J Biol Rhythms
· 2025 Dec · PMID 40886071
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Biomarkers are valuable tools in a wide range of human health areas including circadian medicine. Valid, low-burden, multivariate molecular approaches to assess circadian phase at scale in people living and working in th...Biomarkers are valuable tools in a wide range of human health areas including circadian medicine. Valid, low-burden, multivariate molecular approaches to assess circadian phase at scale in people living and working in the real world hold promise for translating basic circadian knowledge to practical applications. However, standards for the development and evaluation of these circadian biomarkers have not yet been established, even though several publications report such biomarkers and claim that the methods are universal. Here, we present a basic exploration of some of the determinants and confounds of blood-based biomarker development for suprachiasmatic nucleus (SCN) phase by reanalysing publicly available data sets. We compare performance of biomarkers based on three feature-selection methods: Partial Least Squares Regression, ZeitZeiger, and Elastic Net, as well as performance of a standard set of clock genes. We explore the effects of training sample size and the impact of the experimental protocols from which training samples are drawn and on which performance is tested. Approaches based on small sample sizes used for training are prone to poor performance due to overfitting. Performance to some extent depends on the feature-selection method, but at least as much on the experimental conditions from which the biomarker training samples were drawn. Performance of biomarkers developed under baseline conditions does not necessarily translate to protocols that mimic real-world scenarios such as shiftwork in which sleep may be restricted or desynchronized from the endogenous circadian SCN phase. The molecular features selected by the various approaches to develop biomarkers for the SCN phase show very little overlap although the processes associated with these features have common themes with response to steroid hormones, that is, cortisol being the most prominent. Overall, the findings indicate that establishment of circadian biomarkers should be guided by established biomarker-development concepts and foundational principles of human circadian biology.
J Biol Rhythms
· 2025 Dec · PMID 40879142
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The circadian clock enables organisms to optimize their metabolism, physiology, and behavior with the time-of-day. However, circadian rhythms benefit organisms only if they are properly synchronized with the day/night cy...The circadian clock enables organisms to optimize their metabolism, physiology, and behavior with the time-of-day. However, circadian rhythms benefit organisms only if they are properly synchronized with the day/night cycle; circadian misalignment can have detrimental effects on animals' wellbeing and survival. We previously showed that in , loss of the microRNA advances the phase of circadian evening locomotor activity by several hours under constant darkness conditions. Interestingly, we now report that loss of also delays morning activity under a light/dark cycle with a short photoperiod. We recapitulated these opposite phase phenotypes by eliminating during larval development, but not when this microRNA is lost during pupation to adulthood. The loss of results in significant miswiring within the circadian neural network and severely alters neural activity rhythms in the ventral Lateral Neurons (s-LNvs) and the posterior Dorsal Neurons 1 (DN1ps), which control the timing of morning and evening activity. Silencing the s-LNvs in mutant flies restores the phase of evening activity, while activating the DN1ps rescues the phases of both morning and evening activities. Our findings thus reveal the pivotal role of in sculpting the circadian neural network during development and its long-lasting impact on circuit activity and adult circadian behavior.
J Biol Rhythms
· 2025 Oct · PMID 40832807
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Response to: "Mathematical Analysis of Light-sensitivity Related Challenges in Assessment of the Intrinsic Period of the Human Circadian Pacemaker".Response to: "Mathematical Analysis of Light-sensitivity Related Challenges in Assessment of the Intrinsic Period of the Human Circadian Pacemaker".
