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The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi‐timepoint drug administration strategies across a wide range of drugs. Here, droplet‐engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici‐plate as an in vitro high‐throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time‐dependent variations are demonstrated in toxicity on the Chronotoxici‐plate, highlighting the importance of considering time‐dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing. [ABSTRACT FROM AUTHOR]

Blue light photoreceptor cryptochrome 1 (CRY1) in herbaceous plants plays crucial roles in various developmental processes, including cotyledon expansion, hypocotyl elongation and anthocyanin biosynthesis. However, the function of CRY1 in perennial trees is unclear. In this study, we identified two ortholog genes of CRY1 (PagCRY1a and PagCRY1b) from Populus, which displayed high sequence similarity to Arabidopsis CRY1. Overexpression of PagCRY1 substantially inhibited plant growth and promoted secondary xylem development in Populus, while CRISPR/Cas9‐mediated knockout of PagCRY1 enhanced plant growth and delayed secondary xylem development. Moreover, overexpression of PagCRY1 dramatically increased anthocyanin accumulation. The further analysis supported that PagCRY1 functions specifically in response to blue light. Taken together, our results demonstrated that modulating the expression of blue light photoreceptor CRY1 ortholog gene in Populus could significantly influence plant biomass production and the process of wood formation, laying a foundation for further investigating the light‐regulated tree growth. Summary statement: Light is one of the most important environmental signals that govern plant growth and development. This study demonstrated that the blue light photoreceptor PagCRY1 substantially promotes wood formation and anthocyanin biosynthesis in Populus, and provides evidence showing that HY5 and BBX6 may act downstream of PagCRY1 to activate lignin and anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

Photoreceptor cryptochromes (CRYs) mediate blue‐light regulation of plant growth and development. It has been reported that Arabidopsis CRY1and CRY2 function by physically interacting with at least 84 proteins, including transcription factors or co‐factors, chromatin regulators, splicing factors, messenger RNA methyltransferases, DNA repair proteins, E3 ubiquitin ligases, protein kinases and so on. Of these 84 proteins, 47 have been reported to exhibit altered binding affinity to CRYs in response to blue light, and 41 have been shown to exhibit condensation to CRY photobodies. The blue light‐regulated composition or condensation of CRY complexes results in changes of gene expression and developmental programs. In this mini‐review, we analyzed recent studies of the photoregulatory mechanisms of Arabidopsis CRY complexes and proposed the dual mechanisms of action, including the "Lock‐and‐Key" and the "Liquid‐Liquid Phase Separation (LLPS)" mechanisms. The dual CRY action mechanisms explain, at least partially, the structural diversity of CRY‐interacting proteins and the functional diversity of the CRY photoreceptors. [ABSTRACT FROM AUTHOR]

Objectives: Growing evidence demonstrates a relationship between epilepsy and the circadian system. However, relatively little is known about circadian function in disease states, such as epilepsy. This study aimed to characterize brain and peripheral core circadian clock gene expression in rat models of genetic and acquired epilepsy. Methods: For the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) study, we used 40 GAERS and 40 non‐epileptic control (NEC) rats. For the kainic acid status epilepticus (KASE) study, we used 40 KASE and 40 sham rats. Rats were housed in a 7 am:7 pm light–dark cycle. Hypothalamus, hippocampus, liver, and small intestine samples were collected every 3 h throughout the light period. We then assessed core diurnal clock gene expression of per1, cry1, clock, and bmal1. Results: In the GAERS rats, all tissues exhibited significant changes in clock gene expression (P < 0.05) when compared to NEC. In the KASE rats, there were fewer effects of the epileptic condition in the hypothalamus, hippocampus, or small intestine (P > 0.05) compared with shams. Significance: These results indicate marked diurnal disruption to core circadian clock gene expression in rats with both generalized and focal chronic epilepsy. This could contribute to epileptic symptomology and implicate the circadian system as a viable target for future treatments. [ABSTRACT FROM AUTHOR]

