Circadian Expression Patterns Research Articles

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues

Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroid hormones, exhibit a phase shift in locomotor activity, suggesting altered circadian rhythm. To better understand the physiological and molecular basis of this phenotype, we used Dio3+/+ and Dio3-/- mice of both sexes at different zeitgeber times (ZTs) and analyzed corticosterone and thyroxine (T4) levels, hypothalamic, hepatic, and adipose tissue expression of clock genes, as well as genes involved in the thyroid hormone action or physiology of liver and adipose tissues. Wild type mice exhibited sexually dimorphic circadian patterns of genes controlling thyroid hormone action, including Dio3. Dio3-/- mice exhibited altered hypothalamic expression of several clock genes at ZT12, but did not disrupt the overall circadian profile. Expression of clock genes in peripheral tissues was not disrupted by Dio3 deficiency. However, Dio3 loss in liver and adipose tissues disrupted circadian profiles of genes that determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes in serum T4 and corticosterone as a result of DIO3 deficiency. The circadian alterations manifested sexual dimorphism. Most notable, the time curve of serum corticosterone was flattened in Dio3-/- females. We conclude that Dio3 exhibits circadian variations, influencing the circadian rhythmicity of thyroid hormone action and physiology in liver and adipose tissues in a sex-specific manner. Circadian disruptions in tissue physiology may then contribute to the metabolic phenotypes of DIO3-deficient mice.

Deficiency of circadian clock gene Bmal1 exacerbates noncanonical inflammasome-mediated pyroptosis and lethality via Rev-erbα-C/EBPβ-SAA1 axis

Circadian arrhythmia has been linked to increased susceptibility to multiple inflammatory diseases, such as sepsis. However, it remains unclear how disruption of the circadian clock modulates molecular aspects of innate immune responses, including inflammasome signaling. Here, we examined the potential role of the circadian clock in inflammasome-mediated responses through myeloid-specific deletion of BMAL1, a master circadian clock regulator. Intriguingly, Bmal1 deficiency significantly enhanced pyroptosis of macrophages and lethality of mice under noncanonical inflammasome-activating conditions but did not alter canonical inflammasome responses. Transcriptome analysis of enriched peritoneal myeloid cells revealed that Bmal1 deficiency led to a marked reduction in Rev-erbα expression at steady state and a significant increase in serum amyloid A1 (SAA1) expression upon poly(I:C) stimulation. Notably, we found that the circadian regulator Rev-erbα is critical for poly(I:C)- or interferon (IFN)-β-induced SAA1 production, resulting in the circadian oscillation pattern of SAA1 expression in myeloid cells. Furthermore, exogenously applied SAA1 markedly increased noncanonical inflammasome-mediated pyroptosis of macrophages and lethality of mice. Intriguingly, our results revealed that type 1 IFN receptor signaling is needed for poly(I:C)- or IFN-β-induced SAA1 production. Downstream of the type 1 IFN receptor, Rev-erbα inhibited the IFN-β-induced association of C/EBPβ with the promoter region of Saa1, leading to the reduced transcription of Saa1 in macrophages. Bmal1-deficient macrophages exhibited enhanced binding of C/EBPβ to Saa1. Consistently, the blockade of Rev-erbα by SR8278 significantly increased poly(I:C)-stimulated SAA1 transcription and noncanonical inflammasome-mediated lethality in mice. Collectively, our data demonstrate a potent suppressive effect of the circadian clock BMAL1 on the noncanonical inflammasome response via the Rev-erbα-C/EBPβ-SAA1 axis.

Comprehensive analysis of circadian protein expression patterns in healthy adults

Comprehensive analysis of circadian protein expression patterns in healthy adults

Circadian protein expression patterns in healthy young adults

ObjectivesTo explore how the blood plasma proteome fluctuates across the 24-hour day and identify a subset of proteins that show endogenous circadian rhythmicity. MethodsPlasma samples from 17 healthy adults were collected hourly under controlled conditions designed to unmask endogenous circadian rhythmicity; in a subset of 8 participants, we also collected samples across a day on a typical sleep-wake schedule. A total of 6916 proteins were analyzed from each sample using the SomaScan aptamer-based multiplexed platform. We used differential rhythmicity analysis based on a cosinor model with mixed effects to identify a subset of proteins that showed circadian rhythmicity in their abundance. ResultsOne thousand and sixty-three (15%) proteins exhibited significant daily rhythmicity. Of those, 431 (6.2%) proteins displayed consistent endogenous circadian rhythms on both a sleep-wake schedule and under controlled conditions: it included both known and novel proteins. When models were fitted with two harmonics, an additional 259 (3.7%) proteins exhibited significant endogenous circadian rhythmicity, indicating that some rhythmic proteins cannot be solely captured by a simple sinusoidal model. Overall, we found that the largest number of proteins had their peak levels in the late afternoon/evening, with another smaller group peaking in the early morning. ConclusionsThis study reveals that hundreds of plasma proteins exhibit endogenous circadian rhythmicity in humans. Future analyses will likely reveal novel physiological pathways regulated by circadian clocks and pave the way for improved diagnosis and treatment for patients with circadian disorders and other pathologies. It will also advance efforts to include knowledge about time-of-day, thereby incorporating circadian medicine into personalized medicine.

Novel Insights into the Circadian Rhythms Based on Long Noncoding and Circular RNA Profiling.

Circadian rhythm disorders pose major risks to human health and animal production activity, and the hypothalamus is the center of circadian rhythm regulation. However, the epigenetic regulation of circadian rhythm based on farm animal models has been poorly investigated. We collected chicken hypothalamus samples at seven time points in one light/dark cycle and performed long noncoding RNA (lncRNA), circular RNA (circRNA), and mRNA sequencing to detect biomarkers associated with circadian rhythm. We enhanced the comprehensive expression profiling of ncRNAs and mRNAs in the hypothalamus and found two gene sets (circadian rhythm and retinal metabolism) associated with the light/dark cycle. Noncoding RNA networks with circadian expression patterns were identified by differential expression and circadian analysis was provided that included 38 lncRNAs, 15 circRNAs, and 200 candidate genes. Three lncRNAs (ENSGALT00000098661, ENSGALT00000100816, and MSTRG.16980.1) and one circRNA (novel_circ_010168) in the ncRNA-mRNA regulatory network were identified as key molecules influencing circadian rhythm by regulating AOX1 in retinal metabolism. These ncRNAs were predicted to be related to pernicious anemia, gonadal, eye disease and other disorders in humans. Together, the findings of this study provide insights into the epigenetic mechanisms of circadian rhythm and reveal AOX1 as a promising target of circadian rhythm regulation.

Molecular characterization and dietary regulation of the forkhead box O3 (foxo3a and foxo3b) in grass carp (Ctenopharyngodon idella)

The forkhead box O3 (foxo3) regulates the target genes expression involved in regulating cell cycle and biological metabolism. In the study, foxo3a and foxo3b of grass carp (Ctenopharyngodon idella) were cloned. The foxo3a cDNA have a complete open reading frame (ORF) spanning 1431 bp, which encodes a polypeptide of 476 amino acids. The foxo3b cDNA contains a complete ORF encompassing 1959 bp, encoding a protein consisting of 652 amino acid residues. The analysis of genetic codon preference and phylogenetic relationships revealed that the foxo3a and foxo3b genes in grass carp are closely related to the counterparts of Danio rerio and Megalobrama amblycephala. Tissue expression analysis showed that foxo3a expression reached the maximum in the brain while foxo3b expression in the muscle being the highest. Circadian expression pattern showed that, foxo3a expression presented a periodic upward trend from 3:00–9:00, 12:00–18:00 and 21:00–24:00, and the maximum level of foxo3b was detected at 9:00. Moreover, dietary regulation of foxo3a and foxo3b expression were performed in our research. In the feeding trial with different protein sources, the groups of fishmeal, rapeseed meal and soybean meal are in contrast to each other. Foxo3a expression was significantly higher in fish fed with rapeseed meal compared to those fed with soybean meal and fishmeal at 14th and 21st days of the experiment. Foxo3b expression was highest in the fishmeal group, followed by rapeseed meal, while it was lowest in soybean meal at 14th and 28th days. Analysis of the effect of dietary alanine-glutamine dipeptide on foxo3 expression in grass carp revealed that the expression of foxo3a gradually increased with increasing dietary Ala-Gln dipeptide concentration and reached its highest level at a concentration of 1.5%. Conversely, foxo3b expression significantly decreased when the dipeptide concentration reached 2%. The findings of this study may provide new insights into the molecular properties, spatiotemporal dynamics, and nutritional regulation of foxo3 in fish.

The WD40 domain-containing protein Ehd5 positively regulates flowering in rice (Oryza sativa).

Heading date (flowering time), which greatly influences regional and seasonal adaptability in rice (Oryza sativa), is regulated by many genes in different photoperiod pathways. Here, we characterized a heading date gene, Early heading date 5 (Ehd5), using a modified bulked segregant analysis method. The ehd5 mutant showed late flowering under both short-day and long-day conditions, as well as reduced yield, compared to the wild type. Ehd5, which encodes a WD40 domain-containing protein, is induced by light and follows a circadian rhythm expression pattern. Transcriptome analysis revealed that Ehd5 acts upstream of the flowering genes Early heading date 1 (Ehd1), RICE FLOWERING LOCUS T 1 (RFT1), and Heading date 3a (Hd3a). Functional analysis showed that Ehd5 directly interacts with Rice outermost cell-specific gene 4 (Roc4) and Grain number, plant height, and heading date 8 (Ghd8), which might affect the formation of Ghd7-Ghd8 complexes, resulting in increased expression of Ehd1, Hd3a and RFT1. Taken together, these results demonstrate that Ehd5 functions as a positive regulator of rice flowering and provide insight into the molecular mechanisms underlying heading date.

Soybean Calmodulin-Binding Transcription Activators, GmCAMTA2 and GmCAMTA8, Coordinate the Circadian Regulation of Developmental Processes and Drought Stress Responses.

The calmodulin-binding transcription activators (CAMTAs) mediate transcriptional regulation of development, growth, and responses to various environmental stresses in plants. To understand the biological roles of soybean CAMTA (GmCAMTA) family members in response to abiotic stresses, we characterized expression patterns of 15 GmCAMTA genes in response to various abiotic stresses. The GmCAMTA genes exhibited distinct circadian regulation expression patterns and were differently expressed in response to salt, drought, and cold stresses. Interestingly, the expression levels of GmCAMTA2, GmCAMTA8, and GmCAMTA12 were higher in stem tissue than in other soybean tissues. To determine the roles of GmCAMTAs in the regulation of developmental processes and stress responses, we isolated GmCAMTA2 and GmCAMTA8 cDNAs from soybean and generated Arabidopsis overexpressing transgenic plants. The GmCAMTA2-OX and GmCAMTA8-OX plants showed hypersensitivity to drought stress. The water in the leaves of GmCAMTA2-OX and GmCAMTA8-OX plants was lost faster than that in wild-type (WT) plants under drought-stress conditions. In addition, stress-responsive genes were down-regulated in the GmCAMTA2-OX and GmCAMTA8-OX plants under drought stress conditions compared to WT plants. Our results suggest that GmCAMTA2 and GmCAMTA8 genes are regulated by circadian rhythms and function as negative regulators in development and drought stress responses.

Habitual nappers and non-nappers differ in circadian rhythms of LIPE expression in abdominal adipose tissue explants.