Newly emerging evidence underscores the crucial role of the gut microbiota in regulating various aspects of mammalian physiology and behavior, including circadian rhythms. These rhythms, fundamental to behavioral and phy...Newly emerging evidence underscores the crucial role of the gut microbiota in regulating various aspects of mammalian physiology and behavior, including circadian rhythms. These rhythms, fundamental to behavioral and physiological processes, are orchestrated by a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Extra-SCN oscillators have been identified in brain regions beyond the SCN and in peripheral tissues temporizing wide physiological functions. Under a 12 h light: 12 h dark cycle (12:12 LD), restriction of food access to hours of light in nocturnal animals in a time-restricted feeding (TRF) protocol increases locomotor activity preceding the scheduled daily meal, so-called food anticipatory activity (FAA). This circadian behavior is independent from the SCN and controlled by a food-entrainable oscillator (FEO) dependent on reward-related signals. It is known that signals from the gut microbiota regulate behaviors such as motivation oriented by food reward. Thus, we hypothesized a physiological link between gut microbiota and FEO activity by studying the circadian FAA behavior under TRF and assessing food-oriented motivational behavior. For that aim, C57BL/6J mice treated with antibiotics for generating gut microbiota dysbiosis were subjected to a 3 h TRF protocol at zeitgeber time (ZT) 4-7. Mice treated with antibiotics exhibited greater FAA, lower time for its consolidation, and greater motivation levels for food reward. Moreover, tyrosine hydroxylase (TH) levels were increased in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of antibiotic-treated mice. Finally, changes in the gut microbiota composition-including bacterial diversity and the abundance of certain genera-were observed. These results suggest that gut microbiota has a regulatory role in the circadian motivational output for food reward controlled by the FEO. Understanding this role is important for potential chronotherapeutics targeting gut microbiota in reward-related alterations such as addictions and eating disorders.
The brain is the most energy-demanding organ, yet whether cerebral energy homeostasis exhibits seasonal rhythmicity remains unclear. In this study, 432 healthy men underwent a health checkup program with fasting-state br...The brain is the most energy-demanding organ, yet whether cerebral energy homeostasis exhibits seasonal rhythmicity remains unclear. In this study, 432 healthy men underwent a health checkup program with fasting-state brain [F]fluorodeoxyglucose positron emission tomography (PET) scanning twice: first at the baseline and then at the 5-year follow-up. We analyzed the effect of day length on brain glucose uptake separately for both time points. In both baseline and follow-up scans, day length on the day of imaging significantly predicted glucose uptake in the socio-emotional circuit. A longer day length was associated with increased glucose uptake in the cuneus, precuneus, orbitofrontal cortex, pre- and postcentral gyrus, superior and middle temporal gyrus, posterior cingulate cortex, insula, and frontal pole. This large-scale longitudinal PET study provides landmark evidence for the impact of daylight exposure on brain glucose metabolism. Findings disclose the baseline seasonal variation of brain energy consumption in men.
J Biol Rhythms
· 2025 Oct · PMID 40703065
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The circadian clock maintains oscillations in gene expression with a 24-hour periodicity in nearly every cell of the body and confers rhythmic patterns to many aspects of behavior and physiology. The presence of circadia...The circadian clock maintains oscillations in gene expression with a 24-hour periodicity in nearly every cell of the body and confers rhythmic patterns to many aspects of behavior and physiology. The presence of circadian rhythms in tumors leads to the question of whether tumors may respond differently to chemotherapy given at different times of day. We addressed this question using a male mouse model of hepatoma by treating mice in the morning (ZT2) or evening (ZT14) with cisplatin, and measuring gross effects on body weight, blood counts and chemistry, gene expression, and cellular proliferation. We found that among cisplatin-treated mice, there was a reduction in expression of the proliferation marker protein Ki-67 in tumors of mice treated at ZT14 as compared to ZT2. Corresponding hepatotoxicity, as measured by elevated serum alanine aminotransferase (ALT), and body weight loss were also reduced at ZT14. Overall gene expression at ZT14 was more similar to healthy liver than expression at ZT2. Mitogen-activated protein kinase (MAPK) and Ras-related protein-1 (Rap-1) signaling pathways were specifically downregulated in tumors following treatment at ZT14, which may be related to the decreased proliferation, at this treatment time. These findings align with the possible use of timed chemotherapy to enhance drug efficacy.