The circadian clock plays an integral role in hormone biosynthesis and secretion. However, how the circadian clock precisely coordinates hormonal homeostasis to maintain normal animal development remains unclear. Here, we show that knocking out the core clock gene Cryptochrome 1 (Cry1) significantly delays the developmental time in Bombyx mori. This study focuses on the ecdysone and juvenile hormone signalling pathways of fifth instar larvae with the longest developmental time delay. We found that the mutant reduced prothoracicotropic hormone synthesis in the brain, and could not produce sufficient ecdysone in the prothoracic gland, resulting in a delayed peak of 20‐hydroxyecdysone titre in the hemolymph of fifth instar larvae, prolonging developmental time. Moreover, further investigation revealed that the mutant enhanced juvenile hormone biosynthesis and signalling pathway and that this higher juvenile hormone titre also resulted in prolonged developmental time in fifth instar larvae. Our results provide insights into the molecular mechanisms by which the circadian clock regulates animal development by maintaining hormonal homeostasis. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis cryptochrome 1 (CRY1) is a blue light receptor distributed in the nucleus and cytoplasm. The nuclear CRY1, but not cytoplasmic CRY1, mediates blue light inhibition of hypocotyl elongation. However, the photobiochemical mechanisms distinguishing the CRY1 protein in the two subcellular compartments remains unclear. Here we show that the nuclear CRY1, but not the cytoplasmic CRY1, is regulated by phosphorylation, polyubiquitination and 26S proteasome‐dependent proteolysis in response to blue light. The blue light‐dependent CRY1 degradation is observed only under high fluences of blue light.The nuclear specificity and high fluence dependency of CRY1 explain why this photochemical regulatory mechanism of CRY1 was not observed previously and it further supports the hypothesis that CRY1 is a high light receptor regulating photomorphogenesis.We further show that the nuclear CRY1, but not cytoplasmic CRY1, undergoes blue light‐dependent phosphorylation by photoregulatory protein kinase 1 (PPK1) followed by polyubiquitination by the E3 ubiquitin ligase Cul4COP1/SPAs, resulting in the blue light‐dependent proteolysis. Both phosphorylation and ubiquitination of nuclear CRY1 are inhibited by blue‐light inhibitor of cryptochromes 1 (BIC1), demonstrating the involvement of photo‐oligomerization of the nuclear CRY1.These finding reveals a photochemical mechanism that differentially regulates the physiological activity of the CRY1 photoreceptor in distinct subcellular compartments. See also the Commentary on this article by Batschauer, 234: 1109–1111. [ABSTRACT FROM AUTHOR]

The trimethylation on histone H3 lysine 27 (H3k27me3), a transcriptionally repressive epigenetic mark of permissive chromatin, can be removed by the histone lysine demethylase 6a (Kdm6a). However, the physiological function of H3k27me3 and Kdm6a on circadian genes remains largely elusive. With the ChIP‐Seq and mRNA microarray assays, a critical role is identified for Kdm6a in the regulation of H3k27me3 to impact the expression of Crytochrome 1 (Cry1) in the hypothalamus of diet induced obesity mice. More importantly, both conditional knockout and pharmacological inhibition of Kdm6a reduce body weight and stabilize blood glucose homeostasis. Although a Kdm6a inhibitor fails to decrease body weight in leptin receptor‐deficient db/db mice, it significantly decreases Cry1 expression, enhances sensitivity to exogenous leptin administration, and blocks body weight increases in endo‐leptin‐deficient ob/ob mice. Moreover, gene analysis of the human hypothalamus further reveals a positive correlation between Kdm6a and Cry1. The results show that inhibition of Kdm6a reduces the Cry1 expression and sensitizes leptin signaling to combat obesity‐related disease. Therefore, it implicates Kdm6a as an attractive drug target for obesity and metabolic disorders. [ABSTRACT FROM AUTHOR]

