Circadian clocks orchestrate metabolic processes in the whole body and their response to food. Therefore, not only what and how much, but also when we eat has a significant impact on metabolism. In this context, daytime of carbohydrate and fat intake was recently shown to alter the metabolic state and potentially affect the disease risk. However, molecular mechanisms of this regulation in humans are poorly understood. In this crossover trial, we investigated the effects of two isocaloric 4-week dietary patterns - high carbohydrate in the morning and high fat in the afternoon (HC/HF) and the reverse (HF/HC) - on the subcutaneous adipose tissue (SAT) in overweight non-diabetic men. The SAT transcriptome was assessed using microarrays. Additionally, gene expression in peripheral blood monocytes (PBMCs) was analysed by qPCRs. Analysis of SAT samples collected across the day identified 1386 genes exhibiting diurnal oscillations. In SAT, both oscillatory and non-oscillatory genes related to lipid and glucose metabolism were modulated by the timing of macronutrient intake. Notably, expression of inflammatory response genes in SAT was elevated after HC/HF compared to HF/HC, suggesting that the HC/HF diet might promote an early proinflammatory state in SAT due to higher fat intake in the afternoon. Diet-induced remodelling of the SAT transcriptome was partly reflected in PBMCs. These findings demonstrate that diurnal macronutrient distribution significantly reshapes the SAT transcriptome, underscoring the relevance of eating timing-based (chrononutritional) strategies for prevention of metabolic dysfunction and systemic inflammation associated with obesity and type 2 diabetes.

Circadian clocks regulate essential cellular functions and influence cancer development and treatment outcomes. Aligning therapy with circadian rhythms can improve efficacy and reduce toxicity, yet whether neuroblastoma, a heterogeneous pediatric tumor, maintains circadian function remains unclear. Here, we systematically profiled circadian dynamics across 12 neuroblastoma cell models using long-term bioluminescence assays and computational analysis. Our findings reveal heterogeneous circadian patterns ranging from robust to arrhythmic, which we linked to distinct neuroblastoma genetic features. By integrating drug sensitivity data, we identified candidate compounds whose effectiveness correlates with circadian expression profiles. Moreover, time-of-day treatment assays with the ALK inhibitor lorlatinib and frontline chemotherapeutics revealed distinct temporal drug responses that were more pronounced in circadian-competent than weakly rhythmic cell lines. Together, these findings establish circadian heterogeneity as a previously unrecognized dimension of neuroblastoma biology and highlight the therapeutic potential of chronotherapy approaches for improved treatment efficacy.

Background:Metabolic disorders are closely linked to sleep disturbances. Time-restricted eating (TRE) can improve metabolic disturbances, but its impact on sleep quality is insufficiently studied and recommendations regarding the eating timing in TRE are pending. Our aim was to investigate the impact of early TRE (eTRE) and late TRE (lTRE) on sleep quality in obesity. Methods:This is a secondary analysis of the randomized crossover trial, which included 31 women with overweight and obesity. Following a 2-4 week baseline period, participants were assigned to either a two-week eTRE (eating 8 a.m-4 p.m.) or a two-week lTRE (eating 1 p.m.-9 p.m.), separated by a two-week washout phase. Sleep metrics were assessed objectively by blinded actigraphy and subjectively using Pittsburgh Sleep Quality Index (PSQI) and self-report of sleep quality. Hunger and satiety were examined using a Visual Analogue Scale (VAS). Results:Actigraphy revealed no between-intervention differences in changes in sleep metrics, but improvements were observed within eTRE compared with baseline for sleep efficiency (p = 0.047), sleep fragmentation index (SFI) (p = 0.029), and awakening length (p = 0.043). Individuals with lowest sleep quality at the baseline showed its largest improvements in eTRE. PSQI scores and self-reported sleep quality remained unchanged between and within both interventions. There were no differences in evening hunger and satiety scores between eTRE and lTRE, and no correlations between hunger or satiety and sleep quality. Conclusions:eTRE, but not lTRE, improved objectively assessed sleep quality, and these changes were not related to hunger or satiety. eTRE may be a more effective strategy for improving wellbeing and sleep-related health outcomes. ClinicalTrials.gov number, NCT04351672 (registered on April 17, 2020).