Maternal care is essential for offspring survival. Circadian regulation of maternal behavior and feeding behavior was determined in mice after parturition. The maternal crouching behavior (covering over pups) occurred in...Maternal care is essential for offspring survival. Circadian regulation of maternal behavior and feeding behavior was determined in mice after parturition. The maternal crouching behavior (covering over pups) occurred intensively in the late half of the dark phase (nighttime), referred to as the "crouching-dominant time." Interestingly, a rapid decrease in body temperature preceded the onset of crouching-dominant time. Feeding behavior during lactation increased in line with the increased energy demand. However, during the night, feeding behavior intensively occurred in the first half of the nighttime, as seen during the non-lactation period, and intermittent feeding behavior was added during the daytime. The clock genes expression of the suprachiasmatic nucleus (SCN) showed robust oscillation even during lactation. In contrast, marked changes in expression profiles of peroxisome proliferator-activated receptor α (PPARα)-regulated metabolic genes in the liver during lactation (postpartum 7 to 12 days) were dissociated from the peripheral clock control, compared to those in virgin mice. During lactation, a critical time for the survival of the species, the maternal circadian clock may regulate multiple important behaviors so that they can occur in the appropriate time frame while adapting to energy requirements.
Carpenter JS, Crouse JJ, Shin M
… +8 more, Tonini E, Hindmarsh G, de Haan Z, Iorfino F, Robillard R, Naismith S, Scott EM, Hickie IB
J Biol Rhythms
· 2025 Oct · PMID 40662977
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Despite evidence for links between circadian dysfunction and mood disorders, previous research has largely reported on single biological markers of circadian alignment. The available evidence on relationships between 2 i...Despite evidence for links between circadian dysfunction and mood disorders, previous research has largely reported on single biological markers of circadian alignment. The available evidence on relationships between 2 internal phase markers (e.g., dim light melatonin onset [DLMO] and peak cortisol concentration) suggests these signals may be temporally misaligned in major depressive disorder with greater misalignment associated with more severe depressive symptoms. This study aimed to examine multiple circadian phase markers to determine whether any youth with emerging mood disorders present with clear evidence of internal circadian misalignment, and whether the degree of circadian misalignment is correlated with more severe mood symptoms. Cross-sectional data from 69 youth presenting for mental health care (20.6 ± 3.8 years; 39% male) and 19 healthy controls (24.0 ± 3.6 years; 53% male) included actigraphy monitoring; overnight in-lab measurement of 3 phase markers: DLMO, salivary cortisol peak (CORT), and core body temperature nadir (TEMP); and depressive symptoms (Hamilton Depression Rating Scale). Abnormal phase angles between 2 phase markers were defined as ±2 standard deviations beyond the control mean. In those with emerging mood disorders, earlier TEMP relative to other phase markers (DLMO, CORT, sleep midpoint) was associated with higher depressive symptoms. Sixteen individuals (23%) with emerging mood disorders had abnormal phase angles between at least 1 pair of phase markers, consistent with internal misalignment of the circadian system. The internal misalignment subgroup had later DLMO on average, however presented with a diverse range of individual phase angle abnormalities. Diverse disruptions of circadian alignment occur in youth with mental ill-health. The relative timing of core body temperature and melatonin rhythms may be key circadian features linked to depressive symptoms. Longitudinal research is needed to establish whether correction of circadian misalignment is relevant to treatment of mood syndromes in youth with evidence of disrupted circadian systems.