Napping is a widespread practice worldwide and has in recent years been linked to increased abdominal adiposity. Lipase E or LIPE encodes the protein hormone-sensitive lipase (HSL), an enzyme that plays an important role in lipid mobilization and exhibits a circadian expression rhythm in human adipose tissue. We hypothesized that habitual napping may impact the circadian expression pattern of LIPE, which in turn may attenuate lipid mobilization and induce abdominal fat accumulation. Abdominal adipose tissue explants from participants with obesity (n = 17) were cultured for a 24-h duration and analyzed every 4 h. Habitual nappers (n = 8) were selected to match non-nappers (n = 9) in age, sex, BMI, adiposity, and metabolic syndrome traits. Circadian LIPE expression rhythmicity was analyzed using the cosinor method. Adipose tissue explants exhibited robust circadian rhythms in LIPE expression in non-nappers. In contrast, nappers had a flattened rhythm. LIPE amplitude was decreased in nappers as compared with non-nappers (71% lower). The decrease in amplitude among nappers was related to the frequency of napping (times per week) where a lower rhythm amplitude was associated with a higher napping frequency (r = -0.80; P = 0.018). Confirmatory analyses in the activity of LIPE's protein (i.e., HSL) also showed a significant rhythm in non-nappers, whereas significance in the activity of HSL was lost among nappers. Our results suggest that nappers display dysregulated circadian LIPE expression as well as dysregulated circadian HSL activity, which may alter lipid mobilization and contribute to increased abdominal obesity in habitual nappers.

The evolution and diurnal expression patterns of photosynthetic pathway genes of the invasive alien weed, Mikania micrantha

Mikania micrantha is a fast-growing global invasive weed species that causes severe damage to natural ecosystems and very large economic losses of forest and crop production. It has advantages in photosynthesis, including a similar net photosynthetic rate as C4 plants and a higher carbon fixation capacity. We used a combination of genomics and transcriptomics approaches to study the evolutionary mechanisms and circadian expression patterns of M. micrantha. In M. micrantha, 16 positive selection genes focused on photoreaction and utilization of photoassimilates. In different tissues, 98.1% of the genes associated with photoresponse had high expression in stems, and more than half of the genes of the C4 cycle had higher expression in stems than in leaves. In stomatal opening and closing, 2 genes of carbonic anhydrase (CAs) had higher expression at 18:00 than at 8:00, and the slow anion channel 1 (SLAC1) and high-leaf-temperature 1 kinase (HT1) genes were expressed at low levels at 18:00. In addition, genes associated with photosynthesis had higher expression levels at 7:00 and 17:00. We hypothesized that M. micrantha may undergo photosynthesis in the stem and flower organs and that some stomata of the leaves were opening at night by CO2 signals. In addition, its evolution may attenuate photoinhibition at high light intensities, and enhance more efficient of photosynthesis during low light intensity. And the tissue-specific photosynthetic types and different diurnal pattern of photosynthetic-related genes may contribute to its rapid colonization of new habitats of M. micrantha.

Transcriptional Mechanisms of Thermal Acclimation in Prochlorococcus.

Low temperature limits the growth and the distribution of the key oceanic primary producer Prochlorococcus, which does not proliferate above a latitude of ca. 40°. Yet, the molecular basis of thermal acclimation in this cyanobacterium remains unexplored. We analyzed the transcriptional response of the Prochlorococcus marinus strain MIT9301 in long-term acclimations and in natural Prochlorococcus populations along a temperature range enabling its growth (17 to 30°C). MIT9301 upregulated mechanisms of the global stress response at the temperature minimum (17°C) but maintained the expression levels of genes involved in essential metabolic pathways (e.g., ATP synthesis and carbon fixation) along the whole thermal niche. Notably, the declining growth of MIT9301 from the optimum to the minimum temperature was coincident with a transcriptional suppression of the photosynthetic apparatus and a dampening of its circadian expression patterns, indicating a loss in their regulatory capacity under cold conditions. Under warm conditions, the cellular transcript inventory of MIT9301 was strongly streamlined, which may also induce regulatory imbalances due to stochasticity in gene expression. The daytime transcriptional suppression of photosynthetic genes at low temperature was also observed in metatranscriptomic reads mapping to MIT9301 across the global ocean, implying that this molecular mechanism may be associated with the restricted distribution of Prochlorococcus to temperate zones. IMPORTANCE Prochlorococcus is a major marine primary producer with a global impact on atmospheric CO2 fixation. This cyanobacterium is widely distributed across the temperate ocean, but virtually absent at latitudes above 40° for yet unknown reasons. Temperature has been suggested as a major limiting factor, but the exact mechanisms behind Prochlorococcus thermal growth restriction remain unexplored. This study brings us closer to understanding how Prochlorococcus functions under challenging temperature conditions, by focusing on its transcriptional response after long-term acclimation from its optimum to its thermal thresholds. Our results show that the drop in Prochlorococcus growth rate under cold conditions was paralleled by a transcriptional suppression of the photosynthetic machinery during daytime and a loss in the organism's regulatory capacity to maintain circadian expression patterns. Notably, warm temperature induced a marked shrinkage of the organism's cellular transcript inventory, which may also induce regulatory imbalances in the future functioning of this cyanobacterium.

