Abstract Introduction Evidence suggests that high-sugar diets are linked to increased genomic instability. Further, circadian disturbance can also lead to increased genotoxicity—and subsequent cancer risk—but the influence of a high sugar diet on this observation is poorly understood. By understanding the association between high-sugar diets, genotoxicity, and circadian disturbance, we hope to identify dietary predictors of increased cancer risk. Here, we evaluate the association between shift work status, the consumption of sugars, and DNA damage in immune cells. Methods 30 shift workers (15 night shift (NS); 15 day shift (DS); aged 30-55; 16 females) completed a combined field/laboratory study including 24-hours of continuous wakefulness, hourly isocaloric snacks, and blood draws every 6 hours from which peripheral mononuclear blood cells (PBMCs) were isolated. An alkaline comet assay (CometChip, Cell Array) was used to quantify DNA damage in 1153−4738 PBMCs using scoring software (CometScore, Trevigan), providing total percentage of damaged DNA compared to undamaged DNA. Prior to entering the laboratory, participants completed the General Food Frequency Questionnaire (FHQ) as a metric of past-month food consumption. Total sugar, fructose, carbohydrates, and starch consumption were extracted from the FHQs. Results T-tests revealed no significant differences between DS and NS workers for DNA damage(t(27)=–0.59,p=0.56) or the consumption of carbohydrates, total sugar, glucose, or fructose (t(27)< 1.36,p>0.18). In NS, Pearson correlations revealed a significant, moderate positive relationship between DNA damage and fructose (r=0.55,p=0.04), and non-significant relationships between DNA damage and other food metrics (r< 0.52,p>0.06). For DS, no significant relationships between DNA damage and each food metric were found (r< 0.49,p>0.06). Conclusion We sought to understand how diet contributes to DNA damage in PBMCs and identify how dietary factors are related to genomic instability in shift workers. Our findings align with existing literature, showing a relationship between high intake of fructose and DNA damage. However, this was only the case for night workers, suggesting a potential mechanistic link between diet and cancer risk in this population. Further research is needed to evaluate the utility of these dietary factors as predictors for DNA damage in shift work populations. Support (if any) BCS:Andy Hill CARE Fund,Health Sciences and Services Authority of Spokane County; SG:NIH award R01ES030113

OBJECTIVE:Although Ca2 + signaling and metabolism have been identified as key determinants for the development of Sjögren disease (SjD), the intricate connection between them and salivary gland physiology remains poorly understood. METHODS:Fluorescence-based Ca2+ imaging, RNA sequencing, and mitochondrial activity were used to investigate the effects of circadian rhythm and salivary gland dysfunction. Critical findings were confirmed by studying mouse models of SjD and human salivary gland samples. RESULTS:We identified that Ca2+ entry is essential in modulating Clock genes and circadian rhythm, which also modulate salivary gland secretion and the expression of Stim and Orai genes. Mechanistically, our data show that Bmal2 binding in the promoter region of Stim1 modulates its expression, thereby regulating Ca2+ entry and mitochondrial bioenergetics and providing cyclic rhythm-mediated regulation of cellular physiology. To assess Ca2+-dependent circadian rhythms, we used diet perturbation, in which a Ca2+-deficient diet specifically impacts the Clock gene's rhythm, which was reversed by Ca2+ supplementation. Circadian rhythm-mediated regulation of fluid secretion, as well as STIM1 genes, was also altered in an SjD mouse model. In addition, human patients with SjD also showed dysregulation of CLOCK and STIM1 genes, which could alter salivary physiology, leading to the development of the SjD phenotype. CONCLUSION:Our results provide the first comprehensive evidence of a reciprocal relationship between circadian rhythm, Ca2+ signaling, and metabolism, which is critical for cellular physiology and disease development/progression.

