Guidelines for Genome-Scale Analysis of Biological Rhythms

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them

Guidelines for Genome-Scale Analysis of Biological Rhythms

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding ‘big data’ that is conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them

Therapeutic Implications of Circadian Rhythms

Circadian rhythms are biological processes displaying endogenous and entrainable oscillations of about 24 hours. They are driven by a group of genes called clock genes that have been widely observed in plants, animals and even in bacteria. In mammals, the core clock genes are rhythmically expressed in both the suprachiasmatic nucleus (SCN), the master clock residing in the hypothalamus, and almost all peripheral tissues where they control numerous target genes in a circadian manner, and thus affect many physiological and biochemical processes. Evidence suggests that disruption of the circadian rhythms (or desynchronization) is a significant risk factor for the development of metabolic diseases, cardiovascular diseases, cancer and sleep disorders. Evidence also suggests that the disruption suppresses immune function and increases vulnerability to infectious diseases. Restoring or strengthening the circadian rhythm may be therapeutic for these conditions. This becomes exceptionally important in modern societies because many people are suffering from frequent desynchronization due to shift working, exposure to artificial light, travel by transmeridian air flight, and involvement in social activities. Besides, the temporal variations in the incidence and severity of many diseases, such as the onset of cardiovascular events, chronic obstructive pulmonary disease (COPD), inflammatory diseases and mental disorders have also drawn increasing attention to the circadian clock. The circadian rhythms affect not only the health status, but also the drug efficiency. The effects (and side effects) of many drugs vary with biological timing. The tolerance of many medications displays circadian variation as well. The timing of medical treatment in coordination with the body clock may significantly increase the desired effects of drugs, and lower the dose and toxicity. In addition, circadian rhythms can also be modulated by some therapeutic drugs, for example, melatonin and modafinil, which are used to treat circadian rhythm sleep disorders. In this Research Topic, we assemble a series of critical review and research articles that focus on the therapeutic implications of circadian rhythms. Topics include, but are not limited to: • Circadian disruption caused diseases or disorders and related intervention • Temporal manifestation of diseases or disorders and therapeutic implications • The effects of circadian rhythms on drugs • The effects of drugs on circadian rhythm

Phosphorylation regulating the ratio of intracellular CRY1 protein determines the circadian period

The core circadian oscillator in mammals is composed of transcription/translation feedback loop in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type CRY1 (WT), whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells

Effect of All-Trans-Retinoic Acid on the Development of Chronic Hypoxia-Induced Pulmonary Hypertension

application/pdfBackground: An earlier study showed that all-trans-retinoic acid (ATRA) prevents the development of monocrotalin-induced pulmonary hypertension (PH). The purpose of the present study was to determine the effect of ATRA on another model of chronic hypoxia-induced PH. Methods and Results: Male Sprague – Dawley rats were given 30 mg/kg ATRA or vehicle only by gavage once daily for 14 days during hypobaric hypoxic exposure. Chronic hypoxic exposure induced PH, right ventricular hypertrophy (RVH), and hypertensive pulmonary vascular changes. Quantitative morphometry of the pulmonary arteries showed that ATRA treatment significantly reduced the percentage of muscularized arteries in peripheral pulmonary arteries only with an external diameter between 15 and 50 μm. ATRA treatment also significantly reduced the medial wall thickness in small muscular arteries only with an external diameter between 50 and100 μm. Unfortunately, these reductions did not accompany the lowering of pulmonary artery pressure nor decrease in RVH. Chronic hypoxia-induced PH rats with ATRA had a loss in body weight. Chronic hypoxia increased the expression of endothelial nitric oxide synthase in the lung on western blotting and immunohistochemistry, in which ATRA treatment had no effect. Conclusions: The administration of ATRA might not have a therapeutic role in preventing the development of chronic hypoxia-induced PH, because of body weight loss and the subtle preventable effects of vascular changes.本文/Anesthesiology and Critical Care Medicine, Physiology, Pediatrics, Mie University School of Medicine8

Effect of All-Trans-Retinoic Acid on the Development of Chronic Hypoxia-Induced Pulmonary Hypertension

application/pdf内容の要旨・審査結果の要旨 / 三重大学大学院医学系研究科 臨床医学系講座 麻酔集中治療

Long-term in vivo recording of circadian rhythms in brains of freely moving mice.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle

Long-term in vivo recording of circadian rhythms in brains of freely moving mice.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle