Functioning as a key regulator of circadian rhythms, the PER2 gene exerts a substantial impact on the reproductive traits of animals. However, the effect of the PER2 gene on ovarian development remains unclear. In order to examine the relationship between bovine reproductive trait and the PER2 gene, a total of 901 ovarian samples were collected, categorized into different oestrus cycles (proestrus, oestrus, post-oestrus, anoestrous), and subjected to analysis for two potential insertion/deletions (InDels) in the PER2 gene. Through agarose gel electrophoresis and DNA sequencing, two polymorphic deletion mutations (P2-D5-bp, P3-D13-bp) were identified. Furthermore, a significant association between mature follicle diameter and P2-D5-bp was found (P < 0.05). Additionally, several significant correlations with ovarian length, width, height, and white body diameter were found for P3-D13-bp (P < 0.05). These findings suggested that the bovine PER2 gene plays an important role in above-mentioned reproductive traits, offering new avenues for improving cow fertility through marker-assisted selection (MAS).

METHODS: The aim of this study is to investigate the effect of time-of-day on serum hormones and gene expression in adrenal glands, studying the impact of sex, obesogenic diet, and timing of proanthocyanidins administration, with a focus on glucocorticoids synthesis by this gland.
RESULTS: Female and male rats, assigned to a standard chow or a cafeteria diet-fed group, receive a daily oral dose of a grape seed proanthocyanidin extract (GSPE), or a vehicle (when light is turned on, or when light is turned off). Corticosterone, estradiol, and testosterone serum levels, and the expression analysis of clock genes and genes related to corticosterone synthesis pathway, are assessed. Serum hormone levels exhibited a marked time-of-day effect also see in the expression of scavenger receptor class B member 1 (Scarb1) and cyp11b genes. The correlation between these two genes and period circadian regulator 2 (Per2) is also extended to other clock genes, although to a lesser extent: cryptochrome (Cry) and nuclear receptor subfamily 1 group D member 1 (Rev-erba).
CONCLUSIONS: The strong correlations found suggest an important role of local Per2 (but also of Cry and Rev-erbA) in regulating the expression of the enzymes involved in the corticosterone synthesis pathway. The expression of clock genes in adrenals is influenced by sex and diet but not by GSPE.

BACKGROUND: The incidence and mortality of colorectal cancer (CRC) are persistently higher in men than in women. CRC malignancy is strongly influenced by small non-coding RNAs (miRNAs). Moreover, deregulation of the circadian molecular oscillator has been associated with CRC facilitation. To analyse possible cumulative effects of the above-mentioned factors on CRC progression, we focused on functions of sex-biased miRNAs associated with the clock genes per2 and/or cry2, which are involved in the cell cycle control and DNA damage response.
RESULTS: We identified miR-24, miR-92a, miR-181a, and miR-21 associated with per2 that are up-regulated in transformed colon tissue of men. miR-93, miR-17, miR-20a, and miR-24 with higher expression in males compared to females were linked to cry2. All these miRNAs possess oncogenic potential and exert their effects mainly via inhibition of the tumour suppressors phosphatase and tensin homolog (PTEN) and/or p53. Down-regulation of PTEN and p53 in men was further strengthened by inhibition of tumour suppressor per2. Oncogenic up-regulated miRNAs associated with per2 or cry2 in the transformed colon tissue of women were not detected.
CONCLUSIONS: We conclude that the cancer-promoting, sex-biased miRNAs miR-24, miR-92a, miR-181a, miR-93, miR-17, miR-20a, and miR-21 associated with clock genes per2 and/or cry2 can contribute to the sex-dependent development of CRC via inhibition of the PTEN and p53 pathways.

Cisplatin (CDDP) is a cornerstone chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC) and many solid cancers. However, the mechanisms underlying tumor resistance to CDDP obscure the enhancement of its therapeutic efficacy. In this study, we unveil diminished expression of the biological clock gene PER2 in OSCC, negatively correlated with the expression of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). The overexpression of PER2 suppressed MDR1 and MRP1 expression and increased intracellular CDDP levels and DNA damage, thereby bolstering OSCC cell sensitivity to CDDP. In vivo tumorigenic assays corroborated that PER2 overexpression notably increased OSCC sensitivity to CDDP, augmenting the suppression of OSCC tumorigenesis. Co-immunoprecipitation, GST pull-down, and cycloheximide tracking assays revealed that PER2, via its C-terminal domain, bound to and diminishes PDK1 stability. The degradation of PDK1 was further dependent on the suppression of the AKT/mTOR pathway to enhance the sensitivity of OSCC cells to CDDP. Our study supports PER2 as a target for improving CDDP sensitivity in OSCC, and the combination of PER2 and CDDP is a novel strategy with potential clinical therapeutic value.