Abstract Introduction Chronic stress from sleep deprivation, circadian disruption, and occupational demands among shift workers may increase cumulative wear-and-tear on the body, known as allostatic load (AL). Additionally, individuals with a high prevalence of Adverse Childhood Experiences (ACEs) exhibit heightened vulnerability to disease, and this risk may be particularly pronounced among shift workers with elevated ACE exposure. Mitochondrial DNA (mtDNA) is highly susceptibility to reactive oxygen species-induced damage and therefore has been described as a marker for poor health. Here, we investigate the relationship between stressful early-life events and immune cell mtDNA in shift workers. Methods 12 day-shift (DS) (aged 30-55; 5 females) and 12 night-shift (NS) workers (aged 27-52; 9 females) participated in this combined field/laboratory study. Participants completed their regular shift cycle, slept at home, and then reported to the laboratory as if beginning another shift. Participants completed a strictly controlled 24-hour constant routine with continuous wake, and blood collections via intravenous catheter every 6 hours. Real-time PCR was performed on sheared, genomic DNA extracted from peripheral blood mononuclear cells from each sample (n=4/participant) using primers targeting the mtDNA gene hMito3 (127bp amplicon size). Results were normalized to the nuclear DNA (nDNA) gene B2M1 (188bp amplicon size). The mtDNA to nDNA ratio reflects the mtDNA copy number (mtDNAcn), allowing for comparison of mtDNA across individuals. The ACE questionnaire was administered during a pre-study screening session. Results T-tests revealed no significant differences of mtDNAcn (t(22)=−0.39, p=0.70 or ACE scores (t(22)=−0.41, p=0.69) between NS and DS workers. Pearson’s correlation was used to compare the averaged mtDNAcp to the ACE scores of the NS and DS workers. ACE scores were positively correlated with mtDNAcn (r=0.66, p=0.02) in NS, but not DS workers (r=0.08, p=0.79). Conclusion This combined field/laboratory study revealed significant associations between mtDNAcn and exposure to ACEs. These findings suggest that mtDNA alterations may represent a cellular pathway through which early-life stressors manifest physiologically. This mechanistic link provides a foundation for future research exploring how stress contributes to an accelerated AL, particularly among night shift workers. Support (if any) BCS: Andy Hill CARE Fund & Health Sciences and Services Authority of Spokane County; SG: NIH award R01ES030113

Abstract Introduction The association between body composition and chronic diseases, including certain cancers, is well established. Abdominal adiposity, measured clinically as waist-to-hip ratio, has a strong predictive value for disease development independent of body mass index (BMI). Circadian disturbance exacerbates adiposity-related risk of disease, disproportionately affecting night-shift workers. Though it is understood that circadian disturbance can lead to increased genotoxicity−and subsequent cancer risk−the influence of body composition is poorly understood. Here, we evaluate associations between waist-to-hip ratio, BMI, and shift-work status to DNA damage in immune cells. Methods Non-rotating workers, 15 day-shift (DS) and 15 night-shift (NS) (aged 30-55; 16 females), participated in a controlled, 24-hour laboratory study immediately following their typical workweek. Waist-to-hip ratio and BMI were recorded prior to the study. The laboratory environment involved continuous behavioral monitoring and sustained wakefulness. Peripheral blood mononuclear cells (PBMCs) were collected every 6 hours via intravenous catheter. An alkaline comet assay (CometChip, Cell Array) was used to quantify DNA damage in 1153−4738 PBMCs per participant using comet scoring software (CometScore, Trevigan), providing the percent tail DNA (i.e. total percentage of damaged DNA compared to undamaged DNA). Results T-tests revealed no significant difference between DS and NS workers’ waist-to-hip ratio (t(23)=−0.18, p=0.86) or BMI (t(28)=−1.06, p=0.30). Pearson’s correlations showed that waist-to-hip ratio was strongly associated with DNA damage in NS workers (r=0.79, p< 0.001), but BMI was not (r=0.42, p=0.12). In DS workers, there was no significant correlation between DNA damage and waist-to-hip ratio (r=0.45, p=0.19) or BMI (r=−0.15, p=0.58). Conclusion Our findings are consistent with data showing a relationship between increased waist-to-hip ratio and DNA damage/reduced repair mechanisms in PBMCs, which increases genomic instability and is a hallmark of cancer. However, this was only the case for long-term NS workers, further implicating central adiposity as a risk factor for developing certain cancers in individuals exposed to circadian disturbance. Future research should determine other factors associated with DNA damage in this population. Support (if any) BCS: Andy Hill CARE Fund & Health Sciences and Services Authority of Spokane County; SG: NIH award R01ES030113.

The International Agency for Research on Cancer (IARC) stated that circadian disruption is a potential carcinogen. However, the impact of environmental carcinogens, including sub-erythemal doses of UVB exposure, on circadian rhythms remains unclear. We evaluated the impact of long-term rotating shift, loss of Per1/2 genes, and chronic UVB exposure on the circadian rhythms of SKH-1 mice for up to 7 months. Real-time locomotion and circadian gene expression were measured in these animals. Mice under rotating shift exhibited a longer period of activity of up to 25.20 h, while those under standard light conditions had a clear 24-h rhythm. mPer1/mPer2 mice, conversely, displayed a shortened period of activity of 23.61 h. Interestingly, chronic UVB exposure had no impact on activity rhythms, though it induced skin tumors in all mice. Rotating shift and loss of mPer1/mPer2 led to circadian dysregulation of all core clock genes, with a notable phase difference in Cry1. These findings provide novel insights into environmental and genetic influences on circadian rhythms.