Circadian timing influences human physiology and disease risk, yet scalable measures of molecular circadian phase are lacking. Here we infer circadian phase from circulating blood biomarkers in UK Biobank. Among 3,228 plasma biomarkers, 58% exhibit significant diurnal variation, with harmonic modeling identifying acrophase clustering consistent with canonical circadian patterns and independent constant-routine datasets. Machine-learning models trained on plasma proteomics predict sampling time (R²≈0.68) and retain substantial accuracy with ∼60 proteins. We define a novel construct, circadian acceleration (CA), as deviation from the population-average phase; CA is temporally stable, associates with chronotype and shift work, and responds to environmental perturbation. CA is heritable (h² SNP ≈0.10) and genetically correlated with chronotype and accelerometry-derived sleep traits. These results establish plasma proteomics as a scalable approach for population-level molecular circadian phenotyping.

In the mammalian circadian clock, the transcription factor CLOCK/BMAL1 cycles between an active state recruiting co-activators like MLL1 and repressed states associated with PER1/2 and CRY1/2 clock proteins. The MLL1 complex component WDR5 was also found in a repressive PER complex. Here we show that WDR5 directly binds to the CRY binding domain (CBD) regions of PER1 and PER2. PER2 binds WDR5 via a WBM motif within the PER2/CRY interface, imposing competition between PER2/WDR5-, PER2/CRY- and RbBP5/WDR5 complexes. PER1 binds WDR5 predominantly via a WIN motif outside the CBD, enabling formation of trimeric PER1WIN/WDR5/RbBP5WBM - and PER1/WDR5/CRY complexes. WDR5 WIN site inhibitors weaken PER1/WDR5 interactions and impact cellular Bmal1- and Per2 expression dependent on the PER1 WIN motif, likely modulating the primary BMAL1/CLOCK/PER/CRY- and secondary BMAL1/REV-ERB/ROR circadian feedback loop. Together, our studies revealed a role of WDR5 as a direct PER interaction partner with critical differences between PER1 and PER2. Here the authors reveal that the mammalian clock proteins PERIOD 1 and 2 (PER1, PER2) interact with WDR5 using different WDR5 binding sites and that disrupting the PER1/WDR5 complex with WDR5 WIN site inhibitors affects circadian clock function.

Circadian rhythms-self-sustained, ~24-h oscillations in transcript and protein levels-are generated by a cell-autonomous molecular clock. These rhythms shape how individual cells respond to external signals, influencing key decisions such as differentiation and apoptosis. However, current tools for visualizing circadian rhythms at the single-cell level often rely on genomic engineering and clonal expansion, limiting their accessibility and applicability. We present fluorescent circadian reporters based on the murine REVERBα/Nr1d1 gene, delivered via lentiviral transduction and compatible with time-lapse single-cell microscopy. These reporters produce oscillatory signals that depend on a functional circadian clock and can be used to determine a cell's circadian dynamics parameters, such as circadian phase. Their simple and efficient delivery should make them suitable for a wide variety of cell types, greatly expanding opportunities to study single-cell circadian dynamics and their impact across diverse biological processes and systems.

Keeping cellular circadian clocks synchronised is critical for cellular and physiological health. Glucocorticoid (GC) hormones are the most potent systemic timing signal for cellular clocks, acting via the glucocorticoid receptor (GR), a transcription factor conventionally thought to synchronise cells by translocating into the nucleus and activating the transcription of clock genes. Our findings overturn this model: acute changes in GR translocation and nascent transcription are both dispensable for GC-resetting of the circadian clock. Instead, physiological GC pulses act post-transcriptionally to rapidly increase Period1 mRNA through enhanced RNA processing, not synthesis. This elevates PER1 protein production which, alone, is sufficient to reset the clock. Yet each pulse resets only a subset of cells, explaining why, in vivo , repeated daily hormonal cues are required for full re-entrainment after jet lag or shift work. We propose a new model for hormonal signalling to the circadian clock and identify RNA processing kinetics as a general regulatory lever for nuclear receptor signalling.

Introduction Approximately one in five workers in Europe is engaged in shift work. Studies reveal that night shift work leads to an increased risk of overweight, obesity and related diseases. Yet, the biological and behavioural mechanisms underlying these associations are not fully understood. The cross-sectional and mechanistic studies within the European SHIFT2HEALTH project aim to investigate biological, behavioural and psychosocial key risk factors responsible for the association between night work and obesity across five European countries. Methods A multi-centric cross-sectional study is designed to unravel obesogenic risk factors, eating habits and sleep patterns in night shift workers and day workers from the health- and various industrial sectors. Recruitment takes place in Austria, Denmark, Germany, Poland, and the Netherlands, aiming at 500 night shift workers and 500 day workers. Anthropometric measurements, sensory perception and food preference tests are performed, alongside extensive questionnaires. In addition, biological samples (blood, hair, urine, faeces) are collected for biomarker measurements of inflammation, oxidative stress, glycaemic and lipaemic parameters, for microbiome and metabolomics analyses and chronotype assessment. In a nested mechanistic study, night shift workers (N=200) recruited in Austria and in the Netherlands, additionally collect urine samples from all voids over 24 hours during a day shift and a night shift, as well as dried blood spots and tongue swabs at four time points and undergo continuous sleep, activity and light exposure monitoring through actigraphy. The association between night shift work and its metrics with levels of pre-obesity biomarkers will be evaluated in crude and multivariable-adjusted regression models, adjusting for potential confounders. Stratified analyses by age, gender, sector and chronotype will be conducted. Conclusion In the cross-sectional and mechanistic studies of the SHIFT2HEALTH project, biological, behavioural and psychosocial factors of night shift workers will be compared with those of day workers across sectors. The outcomes of these studies will serve as a basis for future intervention studies and, together, will contribute to the development of strategies to prevent and reduce overweight and obesity with the aim to improve the health and wellbeing of night shift workers. Trial registration: clinicaltrials.gov, ID: NCT06288568

Circadian clocks govern daily physiological and behavioral processes and are crucial for health; disruptions can lead to various diseases. The circadian phase of entrainment-the phase of the internal circadian clock in relation to external environmental cycles-is influenced by both genetic and environmental factors, varies between individuals, and is reflected in daily behaviors such as sleep-wake patterns, cognitive performance, and physical activity. While circadian phase may also fluctuate within individuals, the dynamics and extent of such variation in daily life remain largely unexplored. The gold standard for circadian phase assessment, dim-light melatonin onset (DLMO), is impractical for large-scale studies, and blood-based molecular biomarkers, while promising, are limited in feasibility. To address these challenges, we developed HairTime, a noninvasive assay that estimates circadian phase from a single daytime hair sample. Developed and evaluated in two steps-a training and a validation study-HairTime demonstrated strong predictive power compared to DLMO. Suitable for large-scale studies, it was assessed using over 4,000 samples. Circadian phase estimations showed a normal distribution and were associated with age, sex, and notably, work schedules, with earlier timing on workdays, suggesting that societal factors can modulate internal rhythms. Together, these findings establish HairTime as a promising tool for assessing circadian phase in research and lay the foundation for future applications in personalized chronotherapy.