Summary: Background: Recent findings suggest that circadian time regulates cellular functions in the skin and may affect protection against ultraviolet radiation (UVR). It is not known, however, whether UVR through skin directly affects the expression of circadian genes. We investigated the effect of ultraviolet B (UVB) exposure on cryptochrome circadian clock 1 (CRY1), cryptochrome circadian clock 2 (CRY2), and circadian associated repressor of transcription (CIART) genes. Methods: Healthy volunteers (n = 12) were exposed to narrow‐band UVB radiation of four standard erythemal dose (SED). Epidermal/dermal and subcutaneous adipose tissue samples were obtained by punch biopsies from irradiated and non‐irradiated skin 10 cm away from the irradiated site 24 hours after UVB exposure. Gene expression of CRY1,CRY2, and CIART was measured using RT‐PCR (TaqMan). Results: Ultraviolet B radiation affected mRNA expression in the epidermal/dermal skin and in the subcutaneous adipose tissue. It down‐regulated expression of CRY2 gene in the epidermal/dermal skin, whereas it up‐regulated expression of CRY1 and CIART genes in the subcutaneous adipose tissue. Conclusion: We showed for the first time that UVB radiation affects expression of circadian genes in the subcutaneous adipose tissue. Further studies are warranted to understand the mechanisms in detail. [ABSTRACT FROM AUTHOR]

Cardiotrophin (CT)-1 is a regulator of glucose and lipid homeostasis. In the present study, we analyzed whether CT-1 also acts to peripherally regulate metabolic rhythms and adipose tissue core clock genes in mice. Moreover, the circadian pattern of plasma CT-1 levels was evaluated in normal-weight and overweight subjects. The circadian rhythmicity of oxygen consumption rate (Vo2) was disrupted in aged obese CT-1-deficient (CT-1-/-) mice (12 mo). Although circadian rhythms of Vo2 were conserved in young lean CT-1-/- mice (2 mo), CT-1 deficiency caused a phase shift of the acrophase. Most of the clock genes studied (Clock, Bmal1, and Per2) displayed a circadian rhythm in adipose tissue of both wild-type (WT) and CT-1-/- mice. However, the pattern was altered in CT-1-/- mice toward a lower percentage of the rhythm or lower amplitude, especially for Bmal1 and Clock. Moreover, CT-1 mRNA levels in adipose tissue showed significant circadian fluctuations in young WT mice. In humans, CT-1 plasma profile exhibited a 24-h circadian rhythm in normal-weight but not in overweight subjects. The 24-h pattern of CT-1 was characterized by a pronounced increase during the night (from 02:00 to 08:00). These observations suggest a potential role for CT-1 in the regulation of metabolic circadian rhythms.--López-Yoldi, M., Stanhope, K. L., Garaulet, M., Chen, X. G., Marcos-Gómez, B., Carrasco-Benso, M. P., Santa Maria, E. M., Escoté, X., Lee, V., Nunez, M. V., Medici, V., Martínez-Ansó, E., Sáinz, N., Huerta, A. E., Laiglesia, L. M., Prieto, J., Martínez, J. A., Bustos, M., Havel, P. J., Moreno-Aliaga, M. J. Role of cardiotrophin-1 in the regulation of metabolic circadian rhythms and adipose core clock genes in mice and characterization of 24-h circulating CT-1 profiles in normal-weight and overweight/obese subjects. [ABSTRACT FROM AUTHOR]

Melatonin is an important rhythmic endocrine signal within the circadian system of mammals. The hypophyseal pars tuberalis (PT) is a major target for melatonin and shows a high density of melatonin type 1 receptors (MT1). Melatonin affects expression of clock genes in PT cells which encode for transcriptional regulators of rhythmic gene expression. In this study, microarray analysis was performed to screen for genes coding for transcriptional regulators under the control of MT1 receptors in the mouse PT. Gene expression levels were compared between melatonin-proficient mice deficient for MT1 (MT1−/−) and the corresponding wild-type (WT) during mid-subjective day (CT06, low endogenous melatonin levels) and mid-subjective night (CT18, high endogenous melatonin levels). In situ hybridization was used to validate the data obtained by microarray analysis to analyze the acute effect of daytime melatonin application on gene expression in the wild-type PT. In the wild-type PT, expression of Tim was higher during day as compared to night. These day/night differences in gene expression were not observed in the PT of MT1−/− mice, demonstrating the impact of MT1-mediated signal transduction pathway. Day-time application of melatonin acutely affected Tim and Cry1 expression but not Neurod1 and Npas4 expression. We conclude that melatonin regulates expression of genes coding for transcriptional regulators in the PT through MT1 receptors by either acute or long-term mechanisms. [ABSTRACT FROM AUTHOR]