Genome instability is a major force driving tumorigenesis. The ssDNA-binding protein complex CTC1-STN1-TEN1 (CST) plays a pivotal role in maintaining genome stability by countering replication stress, modulating DNA damage repair, and maintaining telomere integrity. Despite its well-documented role in genome maintenance, the involvement of CST in skin cancer development has yet to be investigated. We recently found that CST localizes at stalled DNA replication sites after UV exposure and may suppress the unwanted repriming activity, suggesting a potential role of CST in suppressing genome instability caused by UV damage. In this study, we first analyzed CST expression and alterations in cutaneous melanoma database and found that the CST genes are frequently altered in cutaneous melanoma and their expression is significantly downregulated in melanoma samples compared to normal tissues. We then generated a conditional knockout (cKO) mouse model with STN1 deficiency specifically in melanocytes to investigate its role in skin cancer formation. Upon chronic exposure to UV irradiation, STN1-deficient mice exhibit no obvious difference in melanoma incidence compared to control littermates, suggesting that STN1 downregulation in mature melanocytes has no significant effect on UV-induced skin cancer development in lab mice.

Abstract Introduction Shift workers experience misalignment between internally-driven circadian rhythms and externally-driven behavioral schedules. Mounting evidence from hormone-, cytokine-, exosome-, proteome-, and metabolome-based biomarker analyses of blood samples predicts disruption of glucose and insulin regulatory pathways during night shift schedules. Here, we quantified circulating glucose and insulin concentrations in a constant routine (CR) protocol to assess functional metabolic outcomes of prior simulated shift work. Methods Healthy adults (N=14; aged 22–34; 4 females) completed a 7-day/6-night in-laboratory study, with randomization to either a 3-day simulated day shift (DS) schedule (n=7) with nighttime sleep (22:00–06:00) and 3 daytime meals (07:30, 13:00, 19:30), or a 3-day simulated night shift (NS) schedule (n=7) with daytime sleep (10:00–18:00) and 3 nighttime meals (19:30, 01:00, 07:30). A 24h CR protocol followed, during which participants stayed awake under constant behavioral and environmental conditions, including hourly isocaloric snacks. Serum collected via intravenous catheter every 6h during the CR was assayed using glucose colorimetric detection and human insulin ELISA. Glucose and insulin concentrations were analyzed with mixed-effects ANOVAs with fixed effects of prior simulated shift condition (day, night) and sampling time (01:30, 07:30, 13:30, 19:30) and their interaction, with a random effect over participants on the intercept. Mixed-effects cosinor regression analyses were conducted to compare endogenous circadian rhythms between conditions. Results For glucose, there was a significant condition*time interaction (F[3,30]=4.99, P=0.006), with a 9.34±2.42h delay (mean±SE) of the endogenous circadian glucose rhythm after simulated NS compared to DS (t[12]=3.85, P=0.002). For insulin, there were significant effects of condition (F[1,30]=5.93, P=0.021) and time (F[3,30]=3.01, P=0.046), with no significant endogenous rhythmicity after simulated DS, but the appearance of a circadian rhythm with reduced insulin concentrations after simulated NS (t[12]=-3.32, P=0.006). Conclusion Under constant routine, we found altered endogenous temporal patterns of glucose and insulin after just 3 days of simulated NS compared to DS. Our results indicate that biomarker and omics-based predictions of disrupted glucose and insulin regulatory pathways have functional significance evident in circulating glucose and insulin concentrations. This may explain the insulin resistance and increased risk of metabolic disorders observed in shift workers. Support (if any) Andy Hill CARE Fund FY22-POP-02 and WSU CPPS start-up funds

This Special Issue (SI) of Environmental and Molecular Mutagenesis (EMM), entitled "Inspiring Basic and Applied Research in Genome Integrity Mechanisms," is to update the community on recent findings and advances on genome integrity mechanisms with emphasis on their importance for basic and environmental health sciences. This SI includes two research articles, one brief research communication, and four reviews that highlight cutting edge research findings and perspectives, from both established leaders and junior trainees, on DNA repair mechanisms. In particular, the authors provided an updated understanding on several distinct enzymes (e.g., DNA polymerase beta, DNA polymerase theta, DNA glycosylase NEIL2) and the associated molecular mechanisms in base excision repair, nucleotide excision repair, and microhomology-mediated end joining of double-strand breaks. In addition, genome-wide sequencing analysis or site-specific mutational signature analysis of DNA lesions from environmental mutagens (e.g., UV light and aflatoxin) provide further characterization and sequence context impact of DNA damage and mutations. This SI is dedicated to the legacy of Dr. Samuel H. Wilson from the U.S. National Institute of Environmental Health Sciences at the National Institutes of Health.