The analysis of long-term variation patterns in heart rate (HR) and heart rate variability (HRV) provides insights into autonomic nervous system function beyond short-term recordings taken under resting or experimental c...The analysis of long-term variation patterns in heart rate (HR) and heart rate variability (HRV) provides insights into autonomic nervous system function beyond short-term recordings taken under resting or experimental conditions. Yet, traditional processing pipelines often require time- and labor-intensive visual inspection of electrocardiography (ECG) data and manual artifact removal. This study evaluated the performance of 3 code-based fully automated batch-processing pipelines-, , and -against the manual gold standard utilizing Kubios for both (diurnal) HR and HRV estimates derived from raw 48-h ECG recordings. Results illustrate that while automated pipelines yield HR estimates in good agreement to the gold standard ( = 0.91-0.99; α = 0.90-0.99), HRV estimates exhibit greater deviations ( = 0.66-0.87; α = 0.76-0.90). Cosinor analyses of diurnal HR patterns indicate strong consistency between Kubios and NeuroKit2 ( = 0.94-0.99; α = 0.97-0.99), but weaker correlations with RHRV and Systole ( = 0.58-0.87; α = 0.63-0.93). HRV cosinor parameters showed even larger discrepancies, with parameter-dependent correlations ranging from = 0.41 to 0.86 and Cronbach's alphas from α = 0.59 to 0.91. Findings suggest that automated batch processing of ECG data for analyzing diurnal variation patterns in HR and HRV produces results that show moderate to good agreement with the gold standard including visual inspection and manual processing. However, caution is warranted, as existing toolboxes and pipelines may lead to different results.
The circadian neuronal network in the brain comprises central pacemaker neurons and associated input and output pathways. These components work together to generate coherent rhythmicity, synchronize with environmental ti...The circadian neuronal network in the brain comprises central pacemaker neurons and associated input and output pathways. These components work together to generate coherent rhythmicity, synchronize with environmental time cues, and convey circadian information to downstream neurons that regulate behaviors such as the sleep/wake cycle. To mediate these functions, neurotransmitters and neuromodulators play essential roles in transmitting and modulating signals between neurons. In , approximately 240 brain neurons function as clock neurons. Previous studies have identified several neurotransmitters and neuromodulators, including the Pigment-dispersing factor (PDF) neuropeptide, along with their corresponding receptors in clock neurons. However, our understanding of the neurotransmitters and receptors involved in the circadian system remains incomplete. In this study, we conducted a T2A-GAL4-based screening for neurotransmitter and receptor genes expressed in clock neurons. We identified 2 neurotransmitter-related genes and 22 receptor genes. Notably, while previous studies had reported the expression of 6 neuropeptide receptor genes in large ventrolateral neurons (l-LN), we also found that 14 receptor genes-including those for dopamine, serotonin, and γ-aminobutyric acid-are expressed in l-LN neurons. These findings suggest that l-LN neurons serve as key integrative hubs within the circadian network, receiving diverse external signals.
J Biol Rhythms
· 2025 Oct · PMID 40579934
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Measuring and analyzing personal light exposure has become increasingly important in circadian and myopia research. Very small measurement values in light exposure patterns, especially zero, are regularly recorded in fie...Measuring and analyzing personal light exposure has become increasingly important in circadian and myopia research. Very small measurement values in light exposure patterns, especially zero, are regularly recorded in field studies. These zero-lux values are problematic for commonly applied logarithmic transformations and should neither be dismissed nor be unduly influential in visualizations and statistical modeling. We compare 4 ways to visualize such data on a linear, logarithmic, hybrid, or symlog scale, and we model the light exposure patterns with a generalized additive model by removing zero-lux values, adding a very small or -1 log lux value to the dataset, or using the Tweedie error distribution. We show that a -transformed visualization, implemented in , displays relevant features of light exposure across scales, including zero-lux, while reducing the emphasis on the small values (<1 lux). is well-suited to visualize differences in light exposure covering heavy-tailed negative values. We further show that small but not negligible value additions to the light exposure data of -1 log lux for statistical modeling allow for acceptable models on a logarithmic scale, while very small values distort results. We also demonstrate the utility of the Tweedie distribution, which does not require prior transformations, models data on a logarithmic scale, and includes zero-lux values, capturing personal light exposure patterns satisfactorily. Data from field studies of personal light exposure require appropriate handling of zero-lux values in a logarithmic context. scales for visualizations and an appropriate addition to input values for modeling, or the Tweedie distribution, provide a solid basis. Beyond light exposure, other time-series data relevant to biological rhythms, such as accelerometry for ambulatory sleep scoring in humans or wheel-running in animal models, exhibit zero inflation and can benefit from the methods introduced here.