Circadian clocks govern daily physiological and behavioral processes and are crucial for health, yet disruptions can lead to various diseases. Chronotype, the state of circadian timing, varies between individuals and is reflected in behaviors such as sleep-wake patterns, cognitive performance, and physical activity. This interindividual variability is influenced by both genetic factors and environmental cues, but the relative contributions of each remain unclear, particularly in terms of plasticity - how much chronotype can shift in response to lifestyle and environmental factors. The gold standard for chronotype assessment, dim-light melatonin onset (DLMO), is invasive and impractical for large-scale studies, while blood-based molecular biomarker tests, which estimate internal time, show promise but are limited by practicality. Here, we introduce HairTime, a novel assay that estimates chronotype from a single hair sample collected at one point during the day. HairTime was developed and evaluated in two studies: a training study and a validation study, where it demonstrated a strong ability to predict chronotype, with DLMO as the comparison. This non-invasive method is suitable for large-scale, longitudinal studies and clinical practice. We assessed HairTime using over 4,000 samples, observing a normal distribution of chronotype across the population, with its estimation associating with age, sex, and notably, work schedules. The correlation with work schedules reveals the plasticity of chronotype, as workdays shift circadian timing earlier, highlighting how societal factors can influence and modify an individual's internal rhythm. Additionally, we explore the concept of circadian amplitude, finding that lower amplitude rhythms in hair follicle cells are linked to reduced chronotype prediction accuracy. Our results highlight how both intrinsic circadian mechanisms and external factors, such as lifestyle and work schedules, shape chronotype. HairTime offers an innovative tool for understanding circadian rhythms, facilitating personalized chronotherapy, and improving health outcomes by aligning treatments with an individual's biological rhythms. ### Competing Interest Statement Authors B.M and A.K. are shareholders of BodyClock Technologies GmbH, which provided data for this study. The company's business model is based on intellectual property described in this publication. All other authors declare that they have no competing financial or personal interests that could have influenced the research presented in this article.

Time-restricted eating (TRE) is a promising strategy against metabolic disorders, but its effects on lipid metabolism remain controversial. The present research assesses and compares the impact of early (eTRE) versus late (lTRE) TRE on the plasma lipidomic profile. This is an exploratory outcome of the previously published randomized crossover trial, which examines 31 women with overweight or obesity who follow a two-week eTRE and a two-week lTRE in an intended isocaloric setting. Blood plasma and subcutaneous adipose tissue biopsies are analyzed using shotgun lipidomics and transcriptomics, respectively. Between interventions and within the lTRE, lipid species and classes, as well as enzyme activity indices, are not substantially changed. Within the eTRE, changes are observed for 103 lipid species, including a reduction of ceramide and phosphatidylcholine classes, and for the desaturation indices D5D, D6D, and D9D, as well as the elongation index ELOVL6. Combined analysis of plasma lipidome and adipose tissue reveals alterations in the glycerophospholipid pathway and in the expression of phospholipase enzymes PLB1, PLA2G6, and PLAG4B, dependent on TRE timing. These results suggest that eating timing during TRE may be crucial for remodeling the plasma lipidome and adipose tissue transcriptome and highlight the need of future lipidomic research in TRE.

Single-cell circadian oscillators exchange extracellular information to sustain coherent circadian rhythms at the tissue level. The circadian clock and the cell cycle couple within cells but the mechanisms underlying this interplay are poorly understood. We show that the loss of extracellular circadian synchronization disrupts circadian and cell cycle coordination within individual cells, impeding collective tissue growth. We use the theory of coupled oscillators combined with live population, and single-cell recordings and precise experimental perturbations. Coherent circadian rhythms yield oscillatory growth patterns, which unveil a global timing regulator of tissue dynamics. Knocking out core circadian elements abolishes the observed effects, highlighting the central role of circadian clock regulation. Our results underscore the role of tissue-level circadian disruption in regulating proliferation, thereby linking disrupted circadian clocks with oncogenic processes. These findings illuminate the intricate interplay between circadian rhythms, cellular signalling and tissue physiology and enhance our understanding of tissue homeostasis and growth regulation in the context of both health and disease.

The circadian clock regulates key physiological processes, including cellular responses to DNA damage. Circadian-based therapeutic strategies optimize treatment timing to enhance drug efficacy and minimize side effects, offering potential for precision cancer treatment. However, applying these strategies in cancer remains limited due to a lack of understanding of the clock's function across cancer types and incomplete insights into how the circadian clock affects drug responses. To address this, we conducted deep circadian phenotyping across a panel of breast cancer cell lines. Observing diverse circadian dynamics, we characterized metrics to assess circadian rhythm strength and stability in vitro. This led to the identification of four distinct circadian-based phenotypes among 14 breast cancer cell models: functional, weak, unstable, and dysfunctional clocks. Furthermore, we demonstrate that the circadian clock plays a critical role in shaping pharmacological responses to various anti-cancer drugs and we identify circadian features descriptive of drug sensitivity. Collectively, our findings establish a foundation for implementing circadian-based treatment strategies in breast cancer, leveraging clock phenotypes and drug sensitivity patterns to optimize therapeutic outcomes.

Because 80%-90% of our time is spent indoors and daylight is the main synchronizer of the central biological clock, the chronic lack of daylight is increasingly considered as a risk factor for metabolic diseases, such as type 2 diabetes. In a randomized crossover design (NCT05263232), 13 individuals with type 2 diabetes were exposed to natural daylight facilitated through windows vs. constant artificial lighting during office hours for 4.5 consecutive days. Continuous glucose monitoring revealed that participants spent more time in the normal glucose range, and whole-body substrate metabolism shifted toward a greater reliance on fat oxidation during daylight. Primary myotubes cultured from skeletal muscle biopsies displayed a phase advance after daylight exposure. Multi-omic analyses revealed daylight-induced differences in serum metabolites, lipids, and monocyte transcripts. Our findings suggest that natural daylight exposure has a positive metabolic impact on individuals with type 2 diabetes and could support the treatment of metabolic diseases.

BACKGROUND:Circadian rhythms are often severely disrupted in critically ill patients in the ICU. The ICU environment, characterized by irregular light-dark signals, continuous nutrition, and round-the-clock interventions, contributes to this disruption by providing weak and conflicting timing cues to the circadian system. Extensive scientific research has demonstrated that circadian rhythms play a vital role in regulating physiology and maintaining overall health. Therefore, integrating circadian principles into critical care may represent a promising strategy to improve patient outcomes in the ICU. RESEARCH QUESTION:What are the key challenges of integrating circadian medicine into critical care, what steps can address these challenges, and which recommendations can guide future study designs and clinical implementation? STUDY DESIGN AND METHODS:We convened a 5-day workshop in September 2024 that brought together 24 international experts with backgrounds in circadian biology, critical care, and implementation science. Each day was organized around a predefined theme, with morning presentations and plenary discussions, and afternoons dedicated to drafting a list of Propositions and Recommendations in breakout groups. Propositions and Recommendations were finalized via a post-workshop survey requiring ≥ 75% agreement. RESULTS:This roadmap summarizes the discussions and outcomes of the workshop, structured around a set of Propositions and Recommendations, and provides a framework for building a robust evidence base for integrating circadian principles into ICU practice. Key recommendations include the development of circadian outcome measures tailored for use in the ICU and using standardized frameworks for evaluating the effect of circadian interventions in clinical trials. INTERPRETATION:Altogether, this roadmap provides an interdisciplinary framework resulting from a collaborative effort of ICU clinicians, circadian biologists, and implementation specialists, for building a robust evidence base for integrating circadian principles into ICU research and practice.

Time-restricted eating (TRE) is a promising strategy to improve metabolic outcomes. However, it remains unclear whether TRE has cardiometabolic benefits in an isocaloric setting and whether its effects depend on the eating timing. We conducted a randomized crossover trial in 31 women with overweight or obesity to directly compare the effects of a 2-week early TRE (eTRE; eating from 8:00 to 16:00) and a 2-week late TRE (lTRE; eating from 13:00 to 21:00) on insulin sensitivity, cardiometabolic risk factors, and the internal circadian phase. During the restricted 8-hour eating period, participants were asked to consume their habitual food quality and quantity. Insulin sensitivity did not differ between (-0.07; 95% CI, -0.77 to 0.62; P = 0.60) or within (eTRE: 0.31; 95% CI, -0.14 to 0.76; P = 0.11; lTRE: 0.19; 95% CI, -0.22 to 0.60; P = 0.25) interventions. Twenty-four-hour glucose, lipid, inflammatory, and oxidative stress markers showed no clinically meaningful between- or within-intervention differences. Participants demonstrated high timely adherence (eTRE, 96.5%; lTRE, 97.7%), unchanged dietary composition and physical activity, minor daily calorie deficit (eTRE, -167 kilocalories/day), and weight loss (eTRE, -1.08 kilograms; lTRE, -0.44 kilograms). In lTRE, the circadian phase in blood monocytes (24 minutes; 95% CI, -5 to 54 minutes; P = 0.10) and sleep midpoint (15 minutes; 95% CI, 7 to 23 minutes; P < 0.001) occurred later compared with eTRE. Overall, in an intended isocaloric setting, neither eTRE nor lTRE improves insulin sensitivity or other cardiometabolic traits, despite a shift of internal circadian clocks.

Aims: Time-restricted eating (TRE) is a promising dietary strategy to prevent and treat obesity and type 2 diabetes. In the past, only few studies have examined the effects of the TRE on plasma lipidome, and no study has investigated possible differences in this regard between early (eTRE) and late TRE (lTRE). Our study aimed to fill this gap to better understand and compare the impact of both eTRE and lTRE on lipid metabolism. Methods: A crossover study was conducted with 31 overweight or obese non-diabetic women (age: 62 (53-65) years). They followed both a two-week eTRE (eating window: 8 a.m. to 4 p.m.) and a two-week lTRE (eating window: 1 p.m. to 9 p.m.). Before and after both interventions, blood plasma was tested for lipids of 14 lipid classes using high throughput shotgun plasma lipidomics. Totally 300 lipid species were quantified. Results: The eTRE intervention affected 103 lipid species significantly reducing ceramide (P = 0.043) and phosphatidylcholine (P = 0.043) classes. After eTRE, the delta-5 desaturase (D5D) (P = 0.002) and stearyl-CoA desaturase 1 (SCD1/D9D for C18) (P = 0.007) activity indices increased, while the delta-6 desaturase (D6D) (P = 0.035) and elongation (ELOVL6) (P < 0.001) indices decreased, showing alterations of the double bond number in 12 lipid classes. lTRE resulted in a D5D increase (P < 0.001), but no other substantial changes of lipid species and classes were observed. Combined analysis of plasma lipidome and subcutaneous adipose tissue biopsies revealed alterations in the glycerophospholipid pathway and the expression of phospholipase enzymes PLB1, PLA2G6, and PLAG4B by the TRE timing. Conclusion: Our investigation revealed that eTRE has more pronounced effects on plasma lipidome compared to lTRE. Our study is the first evidence that the TRE impact on the lipid metabolism depends on the timing of the eating window which has to be considered in future research. ### Competing Interest Statement Kai Simons and Christian Klose are shareholders of Lipotype GmbH. Mathias J. Gerl and Markus Damm are employees of Lipotype GmbH. Other authors declare no competing interests. ### Clinical Trial NCT04351672 ### Funding Statement The study was supported by the German Research Foundation (DFG RA 3340/3-1, project number 434112826 and DFG RA 3340/4-1, project number 530918029 to OP-R), by the German Diabetic Association (Allgemeine Projektfoerderung der DDG 2020 and 2023 to OP-R); and by the European Association for Study of Diabetes 2020 (Morgagni Prize 2020 to OP-R). Funders were not involved in preparation of study design and implementation of the study; data collection, management, data analyzation, and interpretation; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol and informed consent form were approved by the Medical Ethics Committee of the University of Potsdam I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

Circadian clocks are cell-autonomous oscillators that are present in most cells of the body and temporally coordinate their function. Alignment of cellular clocks with each other and the environment is mediated mostly through blood borne signals. Although serum is a potent resetting signal for circadian clocks, the underlying intracellular molecular underpinnings are largely unknown. Here, we employ Circa-SCOPE, a high-throughput single-cell method for constructing Phase Transition Curves (PTCs), to classify intracellular signaling pathways and clock-components that participate in clock resetting by serum. We identify steroid hormone, including sex-hormone receptors as key mediators of serum-induced phase resetting. Unexpectedly, we discover that Cry2 plays a central role in the response to serum and specifically to steroid hormones, irrespectively of its effect on the clock period-length. Furthermore, we find that PTCs are largely unaffected by the period-length. Overall, our findings provide important insight on intracellular determinant of the clock response to serum.