Circadian rhythms, governed by the dominant central clock, in addition to various peripheral clocks, regulate almost all biological processes, including sleep-wake cycles, hormone secretion and metabolism. In certain contexts, the regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle-intrinsic clock-dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period circadian regulator 2 (Per2) and RAR-related orphan receptor alpha (Rorα), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem to be involved in the homeostasis of amine groups. Highly divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the PER2 knockouts and non-oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determining a finely tuned adaptation of muscle tissue to perturbations in health and disease. KEY POINTS: Specific perturbations of the primary and secondary feedback loop of the molecular clock result in specific outcomes on muscle metabolism and function. Ablation of Per2 (primary loop) or Rorα (secondary loop) blunts the amplitude of core clock genes, in absence of a shift in phase. Perturbation of the primary feedback loop by deletion of PER2 primarily affects muscle gene oscillation. Knockout of RORα and the ensuing modulation of the secondary loop results in the aberrant expression of a large number of non-clock genes and proteins. The deletion of PER2 and RORα affects muscle metabolism and contractile function in a circadian manner, highlighting the central role of the molecular clock in modulating muscle plasticity.

Delayed sleep phase disorder and advanced sleep phase disorder cause disruption of the circadian clock and present with extreme morning/evening chronotype with unclear role of the genetic etiology, especially for delayed sleep phase disorder. To assess if genotyping can aid in clinical diagnosis, we examined the presence of genetic variants in circadian clock genes previously linked to both sleep disorders in Slovenian patient cohort. Based on Morning-evening questionnaire, we found 15 patients with extreme chronotypes, 13 evening and 2 morning, and 28 controls. Sanger sequencing was used to determine the presence of carefully selected candidate SNPs in regions of the CSNK1D, PER2/3 and CRY1 genes. In a patient with an extreme morning chronotype and a family history of circadian sleep disorder we identified two heterozygous missense variants in PER3 gene, c.1243C>G (NM_001377275.1 (p.Pro415Ala)) and c.1250A>G (NM_001377275.1 (p.His417Arg)). The variants were significantly linked to Advanced sleep phase disorder and were also found in proband\'s father with extreme morningness. Additionally, a rare SNP was found in PER2 gene in a patient with clinical picture of Delayed sleep phase disorder. The novel variant in PER2 (NM_022817.3):c.1901-218 G>T was found in proband\'s parent with eveningness, indicating an autosomal dominant inheritance. We identified a family with autosomal dominant inheritance of two PER3 heterozygous variants that can be linked to Advanced sleep phase disorder. We revealed also a rare hereditary form of Delayed sleep phase disorder with a new PER2 variant with autosomal dominant inheritance, shedding the light into the genetic causality.

Accumulating evidence indicates that chronic circadian rhythm disruption is associated with the development of neurodegenerative diseases induced by exposure to neurotoxic chemicals. Herein, we examined the relationship between cellular circadian rhythm disruption and cytotoxicity in neural cells. Moreover, we evaluated the potential application of an in vitro cellular circadian rhythm assay in determining circadian rhythm disruption as a sensitive and early marker of neurotoxicant-induced adverse effects. To explore these objectives, we established an in vitro cellular circadian rhythm assay using human glioblastoma (U87 MG) cells stably transfected with a circadian reporter vector (PER2-dLuc) and determined the lowest-observed-adverse-effect levels (LOAELs) of several common neurotoxicants. Additionally, we determined the LOAEL of each compound on multiple cytotoxicity endpoints (nuclear size [NC], mitochondrial membrane potential [MMP], calcium ions, or lipid peroxidation) using a multiparametric high-content screening (HCS) assay using transfected U87 MG cells treated with the same neurotoxicants for 24 and 72 h. Based on our findings, the LOAEL for cellular circadian rhythm disruption for most chemicals was slightly higher than that for most cytotoxicity indicators detected using HCS, and the LOAEL for MMP in the first 24 h was the closest to that for cellular circadian rhythm disruption. Dietary antioxidants (methylselenocysteine and N-acetyl-l-cysteine) prevented or restored neurotoxicant-induced cellular circadian rhythm disruption. Our results suggest that cellular circadian rhythm disruption is as sensitive as cytotoxicity indicators and occurs early as much as cytotoxic events during disease development. Moreover, the in vitro cellular circadian rhythm assay warrants further evaluation as an early screening tool for neurotoxicants.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.

A robust endogenous clock is required for proper function of many physiological processes. The suprachiasmatic nucleus (SCN) constitutes our central circadian clock and allows us to adapt to daily changes in the environment. Aging can cause a decline in the amplitude of circadian rhythms in SCN and peripheral clocks, which contributes to increased risk of several chronic diseases. Strengthening clock function would therefore be an effective strategy to improve health. A high-throughput chemical screening has identified clock-enhancing molecule 3 (CEM3) as small molecule that increases circadian rhythm amplitude in cell lines and SCN explants. It is, however, currently not known whether CEM3 acts by enhancing the amplitude of individual single-cell oscillators or by enhancing synchrony among neurons. In view of CEM3\'s potential, it is of evident importance to clarify the mode of action of CEM3. Here, we investigated the effects of CEM3 on single-cell PERIOD2::LUCIFERASE rhythms in mouse SCN explants. CEM3 increased the amplitude in approximately 80%-90% of the individual cells in the SCN without disrupting the phase and/or period of their rhythms. Noticeably, CEM3\'s effect on amplitude is independent of the cell\'s initial amplitude. These findings make CEM3 a potential therapeutic candidate to restore compromised amplitude in circadian rhythms and will boost the development of other molecular approaches to improve health.