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BACKGROUND. The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing. METHODS. To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes. RESULTS. We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost. CONCLUSION. The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock. FUNDING. This study was supported by the Bundesministerium fur Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany., Introduction Many fundamental aspects of human physiology, metabolism, and behavior display 24-hour rhythms. These are found at all levels--from cells, tissues, and organs to the entire organism (for a review [...]

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1−/−) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day–night differences were dampened in LysMBmal1−/− neutrophils. In LysMBmal1−/− mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1−/− mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI. [ABSTRACT FROM AUTHOR]

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (β-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous β-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of β-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated β-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally. Key words β-TrCP, PERIOD2, protein degradation, F-box protein, CKIζ, mathematical modeling, Circadian clocks are self-sustained oscillators that regulate daily rhythms in physiology, metabolism, and behavior, thus allowing organisms to synchronize with their environment even in the absence of external time cues [...]

Mathematical modeling is used to predict that differential PERIOD (PER) phosphorylation events can result in opposite period phenotypes. The interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts and this concept describes not only the familial advanced sleep phase syndrome phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants in Drosophila.

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (β-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous β-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of β-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated β-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally. [ABSTRACT FROM AUTHOR]

At the core of the mammalian circadian clock is a feedback loop in which the heterodimeric transcription factor CLOCK–Brain, Muscle Arnt-like-1 (BMAL1) drives expression of its negative regulators, periods (PERs) and cryptochromes (CRYs). Here, we provide evidence that CLOCK-Interacting Protein, Circadian (CIPC) is an additional negative-feedback regulator of the circadian clock. CIPC exhibits circadian regulation in multiple tissues, and it is a potent and specific inhibitor of CLOCK–BMAL1 activity that functions independently of CRYs. CIPC–CLOCK protein complexes are present in vivo, and depletion of endogenous CIPC shortens the circadian period length. CIPC is unrelated to known proteins and has no recognizable homologues outside vertebrates. Our results suggest that negative feedback in the mammalian circadian clock is divided into distinct pathways, and that the addition of new genes has contributed to the complexity of vertebrate clocks. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.

Objectives: The circadian rhythm of clinical symptoms in rheumatoid arthritis (RA) has been primarily attributed to circadian variations in humoral factors and hormones. In this study, we investigated circadian rhythms of cellular immunity in RA (CiRA study)., Methods: Peripheral blood of female postmenopausal patients with active RA (DAS 28 ≥ 4.2) (n=5) and female postmenopausal non-RA controls (n=5) was collected every 2 hours for 24 hours and analysed by flow cytometry, cytokine multiplex suspension array and quantitative RT-PCR of clock gene expression in isolated CD14+ monocytes. Endogenous circadian rhythms of macrophages were investigated by BMAL1-luciferase bioluminescence. Significance of circadian rhythms was tested by Cosinor analysis., Results: We found (i) circadian rhythms in the relative frequency of peripheral blood cell populations that were present in postmenopausal non-RA controls but absent in patients with active RA, (ii) circadian rhythms that were absent in non-RA controls but present in patients with RA and (iii) circadian rhythms that were present in both groups but with differences in peak phase or amplitude or amplitude/magnitude. The circadian rhythm in expression of the clock genes PER2 and PER3 in CD14+ monocytes was lost in patients with RA. The amplitude of BMAL1-luciferase bioluminescence tended to be lower in patients with RA than in non-RA controls., Conclusions: We conclude that (i) in RA some immune cell populations lose their normal circadian rhythms whereas others establish new 'inflammatory' circadian rhythms and (ii) these findings provide a good basis for further identifying pathophysiological aspects of RA chronobiology with potential therapeutic implications.

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (beta-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous beta-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of beta-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated beta-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.