Circadian period is compensated for repressor protein turnover rates in single cells.

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

Time-of-day effects of cancer drugs revealed by high-throughput deep phenotyping.

The circadian clock, a fundamental biological regulator, governs essential cellular processes in health and disease. Circadian-based therapeutic strategies are increasingly gaining recognition as promising avenues. Aligning drug administration with the circadian rhythm can enhance treatment efficacy and minimize side effects. Yet, uncovering the optimal treatment timings remains challenging, limiting their widespread adoption. In this work, we introduce a high-throughput approach integrating live-imaging and data analysis techniques to deep-phenotype cancer cell models, evaluating their circadian rhythms, growth, and drug responses. We devise a streamlined process for profiling drug sensitivities across different times of the day, identifying optimal treatment windows and responsive cell types and drug combinations. Finally, we implement multiple computational tools to uncover cellular and genetic factors shaping time-of-day drug sensitivity. Our versatile approach is adaptable to various biological models, facilitating its broad application and relevance. Ultimately, this research leverages circadian rhythms to optimize anti-cancer drug treatments, promising improved outcomes and transformative treatment strategies.

The Ecology of Human Sleep (EcoSleep) Cohort Study: Protocol for a longitudinal repeated measurement burst design study to assess the relationship between sleep determinants and outcomes under real-world conditions across time of year.

The interplay of daily life factors, including mood, physical activity, or light exposure, influences sleep architecture and quality. Laboratory-based studies often isolate these determinants to establish causality, thereby sacrificing ecological validity. Furthermore, little is known about time-of-year changes in sleep and circadian-related variables at high resolution, including the magnitude of individual change across time of year under real-world conditions. The Ecology of Human Sleep (EcoSleep) cohort study will investigate the combined impact of sleep determinants on individuals' daily sleep episodes to elucidate which waking events modify sleep patterns. A second goal is to describe high-resolution individual sleep and circadian-related changes across the year to understand intra- and inter-individual variability. This study is a prospective cohort study with a measurement-burst design. Healthy adults aged 18-35 years (N = 12) will be enrolled for 12 months. Participants will continuously wear actimeters and pendant-attached light loggers. A subgroup will also measure interstitial fluid glucose levels (six paticipants). Every 4 weeks, all participants will undergo three consecutive measurement days of four ecological momentary assessments each day ('bursts') to sample sleep determinants during wake. Participants will also continuously wear temperature loggers (iButtons) during the bursts. Body weight will be captured before and after the bursts in the laboratory. The bursts will be separated by two at-home electroencephalogram recordings each night. Circadian phase and amplitude will be estimated during the bursts from hair follicles, and habitual melatonin onset will be derived through saliva sampling. Environmental parameters (bedroom temperature, humidity, and air pressure) will be recorded continuously.

Network switches and their role in circadian clocks.

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.

Coupling allows robust mammalian redox circadian rhythms despite heterogeneity and noise.

Circadian clocks are endogenous oscillators present in almost all cells that drive daily rhythms in physiology and behavior. There are two mechanisms that have been proposed to explain how circadian rhythms are generated in mammalian cells: through a transcription-translation feedback loop (TTFL) and based on oxidation/reduction reactions, both of which are intrinsically stochastic and heterogeneous at the single cell level. In order to explore the emerging properties of stochastic and heterogeneous redox oscillators, we simplify a recently developed kinetic model of redox oscillations to an amplitude-phase oscillator with 'twist' (period-amplitude correlation) and subject to Gaussian noise. We show that noise and heterogeneity alone lead to fast desynchronization, and that coupling between noisy oscillators can establish robust and synchronized rhythms with amplitude expansions and tuning of the period due to twist. Coupling a network of redox oscillators to a simple model of the TTFL also contributes to synchronization, large amplitudes and fine-tuning of the period for appropriate interaction strengths. These results provide insights into how the circadian clock compensates randomness from intracellular sources and highlight the importance of noise, heterogeneity and coupling in the context of circadian oscillators.

Circadian protein expression patterns in healthy young adults.

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

Are circadian amplitudes and periods correlated? A new twist in the story.

Three parameters are important to characterize a circadian and in general any biological clock: period, phase and amplitude. While circadian periods have been shown to correlate with entrainment phases, and clock amplitude influences the phase response of an oscillator to pulse-like zeitgeber signals, the co-modulations of amplitude and periods, which we term twist, have not been studied in detail. In this paper we define two concepts: parametric twist refers to amplitude-period correlations arising in ensembles of self-sustained clocks in the absence of external inputs, and phase space twist refers to the co-modulation of an individual clock's amplitude and period in response to external zeitgebers. Our findings show that twist influences the interaction of oscillators with the environment, facilitating entrainment, fastening recovery to pulse-like perturbations or modifying the response of an individual clock to coupling. This theoretical framework might be applied to understand the emerging properties of other oscillating systems.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).

Circadian rhythm disruption in critically ill patients.

Patients admitted to the intensive care unit (ICU) are in need of continuous organ replacement strategies and specialized care, for example because of neurological dysfunction, cardio-pulmonary instability, liver or kidney failure, trauma, hemorrhagic or septic shock or even preterm birth. The 24-h nursing and care interventions provided to critically ill patients significantly limit resting and/or recovery phases. Consecutively, the patient's endogenous circadian rhythms are misaligned and disrupted, which in turn may interfere with their critical condition. A more thorough understanding of the complex interactions of circadian effectors and tissue-specific molecular clocks could therefore serve as potential means for enhancing personalized treatment in critically ill patients, conceivably restoring their circadian network and thus accelerating their physical and neurocognitive recovery. This review addresses the overarching issue of how circadian rhythms are affected and disturbed in critically ill newborns and adults in the ICU, and whether the conflicting external or environmental cues in the ICU environment further promote disruption and thus severity of illness. We direct special attention to the influence of cell-type specific molecular clocks on with severity of organ dysfunctions such as severity of brain dysfunction, pneumonia- or ventilator-associated lung inflammation, cardiovascular instability, liver and kidney failure, trauma, and septic shock. Finally, we address the potential of circadian rhythm stabilization to enhance and accelerate clinical recovery.