Time-restricted feeding (TRF) can improve metabolic outcomes. Rodents experiencing TRF exhibit an increase in spontaneous locomotor activity before mealtime and show a phase shift in the rhythm of clock gene expression i...Time-restricted feeding (TRF) can improve metabolic outcomes. Rodents experiencing TRF exhibit an increase in spontaneous locomotor activity before mealtime and show a phase shift in the rhythm of clock gene expression in peripheral organs, particularly in the liver. Because activation of the transient receptor potential vanilloid-1 (TRPV1) channel produces similar beneficial effects on metabolism as TRF, we hypothesized that this channel mediates the metabolic changes induced by TRF. To assess the role of TRPV1 in metabolism and circadian responses, we utilized the agonist resiniferatoxin (RTX), which at a dosage of 20 µg/kg desensitizes TRPV1. After treatment with RTX or its vehicle, adult male rats were exposed to 21 days of TRF during the light phase. RTX-treated rats show some effects of TRF similar to vehicle-treated controls, with increased locomotor activity and body temperature at the beginning of the light phase, decreased body weight gain and food intake relative to -fed controls. However, RTX-treated rats did not show a decrease in VO consumption or an improvement in glucose tolerance induced by TRF. In addition, RTX treatment eliminated the temporal changes in the expression of clock genes and in the liver as well as leptin blood levels. In addition, RTX abolished the temporal alterations of the gene in the liver, which encodes a protein that negatively modulates insulin signaling without affecting the expression of insulin, , or other clock genes in the liver. In conclusion, TRPV1 may participate in the TRF-induced alterations in metabolism, most likely through its regulation of the temporal changes in , , and expressions in the liver, along with leptin secretion.
Nthlane RA, Scheuermaier K, Mkhize SA
… +1 more, Michel FS
J Biol Rhythms
· 2025 Aug · PMID 40537446
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Postmenopausal shift workers face increased cardiometabolic risk due to estrogen decline and shift work-induced circadian misalignment. Yet, their combined effects remain poorly understood, especially in hypertensive ind...Postmenopausal shift workers face increased cardiometabolic risk due to estrogen decline and shift work-induced circadian misalignment. Yet, their combined effects remain poorly understood, especially in hypertensive individuals. This study investigated whether circadian misalignment worsens cardiometabolic parameters in a hypertensive ovariectomized rat model. Female spontaneously hypertensive rats (SHR) were ovariectomized or sham-operated (7-week-old), and then exposed to a chronic phase shift (CPS) protocol or a control light schedule for 10 weeks ( = 9 per group). Measurements included body mass, food and water intake, blood pressure (BP), fasting glucose, glucose tolerance, organ masses, and low-density lipoprotein (LDL) concentration. Ovariectomized rats were heavier and had greater food intake and organ masses than sham-operated rats. However, food intake and organ masses were reduced relative to body mass. CPS rats had greater water intake and reduced liver mass than control light rats. In addition, ovariectomized rats showed lower glucose concentration than sham-operated rats, whereas CPS rats showed higher glucose concentration than control light rats during the oral glucose tolerance test. Moreover, the CPS rats had higher systolic BP. The LDL and fasting glucose concentrations were similar. No interaction between ovariectomy and CPS was observed. These findings suggest that estrogen deficiency increases body mass, but does not worsen cardiometabolic parameters in female SHR. CPS-induced circadian misalignment altered water intake, liver mass, systolic BP and glucose tolerance in the CPS condition in female SHR. This study was unable to monitor physiological or behavioral indicators to confirm circadian misalignment by the CPS protocol. However, the findings provide novel insights into how CPSs independently impair cardiometabolic outcomes in female SHR, with implications for understanding risk in postmenopausal shift workers.
J Biol Rhythms
· 2025 Oct · PMID 40536001
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In the cyanobacterial circadian clock, a core oscillator comprising the proteins KaiA, KaiB, and KaiC keeps time based on a rhythmic phosphorylation of KaiC, and histidine protein kinases relay temporal information from...In the cyanobacterial circadian clock, a core oscillator comprising the proteins KaiA, KaiB, and KaiC keeps time based on a rhythmic phosphorylation of KaiC, and histidine protein kinases relay temporal information from the KaiABC complex to regulate gene expression. The kinases SasA and CikA engage directly with the oscillator and are responsible for modulating the phosphorylation and dephosphorylation throughout the circadian day of the response-regulator transcription factor RpaA; the phosphorylation state of RpaA in turn determines circadian gene expression. We recently showed that either CikA or SasA can drive rhythmic phosphorylation and DNA binding of RpaA in an in vitro system. However, when SasA is absent in vivo, a bioluminescence reporter gene shows a very low expression and amplitude rhythm, indicating CikA kinase activity is not sufficient to activate gene expression. We questioned why CikA cannot serve as a robust kinase for RpaA in the absence of SasA in the cell. Here, we investigated post-translational modifications of CikA and found KaiC-dependent phosphorylation sites of CikA that dramatically affect its activity. Phosphomimetic mutants of these sites showed that the phosphorylated version of CikA is not functional. Our data show that inverse correlation of KaiC levels and these inhibitory phosphorylation sites can explain the lower CikA activity in a SasA knockout background. We conclude that these phosphorylation sites act as a rheostat for CikA activity and are regulated by KaiC levels.
Beck AI, Caldart CS, Ben-Hamo M
… +6 more, Weil TA, Perez JG, Kalume F, Brunton BW, de la Iglesia HO, Sanchez REA
J Biol Rhythms
· 2025 Aug · PMID 40476387
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Accurately capturing the temporal distribution of polysomnographic sleep stages is critical for the study of sleep function, regulation, and disorders in higher vertebrates. In laboratory rodents, scoring of electrocorti...Accurately capturing the temporal distribution of polysomnographic sleep stages is critical for the study of sleep function, regulation, and disorders in higher vertebrates. In laboratory rodents, scoring of electrocorticography (ECoG) and electromyography (EMG) recordings is usually performed manually by categorizing 5- to 10-sec epochs as 1 of 3 specific stages: wakefulness, rapid-eye-movement (REM) sleep, and non-REM (NREM) sleep. This process is laborious, time-consuming, and particularly impractical for large experimental cohorts with recordings lasting longer than 24 h, which are critical for the study of the circadian regulation of sleep. To circumvent this problem, we developed an open-source Python toolkit, Sleep Identification Enabled by Supervised Training Algorithms (SIESTA), that automates the detection of these 3 main behavioral stages in mice. We used a supervised machine learning algorithm that extracts features from the ECoG and EMG signals and autonomously scores recordings with a hierarchical classifier based on using logistic regression. We evaluated this approach on data collected from wild-type mice housed under both normal and different lighting conditions, as well as from mutant mouse lines with abnormal sleep phenotypes and from rats. We obtained mean F scores 0.94 for wakefulness, 0.94 for NREM, and 0.74 for REM, and followed up by validating SIESTA with manually scored data from 3 other laboratories. SIESTA has a user-friendly interface that can be used without coding expertise. To our knowledge, this is the first time that such a strategy has been developed using all open-source and freely available resources. Our aim is that SIESTA becomes a useful tool that facilitates further research in sleep on rodent models.