Circadian misalignment due to night work has been associated with an elevated risk for chronic diseases. We investigated the effects of circadian misalignment using shotgun protein profiling of peripheral blood mononuclear cells taken from healthy humans during a constant routine protocol, which was conducted immediately after participants had been subjected to a 3-day simulated night shift schedule or a 3-day simulated day shift schedule. By comparing proteomic profiles between the simulated shift conditions, we identified proteins and pathways that are associated with the effects of circadian misalignment and observed that insulin regulation pathways and inflammation-related proteins displayed markedly different temporal patterns after simulated night shift. Further, by integrating the proteomic profiles with previously assessed metabolomic profiles in a network-based approach, we found key associations between circadian dysregulation of protein-level pathways and metabolites of interest in the context of chronic metabolic diseases. Endogenous circadian rhythms in circulating glucose and insulin differed between the simulated shift conditions. Overall, our results suggest that circadian misalignment is associated with a tug of war between central clock mechanisms controlling insulin secretion and peripheral clock mechanisms regulating insulin sensitivity, which may lead to adverse long-term outcomes such as diabetes and obesity. Our study provides a molecular-level mechanism linking circadian misalignment and adverse long-term health consequences of night work.

View Large Image Figure ViewerDownload Hi-res image Download (PPT) "The core study has uncovered compelling new information regarding age and sex differences in clock gene expression, raising many new questions."Clinical Implications•Disruption of circadian clock genes is potentially associated with aging and disease.•New studies are needed to examine a plethora of sex- and aging-specific differences.•A collaborative research environment and strong study design are essential to tackle all the variables. "The core study has uncovered compelling new information regarding age and sex differences in clock gene expression, raising many new questions." •Disruption of circadian clock genes is potentially associated with aging and disease.•New studies are needed to examine a plethora of sex- and aging-specific differences.•A collaborative research environment and strong study design are essential to tackle all the variables. Day and night have long guided daily activities in our lives. Before time-keeping innovations such as sundials and clocks, humans maintained schedules by merely opening their eyes as the sun rose in the sky. Unbeknownst to them, their internal biological clock was maintaining regulation of their bodily functions throughout the course of the day through the trillions of cells that comprise the human body. This endogenous cellular process is referred to as one's circadian or biological rhythm and oscillates over an approximately 24-hour period, often portrayed as a cosine wave. The master clock, located in the suprachiasmatic nucleus in the hypothalamus, is primarily entrained by sunlight taken in by the retina and is responsible for synchronization to the external environment and relaying information to the peripheral clocks found throughout the body in every organ and tissue. These clocks are vital in regulating functions such as sleep/wake cycles, DNA repair and replication, metabolism, and immune response. A transcriptional–translational feedback mechanism that controls the oscillatory rhythms of clock-controlled genes drives cellular processes such as these regulating functions (Sancar and Van Gelder, 2021Sancar A. Van Gelder R.N. Clocks, cancer, and chronochemotherapy.Science. 2021; 371: eabbo738Crossref Scopus (135) Google Scholar). The core clock proteins, BMAL1, CLOCK (circadian locomotor output kaput), CRY1/2 (cryptochrome), and PER1/2 (period), make up this primary feedback loop, which activates upon BMAL1::CLOCK dimerization. In the primary loop, the heterodimer binds to the E-box, driving transcription of cryptochrome and period genes, which subsequently translocate from the nucleus to the cytoplasm to heterodimerize and bind to the BMAL1::CLOCK heterodimer, impeding their own transcription and other clock-controlled genes. In the secondary feedback loop, binding of BMAL1::CLOCK to the E-box drives the transcription of RORα and REV-ERBα/β (Nr1d1/Nr1d2), which compete to bind to RORE and either activate or repress Bmal1 transcription, respectively. This feedback mechanism, although seemingly simple, has been associated with a wide variety of health conditions ranging from behavioral disorders, metabolic disorders, cancers, and even reproductive disorders. Escalating incidence of disruptive circadian conditions in 24-hour cities and the rising prevalence of diseases have made it imperative to obtain a better understanding of the mechanistic effects of circadian dysregulation in humans owing to its potential association with aging and disease as shown by the Kemp laboratory (Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar). Despite the crucial role of sunlight in resetting circadian rhythm, it is also responsible for increased sunburn and skin carcinogenesis risk, including melanoma (Gaddameedhi et al., 2015Gaddameedhi S. Selby C.P. Kemp M.G. Ye R. Sancar A. The circadian clock controls sunburn apoptosis and erythema in mouse skin.J Invest Dermatol. 2015; 135: 1119-1127Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), which occurs in epidermal melanocytes and is exceptionally lethal in afflicted individuals. Skin cancers arise from exposure to UVR emitted from the sun that induces skin-specific DNA damage—bulky lesions repaired through nucleotide excision repair—a process regulated by the circadian rhythm (Gaddameedhi et al., 2015Gaddameedhi S. Selby C.P. Kemp M.G. Ye R. Sancar A. The circadian clock controls sunburn apoptosis and erythema in mouse skin.J Invest Dermatol. 2015; 135: 1119-1127Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Strong evidence of circadian-regulated genes in mice has led to additional human studies (Wu et al., 2018Wu G. Ruben M.D. Schmidt R.E. Francey L.J. Smith D.F. Anafi R.C. et al.Population-level rhythms in human skin with implications for circadian medicine.Proc Natl Acad Sci USA. 2018; 115: 12313-12318Crossref PubMed Scopus (74) Google Scholar), which have supported this finding and documented the expression of circadian clock genes in human epidermis throughout the 24-hour cycle. However, researchers could not fully investigate sex- and aging-specific differences because the study population was predominantly White and male and because biopsies came from the forearm, a region of the skin commonly exposed to sunlight and therefore susceptible to more exposures. This knowledge gap made it possible for Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar to investigate age and sex differences in core clock gene expression in human epidermis, a sample that is more accessible than other tissues (Wu et al., 2018Wu G. Ruben M.D. Schmidt R.E. Francey L.J. Smith D.F. Anafi R.C. et al.Population-level rhythms in human skin with implications for circadian medicine.Proc Natl Acad Sci USA. 2018; 115: 12313-12318Crossref PubMed Scopus (74) Google Scholar). In contrast to previous studies that used whole skin or samples without a documented collection time, Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar did both and performed a pilot study involving 24 participants. From these subjects, they obtained skin punch biopsies from the lower back/upper buttock, recorded the collection time, and isolated the epidermis through heat shock. Selecting skin from this anatomical region was an excellent choice because it minimizes external confounders such as UVB exposure, which is common to regions such as the face, neck, and hands—areas typically exposed to sunlight in routine daily tasks. In their study, Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar performed TaqMan-based qRT-PCR on isolated epidermis from 12 subjects aged between 20 and 29 years and >65 years, a sufficient age distribution to address their research question. Their analysis of 10 core clock genes, Arntl, Clock, Npas2, Cry1, Cry2, Per1, Per2, Nr1d1, Nr1d2, and RORα, revealed greater relative expression of Npas2 in all females than in all males, regardless of age, and in all older individuals than in all younger ones, regardless of sex. They also found that among young adults, females exhibited higher relative expression of Arntl and Cry1 and lower relative expression of Cry2 and Per2 than males. Interestingly, in older adults, females had a higher relative expression of 3 genes, Npas2, Nr1d1, and RORα, than males, but males did not have a higher relative expression of any clock genes than females. Comparisons between younger and older adults of each sex indicated that older females had a higher relative expression of Nr1d1 and Per1 but lower Arntl expression than younger females, and younger males had higher expression of Cry2 than older males. The authors speculate that the absence of significant differences in clock gene expression between certain groups may be because all biopsies collected from young females were collected in the afternoon. This could be true if researchers collected the other biopsies over a broader range of time points. Understandably, Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar have a limited sample size, and analysis requires averaging multiple time points from subjects of each group. This potentially reduces or even misses significant differences that may exist in time-of-day oscillations of clock genes, which are known to oscillate dynamically in both mice and humans over the 24-hour day, and because expression at a single time point can vary considerably compared with an average combined expression value of all circadian time points (Koritala et al., 2021Koritala B.S.C. Porter K.I. Arshad O.A. Gajula R.P. Mitchell H.D. Arman T. et al.Night shift schedule causes circadian dysregulation of DNA repair genes and elevated DNA damage in humans.J Pineal Res. 2021; 70e12726Crossref PubMed Scopus (42) Google Scholar; Wu et al., 2018Wu G. Ruben M.D. Schmidt R.E. Francey L.J. Smith D.F. Anafi R.C. et al.Population-level rhythms in human skin with implications for circadian medicine.Proc Natl Acad Sci USA. 2018; 115: 12313-12318Crossref PubMed Scopus (74) Google Scholar). If investigators controlled for this variation in a larger study, it would be interesting to see whether these clock genes do indeed maintain their rhythmicity and robustness throughout the day in the epidermis between sexes and as the subjects age because expression at a single timepoint and an averaged expression value can vary considerably. As a pilot study, the findings are more than sufficient to draw preliminary conclusions regarding differences between sex and age, providing ample evidence for a full study that potentially contains samples from more times throughout the day. The addition of demographics; occupation; and any long-term health concerns such as smoking, obesity, or cancer could bolster any findings because they could account for confounding variables that may not have been possible to correct for in the pilot. The aging effect on circadian gene expression in this study is not only a fascinating observation but also reveals more aging-related questions. In this study, the older adult group is designated as those aged ≥65 years, leading one to question whether aging-specific differences in clock gene expression are directly attributable to aging or whether it could be caused by lifestyle changes after retirement (Baxter et al., 2021Baxter S. Blank L. Cantrell A. Goyder E. Is working in later life good for your health? A systematic review of health outcomes resulting from extended working lives.BMC Public Health. 