Abstract 1456: An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor

Abstract The circadian clock lies in the suprachiasmatic nucleus of the hypothalamus and is regulated directly by the daily light/dark cycles, generating rhythmic expression of core circadian-controlled genes (CCCG) some belonging to the nuclear receptors (NR) superfamily. Recent advances show that the disruption of the circadian clock is identified as a risk factor for endocrine cancers, such as prostate cancer which is the most common hormone-related male cancer globally. Whether the prostate gland has its own circadian expression of CCCG and NR, and how the dysregulated circadian clock would influence the fate of the prostate tumors is not well studied. Thus, the present study aims to determine the circadian expression patterns of CCCG and NR in normal mouse prostate and further validate their disrupted circadian expression in a transgenic mouse adenocarcinoma prostate cancer model (TGMAP) with neuroendocrine phenotypes and suppressed androgen gene expression (AR), over the 12 hours light/dark cycle. We establish the rhythmic expression patterns of multiple CCCG and the full class of NR by quantitative polymerase-chain reaction analysis in normal mouse prostate (C57BL/6 strain) and advanced prostate tumors derived from TGMAP in a 12-hours light/dark cycle with 4-hours interval. Our results showed that 30 out of 49 NR and several CCCG follow rhythmic expression patterns, followed by 10 NR with unidentifiable expression patterns and 9 NR showing very low expression in the mouse prostate tissue. The circadian expression of these transcription factors including the AR gene, could be divided into 2 categories with single peak or double peaks at specific time points throughout the light/dark cycle, linking them to the regulating function of the circadian clock in the normal prostate gland. In the advanced prostate tumors derived from TGMAP model that exhibit androgen independence and neuroendocrine trans-differentiation phenotype, the rhythmic cycles of the NR and CCCG showed disruption, and several transcription factors were upregulated in overall circadian expression in comparison to the normal prostate tissue. In summary, this study establishes two categories of rhythmic expression patterns in normal mouse prostate tissue that link the circadian expression of these transcription factors in the peripheral clock to that of androgen signaling axis that is responsible for the maintenance of prostate gland while disruption of such circadian expression patterns was demonstrated in the TGMAP tumors of which the AR signaling was either lost or disrupted. The upregulated circadian expression patterns as revealed in TGMAP model also provide insight into the significance of the dysregulation of circadian rhythm as regulated by CCCG and NR in the advanced prostate cancer. (Study supported by a Direct Grant for Research 2021-2022 Project No. 2021.062, CUHK) Citation Format: Ria Chopra, Franky Leung Chan. An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1456.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

Is obstructive sleep apnea a circadian rhythm disorder?

Obstructive sleep apnea is the most common sleep-related breathing disorder worldwide and remains underdiagnosed. Its multiple associated comorbidities contribute to a decreased quality of life and work performance as well as an increased risk of death. Standard treatment seems to have limited effects on cardiovascular and metabolic aspects of the disease, emphasising the need for early diagnosis and additional therapeutic approaches. Recent evidence suggests that the dysregulation of circadian rhythms, processes with endogenous rhythmicity that are adjusted to the environment through various cues, is involved in the pathogenesis of comorbidities. In patients with obstructive sleep apnea, altered circadian gene expression patterns have been demonstrated. Obstructive respiratory events may promote circadian dysregulation through the effects of sleep disturbance and intermittent hypoxia, with subsequent inflammation and disruption of neural and hormonal homeostasis. In this review, current knowledge on obstructive sleep apnea, circadian rhythm regulation, and circadian rhythm sleep disorders is summarised. Studies that connect obstructive sleep apnea to circadian rhythm abnormalities are critically evaluated. Furthermore, pathogenetic mechanisms that may underlie this association, most notably hypoxia signalling, are presented. A bidirectional relationship between obstructive sleep apnea and circadian rhythm dysregulation is proposed. Approaching obstructive sleep apnea as a circadian rhythm disorder may prove beneficial for the development of new, personalised diagnostic, therapeutic and prognostic tools. However, further studies are needed before the clinical approach to obstructive sleep apnea includes targeting the circadian system.

Repetitive mild traumatic brain injury alters central and peripheral clock gene expression in the adolescent rat

Mild traumatic brain injury (mTBI) or concussion is a common injury worldwide leading to substantial medical costs and a high burden on society. In adolescents, falls and sports related trauma are often the causes of mTBI. Importantly, critical brain growth and development occurs during this sensitive period making the prospect of a brain injury a worrying phenomenon. Upwards of 70% of patients report circadian disruption following these injuries and this has been shown to impede recovery. Therefore, we sought to determine if core circadian clock gene expression was disrupted in rat model of repetitive mTBI (RmTBI). Male and female adolescent rats (n = 129) received sham or RmTBI. The animals were then euthanized at different times throughout the day and night. Tissue from the hypothalamus, cerebellum, hippocampus, liver, and small intestine were evaluated for the expression of per1, per2, cry1, clock, bmal1 and rev-erb-α. We found most clock genes varied across the day/night indicating circadian expression patterns. In the hypothalamus we found RmTBI altered the expression of cry1 and bmal1 in addition to sex differences in per2, cry1, clock, bmal1 and rev-erb- α. In the cerebellum, per1, per2, cry1, clock, bmal1 and rev-erb-α rhythms were all knocked out by RmTBI in addition to sex differences in cry1, clock and bmal1 expression. We also detected a significant decrease in overall expression of all clock genes in males in the middle of the night. In the hippocampus we found that RmTBI changed the rhythm of rev-erb-α expression in addition to sex differences in bmal1 expression. In the liver we detected strong rhythms in all genes examined, however only per2 expression was knocked out by RmTBI, in addition we also detected sex differences in per2 and cry1. We also detected an overall decrease in female clock gene expression in the early night. In the small intestine, RmTBI altered cry1 expression and there were sex differences in rev-erb-α. These results indicate that RmTBI alters core circadian clock gene expression in the central and peripheral nervous system in a time, tissue and sex dependent manner. This may be disrupting important phase relationships between the brain and peripheral nervous system and contributing to post-injury symptomology and also highlights the importance for time and sex dependent assessment of injury outcomes.

Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair.

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.

Structural characteristics of orexin receptor type 2 in Pacific abalone and its diurnal expression pattern after fasting and re-feeding

Pacific abalone (Haliotis discus hannai) is a typical nocturnal organism. To examine the circadian expression pattern of orexin receptor type 2 (OX2R) and its potential effect on the feeding behavior of abalone, the coding region sequence of OX2R that is 1215 bp in length and encodes 404 amino acids was first cloned using the rapid amplification of cDNA ends technique. A recombinant expression vector was constructed for H. discus hannai based on the OX2R protein, obtaining a recombinant protein with a molecular weight of 46 kDa. Polyclonal antibody was prepared with the purified recombinant protein used as the antigen, and the antibody titer of ≥512 K was detected by enzyme-linked immunosorbent assay. The expression levels of OX2R determined using western blotting were highest in the intestinal tract (P < 0.05), but they were not significantly different from the levels in the pedal. Immunofluorescence experiments affirmed that OX2R was widely expressed in the columnar cells of the intestinal mucosal epithelium. To further account for the relationship between the onset of feeding behavior and the expression level of OX2R in abalone, the circadian expression characteristics of OX2R were analyzed by dissecting the intestinal tissues after three days of normal feeding and fasting and following the refeeding treatment. The expression levels of OX2R in the refeeding group were significantly higher than those in the normal feeding and fasting groups at any time point (P < 0.05). The cosine curve analysis revealed that the expression levels of OX2R lost rhythmicity after fasting. Based on the quantification of behavioral data for abalone after fasting and refeeding, the cumulative movement distance and movement duration in each group followed a significant cosine rhythm (P < 0.05), which is consistent with abalone's nocturnal ecological habits. However, the cumulative movement distance and movement duration in the fasting group were significantly lower than those in the normal feeding and refeeding groups (P < 0.05). The peak phases of the cumulative movement distance and movement duration in the refeeding group (ZT08:22 and ZT08:44) shifted backward compared to the normal feeding group (ZT07:33 and ZT07:39). The above results first identified the structural characteristics and circadian expression patterns of OX2R in the marine mollusk abalone, which may reveal the molecular mechanism behind the generation of a feeding rhythm in marine nocturnal organisms and serve as a tool helping to maintain the diversity of marine benthic resources.

Circadian Genes Expression Patterns in Disorders Due to Enzyme Deficiencies in the Heme Biosynthetic Pathway.

Heme is a member of the porphyrins family of cyclic tetrapyrroles and influences various cell processes and signalling pathways. Enzyme deficiencies in the heme biosynthetic pathway provoke rare human inherited metabolic diseases called porphyrias. Protein levels and activity of enzymes involved in the heme biosynthetic pathway and especially 5'-Aminolevulinate Synthase 1 are featured by 24-h rhythmic oscillations driven by the biological clock. Heme biosynthesis and circadian pathways intermingle with mutual modulatory roles. Notably, heme is a ligand of important cogs of the molecular clockwork, which upon heme binding recruit co-repressors and inhibit the transcription of numerous genes enriching metabolic pathways and encoding functional proteins bringing on crucial cell processes. Herein, we assessed mRNA levels of circadian genes in patients suffering from porphyrias and found several modifications of core clock genes and clock-controlled genes expression, associated with metabolic and electrolytic changes. Overall, our results show an altered expression of circadian genes accompanying heme biosynthesis disorders and confirm the need to deepen the knowledge of the mechanisms through which the alteration of the circadian clock circuitry could take part in determining signs and symptoms of porphyria patients and then again could represent a target for innovative therapeutic strategies.

Melanopsin expression in the retinas of owls with different daily activity patterns

Melanopsin is a photopigment found in a subset of retinal ganglion cells that is responsible for generating a series of responses to light in organisms, such as circadian rhythm regulation, pupillary light reflex, and body temperature control. The role of each melanopsin gene in the vertebrate retina is still not fully elucidated and a diversity of expression patterns of this photopigment can be observed in retinas of different vertebrate species. Owls are an excellent model for studying the role of melanopsin due to the diversity of species, which may present diurnal, nocturnal, or cathemeral habits. The purpose of this study was to characterize the expression of melanopsin genes in the retina of four different owl species from the Strigidae family (Athene cunicularia, Asio clamator, Glaucidium brasilianum, and Megascops choliba) through genetic and phylogenetic analysis. The specimens were euthanized, and the retinas were collected for RNA extraction and cDNA transcription. cDNA was used in the polymerase chain reaction (PCR) with subsequent sequencing to identify melanopsin genes expressed in the retina of owls. For the quantitative analysis of gene expression, real-time PCR was performed. The phylogenetic reconstruction was obtained by the maximum likelihood. The results showed that owls express both melanopsin genes, Opn4x and Opn4m, with different patterns of expression among the species. The expression of the Opn4x gene was two times higher in Asio clamator (nocturnal) compared to A. cunicularia (cathemeral), Glaucidium brasilianum (diurnal), and Megascops choliba (nocturnal). On the other hand, the expression of Opn4m was about two times lower in the cathemeral, A. cunicularia compared to the other three species. These results might indicate functional differences of the Opn4x and Opn4m genes among species, related to circadian rhythm regulation. Further investigation in owls at other times of the day will bring light to the circadian pattern of melanopsin expression in these species and its correlation with the different patterns of daily activity.