liver-related diseases.

Daily running enhances molecular and physiological circadian rhythms in skeletal muscle

ObjectiveExercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise. MethodsWildtype C57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise. ResultsWe demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed in mice, and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the murine muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the time-dependent response to moderate daily exercise in mice. ConclusionsCollectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of murine muscle gene expression, which in turn support daily fluctuations in exercise performance.

The hepatocyte insulin receptor is required to program the liver clock and rhythmic gene expression.

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression.

Pleiotropic Roles of Scavenger Receptors in Circadian Retinal Phagocytosis: A New Function for Lysosomal SR-B2/LIMP-2 at the RPE Cell Surface.

The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway

Context The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampō, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. Objective We examine effective crude drugs, which cover at least one or two of Kampō, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. Materials and methods We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 μg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. Results We found that Polygalae Radix (ED50: 24.01 μg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. Discussion and conclusions Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

A peroxisomal heterodimeric enzyme is involved in benzaldehyde synthesis in plants

Benzaldehyde, the simplest aromatic aldehyde, is one of the most wide-spread volatiles that serves as a pollinator attractant, flavor, and antifungal compound. However, the enzyme responsible for its formation in plants remains unknown. Using a combination of in vivo stable isotope labeling, classical biochemical, proteomics and genetic approaches, we show that in petunia benzaldehyde is synthesized via the β-oxidative pathway in peroxisomes by a heterodimeric enzyme consisting of α and β subunits, which belong to the NAD(P)-binding Rossmann-fold superfamily. Both subunits are alone catalytically inactive but, when mixed in equal amounts, form an active enzyme, which exhibits strict substrate specificity towards benzoyl-CoA and uses NADPH as a cofactor. Alpha subunits can form functional heterodimers with phylogenetically distant β subunits, but not all β subunits partner with α subunits, at least in Arabidopsis. Analysis of spatial, developmental and rhythmic expression of genes encoding α and β subunits revealed that expression of the gene for the α subunit likely plays a key role in regulating benzaldehyde biosynthesis.

Old macrophages lose their (circadian) rhythm

Blacher and colleagues have discovered that the circadian patterns of macrophage gene expression and immune function that exist in young mice are disrupted in aging mice. KLF4 was identified as a key transcription factor (TF) regulating rhythmic expression of immune genes, which is lost in old macrophages.

Hypomorphic expression of parathyroid Bmal1 disrupts the internal parathyroid circadian clock and increases parathyroid cell proliferation in response to uremia

The molecular circadian clock is an evolutionary adaptation to anticipate recurring changes in the environment and to coordinate variations in activity, metabolism and hormone secretion. Parathyroid hyperplasia in uremia is a significant clinical challenge. Here, we examined changes in the transcriptome of the murine parathyroid gland over 24 hours and found a rhythmic expression of parathyroid signature genes, such as Casr, Vdr, Fgfr1 and Gcm2. Overall, 1455 genes corresponding to 6.9% of all expressed genes had significant circadian rhythmicity. Biological pathway analysis indicated that the circadian clock system is essential for the regulation of parathyroid cell function. To study this, a novel mouse strain with parathyroid gland-specific knockdown of the core clock gene Bmal1 (PTHcre;Bmal1flox/flox) was created. Dampening of the parathyroid circadian clock rhythmicity was found in these knockdown mice, resulting in abrogated rhythmicity of regulators of parathyroid cell proliferation such as Sp1, Mafb, Gcm2 and Gata3, indicating circadian clock regulation of these genes. Furthermore, the knockdown resulted in downregulation of genes involved in mitochondrial function and synthesis of ATP. When superimposed by uremia, these PTHcre;Bmal1flox/flox mice had an increased parathyroid cell proliferative response, compared to wild type mice. Thus, our findings indicate a role of the internal parathyroid circadian clock in the development of parathyroid gland hyperplasia in uremia.

Comparative analysis of the daily brain transcriptomes of Asian particolored bat

Daily rhythms are found in almost all organisms, and they comprise one of the most basic characteristics of living things. Daily rhythms are generated and mainly regulated by circadian clock. Bats have attracted interest from researchers because of their unique biological characteristics. However, little is known about the molecular underpinnings of daily rhythms in bats. In this study, we used RNA-Seq to uncover the daily rhythms of gene expression in the brains of Asian particolored bats over the 24-h day. Accordingly, four collected time points corresponding to four biological states, rest, sleep, wakefulness, and active, were selected. Several groups of genes with different expression levels in these four states were obtained suggested that different physiological processes were active at various biological states, including drug metabolism, signaling pathways, and the circadian rhythm. Furthermore, downstream analysis of all differentially expressed genes in these four states suggested that groups of genes showed daily rhythms in the bat brain. Especially for Per1, an important circadian clock gene was identified with rhythmic expression in the brain of Asian particolored bat. In summary, our study provides an overview of the brain transcriptomic differences in different physiological states over a 24-h cycle.

Lack of food intake during shift work alters the heart transcriptome and leads to cardiac tissue fibrosis and inflammation in rats

BackgroundMany epidemiological studies revealed that shift work is associated with an increased risk of a number of pathologies, including cardiovascular diseases. An experimental model of shift work in rats has additionally been shown to recapitulate aspects of metabolic disorders observed in human shift workers, including increased fat content and impaired glucose tolerance, and used to demonstrate that restricting food consumption outside working hours prevents shift work-associated obesity and metabolic disturbance. However, the way distinct shift work parameters, such as type of work, quantity, and duration, affect cardiovascular function and the underlying mechanisms, remains poorly understood. Here, we used the rat as a model to characterize the effects of shift work in the heart and determine whether they can be modulated by restricting food intake during the normal active phase.ResultsWe show that experimental shift work reprograms the heart cycling transcriptome independently of food consumption. While phases of rhythmic gene expression are distributed across the 24-h day in control rats, they are clustered towards discrete times in shift workers. Additionally, preventing food intake during shift work affects the expression level of hundreds of genes in the heart, including genes encoding components of the extracellular matrix and inflammatory markers found in transcriptional signatures associated with pressure overload and cardiac hypertrophy. Consistent with this, the heart of shift worker rats not eating during work hours, but having access to food outside of shift work, exhibits increased collagen 1 deposition and displays increased infiltration by immune cells. While maintaining food access during shift work has less effects on gene expression, genes found in transcriptional signatures of cardiac hypertrophy remain affected, and the heart of shift worker rats exhibits fibrosis without inflammation.ConclusionsTogether, our findings unraveled differential effects of food consumption on remodeled transcriptional profiles of the heart in shift worker rats. They also provide insights into how shift work affects cardiac function and suggest that some interventions aiming at mitigating metabolic disorders in shift workers may have adverse effects on cardiovascular diseases.

Wheel-Running Facilitates Phase Advances in Locomotor and Peripheral Circadian Rhythm in Social Jet Lag Model Mice.

The circadian clock maintains our health by controlling physiological functions. Social jet lag is one factor that can disrupt the body clock. This is caused by the difference in sleeping hours between weekdays when we live according to social time and holidays when we live according to our body clock. The body clock can be altered by exercise, nutrition, and stress, and several studies have reported that these factors can be used to improve a disturbed body clock. Here we focused on exercise and examined whether continuous wheel-running could improve the disordered body clock in a mouse model that mimics social jet lag. The results showed that the wheel-running exercise group showed faster synchronization of the onset of activities on weekdays which had been delayed by social jet lag and the results were even more pronounced in the high-fat diet feeding condition. Also, when the expression rhythms of the clock genes were examined, they experienced a sudden time shift in the advance light condition or social jet lag condition, it was found that the wheel-running group had a higher ability to adapt to the advance direction. Thus, it is possible that the effective inclusion of exercise in human, especially those who eat high-fat foods, life can improve the disordered body clock in terms of social jet lag.

Chronic Treatment with Metformin Has No Disrupting Effect on the Hepatic Circadian Clock in Mice.

Background and Objectives: The antidiabetic agent metformin is known to activate AMP-activated protein kinase (AMPK) in various tissues. Because AMPK can modulate intracellular circadian clocks through regulating the stability of clock components, a single dose of metformin has been reported to affect circadian clocks in the peripheral tissues. In this study, therefore, we investigated whether chronic treatment with metformin causes the impairment of circadian clocks, especially if given at an inappropriate time. Materials and Methods: Non-diabetic C57BL/6J mice were allowed access to food only during 4 h at the beginning of the dark period, and repeatedly i.p. injected with a nearly maximum non-toxic dose of metformin, once daily either at 4 h after the beginning of the dark period or at the beginning of the light period. Diabetic ob/ob mice were given free access to food and treated with metformin in drinking water. Results: Under the controlled feeding regimen, 8-day treatment with metformin did not alter the mRNA expression rhythms of clock genes in both liver and adipose tissue of C57BL/6J mice, regardless of dosing time. In addition, chronic treatment with metformin for 2 weeks affected hepatic AMPK activation rhythm but did not disrupt the circadian clocks in the liver and adipose tissues of the ob/ob mice. Conclusions: These results mitigate concerns that treatment with metformin impairs peripheral circadian clocks, although confirmation is needed in humans.

Effects of varying temperature on rhythmic expression of abiotic stress-responding genes in Tibetan hulless barley

Effects of varying temperature on rhythmic expression of abiotic stress-responding genes in Tibetan hulless barley

Oak extracts modulate circadian rhythms of clock gene expression in vitro and wheel-running activity in mice.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which coordinates the circadian rhythm and controls locomotor activity rhythms. In addition to SCN cells, the peripheral tissues and embryonic fibroblasts also have clock genes, such as Per1/2 and Bmal1, which generate the transcriptional-translational feedback loop to produce an approximately 24-h cycle. Aging adversely affects the circadian clock system and locomotor functions. Oak extract has been reported to improve age-related physiological changes. However, no study has examined the effect of oak extract on the circadian clock system. We examined the effects of oak extract and its metabolites (urolithin A [ULT] and ellagic acid [EA]) on clock gene expression rhythms in mouse embryonic fibroblasts (MEFs) and SCN. Furthermore, locomotor activity rhythm was assessed in young and aged mice. Chronic treatment with EA and ULT delayed the phase of PER2::LUC rhythms in SCN explants, and ULT prolonged the period of PER2::LUC rhythms in MEFs in a dose-dependent manner and increased the amplitude of PER2::LUC rhythms in MEFs, though only at low concentrations. Acute treatment with ULT affected the phase of PER2::LUC rhythms in MEFs depending on the concentration and timing of the treatment. In addition, oak extract prolonged the activity time of behavioral rhythms in old mice and tended to increase daily wheel-running revolutions in both young and old mice. These results suggest that oak extract is a novel modulator of the circadian clock in vitro and in vivo. The online version contains supplementary material available at 10.1007/s41105-021-00365-2.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Cortical diurnal rhythms remain intact with microglial depletion

Microglia are subject to change in tandem with the endogenously generated biological oscillations known as our circadian rhythm. Studies have shown microglia harbor an intrinsic molecular clock which regulates diurnal changes in morphology and influences inflammatory responses. In the adult brain, microglia play an important role in the regulation of condensed extracellular matrix structures called perineuronal nets (PNNs), and it has been suggested that PNNs are also regulated in a circadian and diurnal manner. We sought to determine whether microglia mediate the diurnal regulation of PNNs via CSF1R inhibitor dependent microglial depletion in C57BL/6J mice, and how the absence of microglia might affect cortical diurnal gene expression rhythms. While we observe diurnal differences in microglial morphology, where microglia are most ramified at the onset of the dark phase, we do not find diurnal differences in PNN intensity. However, PNN intensity increases across many brain regions in the absence of microglia, supporting a role for microglia in the regulation of PNNs. Here, we also show that cortical diurnal gene expression rhythms are intact, with no cycling gene changes without microglia. These findings demonstrate a role for microglia in the maintenance of PNNs, but not in the maintenance of diurnal rhythms.

Diurnal RNAPII-tethered chromatin interactions are associated with rhythmic gene expression in rice

BackgroundThe daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear.ResultsHere, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs.ConclusionOur findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Identification of circadian clock genes as regulators of immune infiltration in Hepatocellular Carcinoma.

Background: Multiple studies have reported that the immune system is under the control of a circadian clock, especially in cancers, but how circadian clock genes shape tumor immune cell infiltration in hepatocellular carcinoma (HCC) remains unclear.Methods: The rhythmicity of circadian clock genes was investigated using the GETx database. The expression and methylation level of circadian clock genes in HCC and paracancerous was evaluated using the GETx and TCGA databases. The differential expression of circadian clock genes in HCC was analyzed using the “limma” package of the R 4.0.4 software. The prognosis of each circadian clock gene was accessed by Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. Quantitative real-time PCR and immunohistochemistry (IHC) was carried out to confirm the results. The relationship between circadian rhythm and immune infiltration in HCC was evaluated using the TIMER database and the CIBERSORT algorithm.Results: In addition to RORA, RORB, and ARNTL2, there was a rhythmic expression of other circadian clock genes in liver tissue. The correlation between the expression of circadian clock genes differed when comparing HCC and liver tissue. HCC patients who express low levels of PER-1and CRY2 had a poor overall survival (OS). In contrast, patients with higher expression of NPAS2 had a poor prognosis. In HCC, the expression of the PER-1, CRY2, and NPAS2 genes was closely related to immune infiltration.Conclusion: Our study indicated the disruption of the expression of circadian clock-regulated genes in HCC and identified PER-1, CRY2, and NPAS2 as independent predictors of survival. These genes may be applied as candidate molecular targets for diagnosis and therapy of HCC.

Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model.

The circadian clock in murine articular cartilage is a critical temporal regulatory mechanism for tissue homeostasis and osteoarthritis. However, translation of these findings into humans has been hampered by the difficulty in obtaining circadian time series human cartilage tissues. As such, a suitable model is needed to understand the initiation and regulation of circadian rhythms in human cartilage.Methods: We used a chondrogenic differentiation protocol on human embryonic stem cells (hESCs) as a proxy for early human chondrocyte development. Chondrogenesis was validated using histology and expression of pluripotency and differentiation markers. The molecular circadian clock was tracked in real time by lentiviral transduction of human clock gene luciferase reporters. Differentiation-coupled gene expression was assessed by RNAseq and differential expression analysis.Results: hESCs lacked functional circadian rhythms in clock gene expression. During chondrogenic differentiation, there was an expected reduction of pluripotency markers (e.g., NANOG and OCT4) and a significant increase of chondrogenic genes (SOX9, COL2A1 and ACAN). Histology of the 3D cartilage pellets at day 21 showed a matrix architecture resembling human cartilage, with readily detectable core clock proteins (BMAL1, CLOCK and PER2). Importantly, the circadian clocks in differentiating hESCs were activated between day 11 (end of the 2D stage) and day 21 (10 days after 3D differentiation) in the chondrogenic differentiation protocol. RNA sequencing revealed striking differentiation coupled changes in the expression levels of most clock genes and a range of clock regulators.Conclusions: The circadian clock is gradually activated through a differentiation-coupled mechanism in a human chondrogenesis model. These findings provide a human 3D chondrogenic model to investigate the role of the circadian clock during normal homeostasis and in diseases such as osteoarthritis.