Clock and Cry1 expression were examined in the pineal gland of chicken embryos incubated under constant darkness from embryonic day (ED) 0. From ED13, Clock and Cry1 mRNA levels showed episodic alterations. After ED17, circadian pattern of clock gene expression was seen both in vivo and in vitro. Our results support the idea that rhythmic environmental factors are not necessary for the generation of circadian patterns of clock gene expression during development. [ABSTRACT FROM AUTHOR]

Cryptochromes (CRY) are one of the two major classes of photoreceptors that perceive light stimuli in the UV-A to blue light region and they are involved in multiple aspects of plant growth and development. However, knowledge regarding their signaling transduction components and mechanisms remains limited. Here, we report that a MYB transcription factor Blue Insensitive Trait 1 (BIT1), plays an important role in controlling blue light responses. Hypocotyl growth responses indicate that BIT1 functions as a positive element in blue light signaling, since BIT1 antisense and knock-out lines show a reduced light response in blue light. BIT1 controls blue light-dependent expression of various genes such as PsbS, a member of the light-harvesting complex gene family. A transactivation assay showed that BIT1 regulates promoter activity of PsbS in a blue light-dependent manner and that it requires CRY1 for activation of the PsbS promoter. BIT1 undergoes degradation in darkness and CRY1 functions to stabilize BIT1 in a blue light-dependent manner. In contrast, COP1 binds to BIT1 and mediates its degradation. We propose that the PsbS promoter is activated in blue light via the blue light-dependent stabilization of BIT1 by CRY1, while in darkness BIT1 is degraded by COP1-mediated proteolysis. [ABSTRACT FROM AUTHOR]

Background: In rats, alcohol exposure during the period of rapid brain growth produces long-term changes in the free-running period, photoentrainment and phase-shifting responses of the circadian rhythm in wheel-running behavior. To determine whether these alterations in circadian behavior are associated with permanent damage to the circadian timekeeping mechanism or reconfiguration of its molecular components, we examined the long-term effects of neonatal alcohol exposure on clock gene rhythms in the pacemaker located in the suprachiasmatic nucleus (SCN) and in other brain or peripheral tissues of adult rats. Methods: Artificially reared male rat pups were exposed to alcohol (4.5 g/kg/d) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4 to 9. At 3 months of age, animals were exposed to constant darkness and then SCN, cerebellum, and liver tissue were harvested at 6-hour intervals for subsequent analysis of Period1 ( Per1), Per2, Cryptochrome1 ( Cry1), Bmal1, and Rev-erbα mRNA levels by quantitative PCR. Results: In the SCN, cerebellum and liver, Per1, Per2, Cry1, Bmal1, and Rev-erbα expression oscillated with a similar amplitude (peak-to-trough differences of 2- to 9-fold) and phase in the suckle control (SC) and GC groups. These clock gene rhythms in control animals were marked by peak expression of Per1, Per2, Cry1, and Rev-erbα during the subjective day and of Bmal1 during the subjective night. The EtOH group was distinguished by altered rhythms in the expression of specific clock genes within the SCN, cerebellum and liver. In EtOH-treated rats, the SCN rhythm in Cry1 expression was strongly damped and the Per2 rhythms in the cerebellum and liver were phase-advanced such that peak expression occurred during the mid-subjective day. Conclusions: These results demonstrate alcohol exposure during the brain growth spurt alters the circadian regulation of some molecular components of the clock mechanism in the rat SCN, cerebellum, and liver. The observed alterations in the temporal configuration of essential “gears” of the molecular clockworks may play a role in the long-term effects of neonatal alcohol exposure on the regulation of circadian behavior. [ABSTRACT FROM AUTHOR]