2021; 21: 1356Crossref PubMed Scopus (19) Google Scholar). Drastic change or loss of one's schedule could potentially affect peripheral clock entrainment, and researchers must consider this when interpreting the results. To mitigate such confounders, including participants aged between 30 and 65 years could be used to show a linear relationship between age and clock gene expression. Alternatively, investigators may use epigenetic clocks to perform quantitative measurements of a subject's biological age and verify whether their chronological age aligns or is relatively close, thus removing retirement as a contributing factor. Although expensive, it also provides the opportunity to investigate the associations with disease outcomes, given the recent implications between cancer and epigenetic modifications. Recently, circadian disruption through night shift work has been classified as a Group 2A carcinogen by International Agency for Research on Cancer. This type of work is highly prevalent in the United States, accounting for about 25% of the United States workforce, given its necessity in health care, transportation, and security sectors. Although none of the participants were night shift workers, circadian disruption through inconsistent work and sleep schedules is an extremely common phenomenon in younger adults; hence, determining the consistency of the subjects' sleep schedules could yield interesting results. Because circadian disruption is so strongly associated with altered regulation of biological processes, these samples provide an opportunity to investigate whether healthy rhythms are detectable in the epidermis to measure an individual's health status or risk of disease onset. Within the last 2 decades of research, several studies have found robust circadian activity in keratinocytes, suggesting that individual cell types might be driving altered gene rhythmicity. Further research into cell type and variation in physiological states could provide more insight (Plikus et al., 2015Plikus M.V. Van Spyk E.N. Pham K. Geyfman M. Kumar V. Takahashi J.S. et al.The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.J Biol Rhythms. 2015; 30: 163-182Crossref PubMed Scopus (124) Google Scholar). As the first line of defense in the immune system, skin dysregulation is likely to increase susceptibility to both acquired and autoimmune disorders as well as facilitate aging due to altered circadian-mediated checkpoint (both G1/S and G2/M) regulation, which affects the cell cycle and therefore cell proliferation (Plikus et al., 2015Plikus M.V. Van Spyk E.N. Pham K. Geyfman M. Kumar V. Takahashi J.S. et al.The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.J Biol Rhythms. 2015; 30: 163-182Crossref PubMed Scopus (124) Google Scholar). Researchers observed effects on immune function in old and young murine skin in which skin antiviral response was compromised, possibly owing to reduced clock gene expression in aging skin (Kirchner et al., 2023Kirchner S.J. Lei V. Kim P.T. Patel M. Shannon J.L. Corcoran D. et al.An aging-susceptible circadian rhythm controls cutaneous antiviral immunity.JCI Insight. 2023; 8e171548Crossref PubMed Scopus (3) Google Scholar). Another recent murine study demonstrated age-related metabolic effects in overall gene expression as well as extended lifespans upon changes in food consumption time (Acosta-Rodríguez et al., 2022Acosta-Rodríguez V. Rijo-Ferreira F. Izumo M. Xu P. Wight-Carter M. Green C.B. et al.Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.Science. 2022; 376: 1192-1202Crossref PubMed Scopus (129) Google Scholar). Although researchers have measured clock gene expression in a variety of tissues, impacts on tissues unrelated to immune or metabolic function are surprisingly limited, leaving ample room for investigation into differences in expression across organs affected through immune or endocrine mechanisms (Plikus et al., 2015Plikus M.V. Van Spyk E.N. Pham K. Geyfman M. Kumar V. Takahashi J.S. et al.The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.J Biol Rhythms. 2015; 30: 163-182Crossref PubMed Scopus (124) Google Scholar). Researchers have realized the importance of sex-specific studies in recent years, and Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar have provided additional evidence to support this. Unfortunately, research has underrepresented women in much of scientific history, and the study by Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar is no exception despite best efforts. Increasing the number of female subjects not only ensures equal representation between sexes but also allows for additional analysis of the effect of the endocrine system on gene expression because it incorporates an adequate sample size into the study design. If the number of epidermis samples is insufficient, collecting hair follicles instead could be an option because it also has a prominent clock and is much easier to attain (Plikus et al., 2015Plikus M.V. Van Spyk E.N. Pham K. Geyfman M. Kumar V. Takahashi J.S. et al.The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.J Biol Rhythms. 2015; 30: 163-182Crossref PubMed Scopus (124) Google Scholar). Differences in clock gene expression between males and females may be related to the menstrual cycle in females and would be fascinating to investigate, given the opportunity. Along the same lines, if the menstrual cycle is associated, experiences with pregnancy or menopause might contribute to the differences in older and younger females. Overall, more information about the participants, such as health history, sleep pattern, chronotype, and demographics, presents an intriguing angle to investigate and expand the scope of the pilot. Doing so would incorporate more diverse populations found across the globe to yield results that encompass lifestyle differences as well as melanin and skin tone variation caused by Fitzpatrick skin types. There is also the option to include additional circadian time points, which would permit greater power and provide an exciting opportunity to compare expression at hourly intervals instead of just morning and afternoon. It may also uncover more differences between age and sex, such as maintenance or loss of oscillation robustness or differences in circadian measurements of amplitude, mesor, acrophase, or phase (Koritala et al., 2023Koritala B.S.C. Dakup P.P. Porter K.I. Gaddameedhi S. The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study.F1000Res. 2023; 12: 762Crossref PubMed Google Scholar). Cvammen et al., 2023Cvammen W. Rider Jr., S.D. Travers J.B. Kemp M.G. Effects of age and sex on the expression of core circadian clock genes in human skin epidermis.J Invest Dermatol. 2024; 144: 1172-1175Abstract Full Text Full Text PDF Scopus (1) Google Scholar's study has uncovered compelling new information regarding age and sex differences in clock gene expression, raising many new questions. These findings, combined with an increasingly light-polluted society at night, make it pertinent to better understand how the molecular mechanism responsible for circadian disruption works. More aggressive forms of environmental circadian disruption include night shift work and chronic jet lag, both of which research has associated with aging-related neurodegeneration such as Alzheimer's disease (Acosta-Rodríguez et al., 2022Acosta-Rodríguez V. Rijo-Ferreira F. Izumo M. Xu P. Wight-Carter M. Green C.B. et al.Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.Science. 2022; 376: 1192-1202Crossref PubMed Scopus (129) Google Scholar) (Figure 1). Despite a growing human population, obtaining many human samples remains extremely challenging. This difficulty and the inability to control variation in life experiences are the most common limitations in such studies. Therefore, once gaining consent, a strong study design could address multiple questions and extract as much information as possible to counteract the bottleneck caused by the lack of human samples. An increasingly collaborative research community will hopefully aid in this endeavor, but until then, research requires other mammalian models to answer questions pertaining to human health. The authors state no conflict of interest. SG is supported by the National Institutes of Health National Institute of Environmental Health Sciences grant R01ES030113. Effects of Age and Sex on the Expression of Core Circadian Clock Genes in Human Skin EpidermisJournal of Investigative DermatologyVol. 144Issue 5PreviewMany aspects of skin biochemistry and physiology, including wound healing, immune defense, and responses to stress, exhibit circadian rhythmicity. Much of our understanding of circadian rhythms in the skin has been derived from studies in nocturnal mice (Mohawk et al, 2012), and thus work with diurnal human subjects is important to extend and validate this knowledge to humans. Although two previous studies confirmed the rhythmic expression of core clock genes in the epidermis (Spörl et al, 2012; Wu et al, 2018), these works were limited to younger adults and primarily to males. Full-Text PDF

<p>PDF file - 114K, Downregulation of Egr1 by pooled or single siRNA is specific and does not affect the E2F1 level and vise versa</p>

Over the past decades, studies of the TRPM8 channel, a non-selective cation channel, have provided much insight into the fundamental mechanisms of sensory neuron function that lead to the detection of cold sensors. In the current issue of Acta Physiologica, Reimundez et al1 provide evidence of a novel role of TRPM8 in circadian function in mice. The TRPM (transient receptor potential ion channels, where "m" stands for melastatin) is a family of eight different channels, TRPM1-TRPM8.2 The TRPM8 is a cold sensor and is also activated by chemical ligands such as menthol and icilin, but recently, their role in maintaining core body temperature has also been recognized.3, 4 TRPM8 is expressed in sensory neurons whose axonal afferents innervate peripheral tissues such as skin and oral cavity. Still, it has not been detected in the brain and spinal cord. Besides nerve tissues, TRPM8 is expressed in tissues such as the prostate, bladder, lungs, and urogenital tract. However, other than the sensory nervous system, the functional role of TRPM8 channels is not well understood. In this issue of Acta Physiologica, Reimundez et al1 provide solid experimental evidence for such a novel mechanism—where they demonstrate that TRPM8 might regulate Period 2 (Per2) mRNA levels in central clock (SCN) and peripheral clock (liver and white adipose tissue) and the circadian regulation of core body temperature. Their publication is among the most significant advances in the field of circadian regulation using cold sensors—it will probably set up the stage for several future research activities in which Reimundez and colleagues' ideas will be rigorously dissected and probed in circadian biology research. Previously, Ordás et al5 demonstrated the presence of TRPM8 fibers in the suprachiasmatic nucleus of the hypothalamus (SCN) of the brain, which is the principal circadian pacemaker in mammals and host a range of other physiological processes such as circadian oscillation, autonomic/peripheral and central nervous system function, regulating core body temperature and sleep–wake cycle. Since SCN receives axonal projections mainly from intrinsically photosensitive retinal ganglion cells (ipRGCs) that are responsible for resetting the circadian clock through SCN neuron activity with light as a primary zeitgeber.6 Here, authors used conventional PCR and mouse reporter lines to show the expression of TRPM8 in the inner retina, specifically in the ganglion cell layer (GC) and in the inner nuclear layer (INL). Next, they used the colocalization technique to demonstrate that melanopsin, a marker of ipRGCs7 and using mouse reporter lines, that TRPM8 expressing cells are indeed expressed in a subset of ipRGCs cells. Furthermore, they showed that TRPM8 is also expressed in cholinergic amacrine cells, characterized by releasing two neurotransmitters, GABA and acetylcholine. Injection of the fluorescent anterograde tracer cholera toxin subunit B (CTB-594) into both eyes in two TRPM8 reporter lines indicates the existence of ipRGCs expressing TRPM8 and projecting to the SCN (Figure 1). The choroid is an essential high blood flow vascular structure in the eye and is known to regulate ocular and retinal temperature8 and is richly innervated by sensory trigeminal nerve fibers; therefore, the authors next examined the role of TRPM8 in regulation of eye temperature (Teye) using infrared (IR) thermography. Interestingly, they discovered mice lacking TRPM8 started to decline at temperatures below 25°C, and Trpm8−/− mice displayed a significantly lower Teye than WT littermates when the temperature plate was around 15°C. This suggests a function of TRPM8 and ambient temperature in regulating choroid and ciliary body blood flow and, therefore, in controlling internal ocular temperature. The ability of mice to regulate homeostatic temperature control and the expression of TRPM8 in the ipRGCs is indicative but does not directly link that to central clock regulation. The authors performed expression and functional studies to investigate a direct link between TRPM8 and circadian clockwork at the SCN. First, they determined the expression of the Per2 gene, which is one of the essential components of core circadian clocks in the SCN.9 Authors found in the TRPM8 deficient mouse SCN a significant increase of ~40% in Per2 mRNA content in relation to WT littermates during the daytime (ZT4). Next, they examined one of the most relevant clock-controlled neuropeptide Arginine Vasopressin (AVP) in the SCN. AVP-expressing neurons in the SCN constitute the main output of this nucleus and are known to be involved in core temperature (Tc) circadian regulation.10 Like Per2, Trpm8−/− mice displayed greater AVP expression than control mice during the lights-on phase (~50% higher) but conserved the oscillation between day and nighttime. Briefly, these results suggest that the absence of TRPM8 does not prevent oscillatory expression of Per2 and AVP but involves modifying clockwork and neuropeptide levels in this hypothalamic nucleus, contributing to the regulation of the circadian oscillation of Tc. Second, Reimundez et al1 found that TRPM8 plays a significant role in regulating core body temperature where TRPM8 deficient mice show a significantly reduced temperature and increased amplitude both in light/dark (LD) and dark/dark (DD) conditions compared with its wild-type controls. Future studies investigating TRPM8 regulation on circadian rhythm properties such as period, acrophase, and mesor would be interesting. Third, authors extended the TRPM8 role in peripheral clocks liver and gonadal WAT by measuring Per2 mRNA levels during the day (ZT4) and night (ZT16). TRPM8 deficient mice liver has a significantly elevated Per2 levels during day and dampened levels during night compared to WT control. Interestingly, in gonadal WAT tissue, Per2 levels were significantly lower both in day and night compared to WT control. Future studies, investigating with more circadian timepoints (every 3 hours in a 24-hour cycle) on whether Per2 levels were significantly rhythmic would be interesting. The authors provide strong evidence of the TRPM8 channel as a connecting link between temperature and central and peripheral clocks, regulating the circadian oscillations of body temperature. However, the presented evidence is scant and indirect, especially using the global TRPM8 deficient mice and knowing that TRPM8 is also a peripheral sensor in the skin, which might play an indirect, yet a meaningful role in mediating this effect. Most importantly, extending the TRPM8 role to other core clock genes such as Cry1, Cry2, Per1, Bmal1, Clock, and Npas2 oscillation and circadian rhythmicity will be interesting. No conflict of interest to declare.

Supplementary Figures 1-6 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Supplementary Table 2 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Supplementary Table 1 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells